Mutations In Calreticulin Gene Research Articles

The Progress of Growth and Apoptosis Are Associated with Both of AURKA and Aurkb in Marimo Cells with Calr Gene Mutation

Objective: Mutations of calreticulin (CALR) are a breakthrough discovery in exon 9 as most common driver mutations in janus kinase 2 (JAK2) or thrombopoietin receptor (MPL)-negative mutant essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Several new inhibitors have displayed a relatively good therapeutic prospect after the JAK inhibitors, but still not completely cure the patients on their own. Thus, improvement of the therapeutic effect of these new inhibitors is particularly important through exploration of the related targets, which is probably compensated the function of original target in patients. Methods: The protein level of aurora was detected in CALR mutant patients, and was quantified by calculating the gray value of WB band. The biological function was examined in CALR mutant cell line (MARIMO) after aurora inhibition by EdU staining, colony assay, western blot, Annexin V, and flow cytometry assay, respectively. Transcriptomic analyzed the enriched genes and related signaling pathway in MARIMO cells with knockdown of Aurora A (AURKA) or Aurora B (AURKB). Results: Here, we found that Aurora B, similarly with Aurora A, was activated in CALR mutant patients. Selective inhibition of AURKA with MLN 8237 induced cell-growth arrest, maturation and apoptosis, and this cell phenotype was further enhanced when the AURKB was also inhibited as the increase of inhibitors' concentration. Specific downregulation of AURKB alone showed obvious inhibition on cell growth, and produced a similar effect on CALR mutant cells treated with MLN 8237 or interfered AURKA. Transcriptomic analyses revealed a replication reaction on cells caused by knockdown of AURKA or AURKB, which also induced a similar inhibition for mitogen-activated protein kinase (MAPK) signaling, but activation for metabolic and apoptosis pathway. Conclusion: These results suggested that both of AURKA and AURKB were associated with the progress of growth and apoptosis in MARIMO cells with CALR gene mutation, and provided a reference for recently proposed complementary therapies with Aurora kinase inhibitors in CALR mutated MPNs.

Myeloproliferative Patients with Calreticulin Gene Mutations: Clinical Manifestations and Outcome, a Single Institution Experience

Myeloproliferative Patients with Calreticulin Gene Mutations: Clinical Manifestations and Outcome, a Single Institution Experience

Impact of Higher Calreticulin Gene Mutation Variant Allele Frequency in Patients with Myelofibrosis

Introduction. Calreticulin (CALR) mutations represent the second most frequent driver mut in myeloproliferative neoplasms (MPN), accounting for 20-30% of patients (pts) with Essential Thrombocythemia (ET) and 25-35% Primary Myelofibrosis (PMF). There are 2 types of CALR mutation, Type 1/like (T1), a del52 bp deletion and similar, and Type 2/like (T2), a 5bp insertion and similar, both in exon 9. Most patients have CALR variant allele frequency (VAF) below 50% (conventionally defined as heterozygous, He), while a few were reported with higher VAF (homozygous, Ho). Homozygosity, that may be associated to LOH at chr19, is phenotypically hallmarked by MPO deficiency in myeloid cells. While well-defined hematologic, clinical and prognostics correlates of the VAF of JAK2V617F mutation were reported in MPN, little information is available regarding CALR. Aim. The aim of the study was to characterize the hematological and clinical correlates of higher CALR VAF in a large population of CALR mut pts with primary (PMF) and PET-MF. Patients and Methods. All patients with a confirmed WHO2016 and IWG-MRT diagnosis of PMF and PET-MF, respectively, available in our database, who were CALR mutated, were included. CALR mut was assayed by PCR amplification and capillary gel electrophoresis starting from peripheral blood granulocytes. The VAF was determined by automated interpolation of the area under the curve, and expressed as the mutant peak area divided by (mutant peak area + wild-type peak area). Mutation analysis of a panel of 40 myeloid neoplasm-associated genes was performed by NGS (Oncomine, ThermoFisher). The nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimate of survival and log-rank test were used as appropriate. Results. A total of 121 pts (42 prePMF, 34.7%; 36 overt PMF, 29.8%; 43 PET-MF, 35.5%) were considered. Overall, T1 was found in 87 pts (71.9%), of which 8 (6.6%) were T1-like; T2 in 34 pts (28-1%), of which 3 (2.5%) T2-like. The median CALR VAF was 51.4+10.4%. For the purposes of the analysis, we considered at Higher VAF (CALR-h) pts having a VAF of >95% confidence interval of the distribution in the population, ie a VAF >55%, compared to Lower VAF (CARL-l; <55%). CALR-h was found in 28 pts, accounting for 23.1% of the study population. The frequency of T1 and T2 variants was similar in CALR-h and CALR-l pts, 71% and 28% and 71% and 29%, respectively. There was a significant trend of increased representation of CALR-h pts from prePMF (n=6, 14.3%) to overt PMF (n=8, 22.2%) and PET-MF (n=14, 32.6%); no difference in T1 and T2 relative representation in the different diagnostic category. ASXL1 mut were enriched among CALR-h pts (13/28, 46.4% vs CALR-l, 24/87, 27.6%; P=0.04) as it was the presence of any >1 mutation in an NGS-panel of myeloid genes (CALR-h 82.5% vs 57.5% in CALR-l, P=0.02). However, limited to HMR mutations, no difference was outlined. There was no significant difference between CALR-h and CALR-l pts as regards gender, age, IPSS score, karyotype abnormalities, BM fibrosis grade, constitutional symptoms, splenomegaly, thrombosis and bleeding events at diagnosis. On the other hand, CALR-h pts had lower Hb level (10.8 g/dl (8.3-14.6) vs 12.1 (7.4-16); P=0.02) and platelet count (466x109/L (102-1053) vs 661 (57-1800; P=0.02), and higher peripheral blood CD34+ cell counts (62.2x106/L (4-3450) vs 16.9 (0-2100); P=0.01). A greater proportion of CALR-h pts developed anemia during the FU (median 5.5y, range 0.3-33y). KM analysis revealed an anemia-free survival (Hb <10g/dL) of 7.9y (5.7-10y) in CALR-h vs 22.1y (13.9-30.3y) in CALR-l pts, P=0.004 (Figure). The HR (95%CI) was 3.1 (1.4-7.0). Also leukocytosis-free survival (leukocytes >25x109/L) was shorter in CALR-h pts (P=0.04), HR 2.55 (1.0-6.6). A CALR-h status did not impact on overall survival nor leukemia-free survival even after stratification based on disease subtype. Discussion. We found that about 20% of CALR mut MF pts harbor a higher VAF, here defined as VAF >55%, that is associated with a more anemic phenotype at diagnosis and greater risk of developing anemia in the FU; however, in spite of anemia development, overall survival was not significantly impacted in the different disease categories. Whether a CALR-h condition has different impact in ET remains to be assessed in a separate study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real world data on myeloproliferative neoplasms in Japan

Since the discovery of the JAK2V617F, MPL gene, and Calreticulin gene mutations, remarkable changes have occurred in the identification of the pathology of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). The diagnostic criteria of polycythemia vera, essential thrombocythemia, and primary myelofibrosis in the world health organization classification systems have also been amended to include these driver gene mutations. Additionally, treatment algorithms for each disease have been reviewed. Following these changes, real world data form several countries based on national surveys have been reported. In Japan, the Japanese Society of Hematology has conducted a prospective study, named the JSH-MPN-15 study, to investigate the overall survival and risk factors of patients with MPNs. Furthermore, the retrospective JSH-MPN-R18 study was conducted and the results have been coming out. In this lecture, the results of these studies will be discussed.

Prevalence of JAK2V617F, CALR in Philadelphia Positive and Negative Myeloproliferative Neoplasm

Background: Myeloproliferative neoplasms (MPNs) are heterogeneous disorders with a variety of genetic abnormalities. We aim to assess the prevalence of Calreticulin (CALR) and JAK2 mutations in Iranian MPNs. Materials and Methods: In a cross-sectional study, CALR and JAK2 mutations among 130 MPNs patients, including 78 Philadelphia chromosome-negative (MPN-) and 52 Philadelphia chromosome-positive (MPN+) as well as 51 healthy control subjects, were investigated by GAP-PCR. Results: In MPN- group JAK2 and CALR gene mutations were found in 64.1% and 7.7%, respectively, that 5.1% were positive for both mutations, and 2.6% had only CALR mutation. In polycythemia vera (PV) patients 90% had JAK2 mutation, which was significantly higher than other MPN- or MPN+ patients. Most of the MPN+ patients had neither mutation in CALR nor JAK2 (70% CALR-/JAK2-). Among all patients’ groups, the prevalence of CALR+ mutation in either rs1450785140 (4 cases) or rs765476509 (5 cases) position was not statistically different. Conclusion: These results showed a low prevalence of CALR mutations in all types of MPNs in the Iranian population that its frequency may influence by ethnicity and genetic diversity. CALR mutation may be seen in JAK2 negative cases, also. The PV had the highest JAK2 mutation with a 90 percent positivity rate among MPNs cases. [GMJ.2021;10:e2127]

Prevalence of JAK2V617F, CALR in Philadelphia Positive and Negative Myeloproliferative Neoplasm

Background: Myeloproliferative neoplasms (MPNs) are heterogeneous disorders with a variety of genetic abnormalities. We aim to assess the prevalence of Calreticulin (CALR) and JAK2 mutations in Iranian MPNs. Materials and Methods: In a cross-sectional study, CALR and JAK2 mutations among 130 MPNs patients, including 78 Philadelphia chromosome-negative (MPN-) and 52 Philadelphia chromosome-positive (MPN+) as well as 51 healthy control subjects, were investigated by GAP-PCR. Results: In MPN- group JAK2 and CALR gene mutations were found in 64.1% and 7.7%, respectively, that 5.1% were positive for both mutations, and 2.6% had only CALR mutation. In polycythemia vera (PV) patients 90% had JAK2 mutation, which was significantly higher than other MPN- or MPN+ patients. Most of the MPN+ patients had neither mutation in CALR nor JAK2 (70% CALR-/JAK2-). Among all patients’ groups, the prevalence of CALR+ mutation in either rs1450785140 (4 cases) or rs765476509 (5 cases) position was not statistically different. Conclusion: These results showed a low prevalence of CALR mutations in all types of MPNs in the Iranian population that its frequency may influence by ethnicity and genetic diversity. CALR mutation may be seen in JAK2 negative cases, also. The PV had the highest JAK2 mutation with a 90 percent positivity rate among MPNs cases. [GMJ.2021;10:e2127]

The Synergistic Inhibitory Effect of Combining MK-2206 and AZD 6244 in MARIMO Cells Harboring a Calreticulin Gene Mutation

Introduction: Somatic mutations in the calreticulin (CALR) gene occur in most myeloproliferative neoplasm (MPN) patients who lack Janus kinase 2 or thrombopoietin receptor (MPL) mutations, but the molecular pathogenesis of MPN with mutated CALR is unclear, which limited the further treatment for CALR gene mutant patients. Objectives: Previous studies showed that CALR mutations not only activated serine/threonine protein kinase (AKT) in primary mouse bone marrow cells but also mitogen-activated protein kinases (MAPKs) in MARIMO cells harboring a heterozygous 61-bp deletion in CALR exon 9, which were responsible for mutant CALR cell survival, respectively. Hence, we aimed to initially explore the mechanism of AKT activation and observe the synergistic inhibitory effect of combining AKT (MK-2206) and MAPK kinase (AZD 6244) inhibitors in MARIMO cells. Methods: We detected the expression of phosphorylated AKT in MARIMO cells treated with inhibitors for 24 or 48 h by western blotting and analyzed cell proliferation, cell cycle, and apoptosis by flow cytometry. We further examined the synergistic inhibitory effect of combining MK-2206 and AZD 6244 in MARIMO cells using the median effect principle of Chou and Talalay. Results: We found that the AKT was activated in MARIMO cells, and blocking its activity significantly inhibited MARIMO cell growth with downregulation of cyclin D and E, and accelerated cell apoptosis by decreasing Bcl-2 but increasing Bax and cleaved caspase-3 levels in a dose-dependent manner. Further analysis showed that AKT activation was dependent on mammalian target of rapamycin but not on the JAK signaling pathway in MARIMO cells, displaying that inhibition of JAK activity by ruxolitinib (RUX) did not decrease the AKT phosphorylation. Furthermore, the combination of MK-2206 and AZD 6244 produced a significantly synergistic inhibitory effect on MARIMO cells. Conclusions: AKT activation is a feature of MARIMO cells and co-targeting of AKT and MAPKs signaling pathways synergistically inhibits MARIMO cell growth.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Frequency of coexistence and kinetics of the BCR-ABL1 transcript level and allele burden of JAK2V617F and CALR Type 1, 2 gene mutations in patients with chronic myeloid leukemia

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.

A Rare Hemoglobinopathy as a Cause of Erythrocytosis in a Patient with Suspected JAK2V617F Negative Polycythemia Vera: A Case Report

Introduction: Differential diagnosis of polyglobulia/erythrocytosis which is caused by deregulated erythropoiesis with an overproduction of red blood cells resulting in elevated hemoglobin and hematocrit levels is a diagnostic challenge. Case Report: A 31-year-old man was referred to us with a suspected diagnosis of polycythemia vera (a clonal myeloproliferative neoplasm) because of erythrocytosis. One year before, he suffered from an ischemic central retinal vein occlusion of unknown cause. Aspirin treatment was initiated. His mother was diagnosed with a JAK2V617F negative polycythemia vera years earlier and treated with phlebotomies as well as aspirin. Apart from erythrocytosis, laboratory analysis showed a normal white blood cell and platelet counts. The differential blood picture and lactate dehydrogenase were within the normal ranges. Molecular testing for BCR-ABL, JAK2V617F and Calreticulin gene mutations by PCR was negative. Bone marrow biopsy was normal without signs for a myeloproliferative neoplasm. As almost all patients with polycythemia vera carry the phenotype-driver mutation JAK2V617F, further genetic testing for congenital causes of erythrocytosis was conducted. Mutations in the EPO-receptor gene were not found, but a very rare heterozygous point mutation in the beta-globin-chain [exon 2 (c.119A>C) leading to a change in codon 40 (CAG>CCG)] was detected by next generation sequencing. This rare variant belongs to the high oxygen affinity hemoglobinopathies leading to a reduction of oxygen supply in tissues and an increase in red cell production. Conclusion: The diagnosis of a JAK2 mutation negative polycythemia vera must always be questioned and congenital causes of erythrocytosis excluded as the therapeutic and prognostic consequences are immense. Rare variants of hemoglobinopathies, particularly those with high oxygen affinity need to be excluded by molecular testing.

Significance of calreticulin gene mutations classification in BCR-ABL1 negative myeloproliferative neoplasms

Objective To investigate calreticulin (CALR) gene mutations classification in BCR-ABL1 negative myeloproliterative neoplasms (MPN), and its relationship with clinical manifestations. Methods Genomic DNA polymerase chain reaction (PCR) amplification product Sanger sequencing method was used to detect the mutation of exon 9 of CALR gene in 236 patients with BCR-ABL1 negative MPN (excluding polycythemia vera and negative CALR mutations) in Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from November 2015 to November 2018. The mutations were classified into 52 bp deletion (type 1) mutation, 5 bp insertion (type 2) mutation and other mutation types according to PCR sequencing analysis. The clinical characteristics of the carriers with two kinds of mutations in 198 patients with essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) were compared. For the types of mutations that could not be determined, they were classified according to the α-helix propensity score of the mutant protein peptide chain or the degree of retention of the negatively charged amino acid residues, and the differences between the two classification methods were also compared. Results Among 236 patients, the CALR gene type 1 or type 2 mutation was detected in 206 cases (87.3%), including 173 ET patients (99 cases of type 1 mutation and 74 cases of type 2 mutation) and 33 PMF patients (28 cases of type 1 mutation and 5 cases of type 2 mutation). The CALR non-type 1 or non-type 2 mutation was detected in 30 cases, including 25 ET patients and 5 PMF patients. Among 173 ET patients with CALR gene mutation, the white blood cell count (WBC) of patients with type 1 mutation was higher than that of patients with type 2 mutation [(8.6±2.7)×109/L vs. (7.6±2.4)×109/L, t = 2.45, P = 0.015]. Among 33 PMF patients with CALR gene mutation, the age of patients with type 1 mutation was older than that of patients with type 2 mutation [(58±13) years old vs. (41±16) years old, t = 2.51, P = 0.018]. According to the α-helix propensity score of mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues, 27 kinds of non-type 1 or non-type 2 mutations were classified by using sequencing method, and there were differences between the two methods. According to the α-helix propensity score of the mutant protein peptide chain, the proportion of type 1/type 1-like mutation in PMF patients was higher than that in ET patients [78.9% (30/38) vs. 56.6% (112/198), P < 0.01]. According to the degree of retention of negatively charged amino acid residues in the mutant protein peptide chain, the isoelectric point (pI) value of the mutant protein peptide chain was higher than that of the wild type sequence. The pI value of the type 1-like mutant protein peptide chain was higher than that of the type 2-like mutation (11.79±0.15 vs. 10.02±0.42, t = 11.51, P < 0.01). Conclusions Type 1 mutated ET patients may be closely related to the high risk of myelofibrosis transformation. The results of the classification of CALR mutations are different according to the α-helix propensity score of the mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues. Further study is necessary to identify the pathogenesis of MPN caused by CALR mutation, and to determine the relationship between mutation type and prognosis of disease. Key words: Myeloproliferative neoplasms; Calreticulin; Thrombocythemia, essential; Primary myelofibrosis; Mutation; DNA mutational analysis

Increased Frequency of Thromboses Among Patients with Philadelphia (Ph) Negative Myeloproliferative Neoplasms in New Mexico: The Role of Prothrombotic Risk Factors

Introduction Thromboses are a common complication in patients with myeloproliferative neoplasms (MPN) and are reported to occur at a rate of 15- 20% at sea level. In the MPN-Thromboses spectrum which includes both arterial and venous thrombosis, cardiovascular disease (CVD) is noted to be the most common thrombotic event. JAK2 V617F is reported to be the most common driver mutation in MPNs and is associated with increased risk of thromboses. CALR and MPL are other mutations whose contribution to the thrombotic phenotype is not known. Chronic hypoxia from living in moderate or high altitude is reported to be an independent prothrombotic risk factor. The average elevation of New Mexico is 5,700 feet (1,740 meters) above sea level. The goal of this study is to evaluate the frequency of thromboses and prothrombotic risk factors in patients with MPN in patients in this distinct population. Methods We reviewed 134 patients, who were diagnosed with MPN in University of New Mexico Comprehensive Cancer Center between 2001 to 2019. A retrospective chart review was conducted to identify demographics, clinical and molecular risk factors for both arterial and venous thromboses. The mutation analyses for Janus Kinase 2 (JAK2), myeloproliferative leukemia (MPL) gene and calreticulin (CALR) gene were performed by polymerase chain reaction (PCR). Contingency table and logistic regression methods were applied to analyze and compare the distribution of the prothrombotic risk factors between the patients with and without thrombosis. Results In this study, 62 patients (47%) were diagnosed with ET, 47 patients (35%) with PV, and 22 patients (17%) with primary myelofibrosis (PMF). Seventy-five patients (56%) were females. Mean age at diagnosis was 62 years. 102 patients (77%) were living in the Albuquerque metropolitan area with an average elevation of 5312 feet above sea level and others were in areas with an elevation of 6000 feet or higher in New Mexico. Forty-four patients (33%) experienced either arterial (29) or venous thromboses (11) or both (4). A significant percentage (70.4%) of thrombotic events were either ischemic stroke or myocardial infarction. The patients with thromboses were predominantly males (21/36, 57%) while most patients without thromboses were females (56/90, 62%) with p=0.003. Twenty-one (53%) patients with thromboses had ET; however, a higher proportion of patients with PV (20/47, 42.5%) developed thromboses compared to ET or PMF (32.2% and 9% respectively). Also, a significant number of patients (32/44, 76%) with thromboses have JAK2 mutations while only 4 patients (9%) have CALR gene mutation. Although not statistically significant, CALR mutation was associated with lesser thrombotic events than other MPN patients. In univariate logistic regression analysis, PV and ET were significantly associated with increased thrombotic events. Patients with PV showed a 7.7-fold increase and patients with ET have a 5.1-fold increase in odds for thrombosis compared to patients with PMF (p=0.0365). Female gender was associated with decreased thrombotic risk with an odds ratio of 0.46 (p=0.0387). There was no significant difference between patients with and without thromboses, regarding other clinical characteristics such as age, previous aspirin use, leukocytosis, diabetes, hypertension, hyperlipidemia, obesity, and smoking. Conclusion An increased frequency of thromboses was observed among patients with Ph negative MPN in New Mexico which is significantly higher than previously reported studies. This strongly suggests the role of mild to moderate hypoxia as a contributing prothrombotic risk factor for MPN. The role of chronic hypoxia and its influence in thrombotic events in MPN need to be further evaluated in prospective studies. The decreased risk of thrombosis in females and patients harboring CALR mutations compared to other common mutations was consistent with other published studies. JAK2 mutation was not associated with an increased risk of thrombosis. Other genetic factors in this population were not evaluated in this study. Disclosures Arana Yi: Jazz Pharmaceuticals: Other: Advisory Board.

Research progress of thrombosis in myeloproliferative neoplasms

Classic Ph negative chronic myeloproliferative neoplasms (MPN) comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are characterized by stem cell-derived clonal myeloproliferative diseases. Thrombotic complications are main reasons of morbidity and mortality in patients with MPN. At present, studies indicate that the most important risk factors of thrombosis in MPN patients are advanced age (>60 years old) and previous history of thrombosis. High white blood cell count and leukocyte activation are risk factors for thrombosis. Studies have pointed out Janus kinase (JAK) 2V617F, calreticulin (CALR), MPL and other genes mutation also affect the thrombosis of MPN. MPN patients need to be assessed the risk of thrombosis before treatment, and received a purposeful and different treatment. This article focuses on thrombosis risks against MPN patients with advanced age (>60 years old), history of thrombosis, cardiovascular events risk factors, JAK2, MPL, CALR gene mutations or leucocytosis and the treatment of MPN. Key words: Thrombosis; Thrombocytosis; Polycythemia; Myeloproliferative neoplasma; Mutation; Leukocyte count

Selective testing for calreticulin gene mutations in patients with splanchnic vein thrombosis: A prospective cohort study

Myeloproliferative neoplasms (MPN) are the leading cause of splanchnic vein thrombosis (SVT). Janus kinase 2 gene (JAK2)V617F mutations are found in 80 to 90% of patients with SVT and MPN. Mutations of the calreticulin (CALR) gene have also been reported. However, as their prevalence ranges from 0 to 2%, the utility of routine testing is questionable. This study aimed to identify a group of patients with SVT at high risk of harboring CALR mutations and thus requiring this genetic testing. CALR, JAK2V617F and thrombopoietin receptor gene (MPL) mutations were analysed in a test cohort that included 312 patients with SVT. Criteria to identify patients at high risk of CALR mutations in this test cohort was used and evaluated in a validation cohort that included 209 patients with SVT. In the test cohort, 59 patients had JAK2V617F, five had CALR and none had MPL mutations. Patients with CALR mutations had higher spleen height and platelet count than patients without these mutations. All patients with CALR mutations had a spleen height ⩾16cm and platelet count >200×109/L. These criteria had a positive predictive value of 56% (5/9) and a negative predictive value of 100% (0/233) for the identification of CALR mutations. In the validation cohort, these criteria had a positive predictive value of 33% (2/6) and a negative predictive value of 99% (1/96). CALR mutations should be tested in patients with SVT, a spleen height ⩾16cm, platelet count >200×109/L, and no JAK2V617F. This strategy avoids 96% of unnecessary CALR mutations testing. Lay summary: Mutations of the CALR gene are detected in 0 to 2% of patients with SVT, thus the utility of systematic CALR mutation testing to diagnose MPN is questionable. This study demonstrates that CALR mutations testing can be restricted to patients with SVT, a spleen height ⩾16cm, a platelet count >200×109/L, and no JAK2V617F. This strategy avoids 96% of unnecessary CALR mutations testing.

CALR gene mutational profile in myeloproliferative neoplasms with non-mutated JAK2 in Moroccan patients: A case series and germline in-frame deletion

BackgroundThe discovery of somatic mutations within the gene encoding calreticulin (CALR) in 2013 represented a major milestone in the molecular diagnosis of BCR-ABL negative myeloproliferative neoplasms (MPN). In fact, exome sequencing revealed that most patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) lacking JAK2 or MPL mutations, harbor somatic insertion and/or deletion in exon 9 of CALR gene. In this study, we identified the first CALR gene mutational landscape in Moroccan patients with MPN nonmutated for the JAK2 gene. MethodsWe performed Sanger sequencing of exon 9 of CALR gene in blood samples obtained from 33 Moroccan patients with ET or PMF non-mutated for JAK2. ResultsOf the 33 patients analyzed, we detected eight distinct variants in 15 patients (45.4%); six indel mutations, five with type 1 recurrent 52bp deletion, four with type 2 recurrent 5bp insertion and one in frame deletion which was found to be a germline variant suggesting a very rare condition in MPN. ConclusionThis is the first cohort reported in CALR gene mutation analysis in Morocco. Our results were concordant with studies reported up to date and very encouraging in promoting the molecular diagnosis of myeloproliferative neoplasms in Moroccan patients. Moreover, the presence of a germline in frame deletion in a symptomatic patient should undermine the effectiveness of sizing assays without DNA sequencing in the diagnosis of CALR mutations.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting

IntroductionRecently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60–80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. MethodsWe examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). ResultsAmong 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. ConclusionOur novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN.

Clinical Manifestation of Calreticulin Gene Mutations in Essential Thrombocythemia without Janus Kinase 2 and MPL Mutations: A Chinese Cohort Clinical Study.

Background:Recently, calreticulin (CALR) gene mutations have been identified in patients with essential thrombocythemia (ET). A high-frequency of ET cases without Janus kinase 2 (JAK2) mutations contain CALR mutations and exhibit clinical characteristics different from those with mutant JAK2. Thus, we investigated the frequency and clinical features of Chinese patients of Han ethnicity with CALR mutations in ET.Methods:We recruited 310 Chinese patients of Han ethnicity with ET to analyze states of CALR, JAK2V617F, and MPLW515 mutations by polymerase chain reaction and direct sequencing. We analyzed the relationship between the mutations and clinical features.Results:CALR, JAK2V617F, and MPLW515 mutations were detected in 30% (n = 92), 48% (n = 149), and 1% (n = 4) of patients with ET, respectively. The mutation types of CALR involved deletion and insertion of base pairs. Most of them were Type 1 (52-bp deletion) and Type 2 (5-bp insertion, TTGTC) mutations, leading to del367fs46 and ins385fs47, respectively. The three mutations were exclusive. Clinically, patients with mutated CALR had a lower hemoglobin level, lower white blood cell (WBC) count, and higher platelet count compared to those with mutated JAK2 (P < 0.05). Furthermore, a significant difference was found in WBCs between wild-type patients (triple negative for JAK2, MPL, and CALR mutations) and patients with JAK2 mutations. Patients with CALR mutations predominantly clustered into low or intermediate groups according to the International Prognostic Score of thrombosis for ET (P < 0.05).Conclusions:CALR mutations were frequent in Chinese patients with ET, especially in those without JAK2 or MPL mutations. Compared with JAK2 mutant ET, CALR mutant ET showed a different clinical manifestation and an unfavorable prognosis. Thus, CALR is a potentially valuable diagnostic marker and therapeutic target in ET.

Diversities of Calreticulin Gene Mutations in Macedonian Patients With Essential Thrombocythemia.

Acquired calreticulin (CALR) gene mutations are one of the molecular hallmarks of essential thrombocythemia (ET). It has been suggested that patients with ET with CALR mutations are associated with a distinct clinical phenotype. We evaluated the clinical and molecular features of 150 patients with ET followed over a period of 15 years. The screening for the presence of insertion/deletion mutations in CALR exon 9 was done with a fluorescent polymerase chain reaction/capillary electrophoresis procedure. Sanger sequencing of CALR exon 9 was used for the characterization of mutations and for the analysis of triple-negative patients. CALR mutations were detected in 42 (28%) patients. The most common CALR mutations were type 1 and type 2, which were present in 11 (26.2%) and 20 (47.6%) patients, respectively. Additionally, 10 different small insertion/deletions (3 known and 7 new) were detected in 11 patients, resulting in an altered calreticulin C-terminal end. The clinical characteristics of all CALR+ patients with ET were in line with previously published data for this subset of patients. Our results showed that a wide range of different CALR mutations are associated with a distinct ET clinical phenotype that is associated with the male gender, younger age at diagnosis, higher platelet and lower leukocyte and erythrocyte counts and lower hemoglobin level, and a milder clinical course. The relatively high frequency of new insertion/deletion mutations indicate that the use of fluorescent polymerase chain reaction followed by capillary electrophoresis is the method of choice for the analysis of these defects.

Relationship between Calreticulin Gene Mutation and JAK2/MPL Negative Myeloproliferative Neoplasms

In 2008, WHO made the JAK2V617F gene mutation as one of the specific molecular diagnostic markers of BCR/ABL-negative myeloproliferative neoplasms (MPN). In 2013 two research teams demonstrated that whole genome sequencing technology (WGS) was used to detect calreticulin gene mutation in essential thrombocythaemia (ET) and primary myelofibrosis (PMF) patients with JAK2V617F⁻ and MPL⁻ mutations. In this review, the relationship of CALR gene mutation with MPN is briefly summarized.

The JAK2V617F mutation disrupts the regulated association between calreticulin and the glucocorticoid receptor observed in normal erythroid cells

Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasmic reticulum that mediates the cellular response to Ca2+ by chaperoning other proteins to their acting sites. Somatic loss-of-function mutations in the CALR gene were recently discovered in 70% of patients with the Philadelphia-negative myeloproliferative neoplasm (MPN) primary myelofibrosis (PMF) who did not harbor gain-of-function mutations of JAK21,2. Nevertheless, the JAK2 pathway is constitutively activated also in patients carrying CALR mutation and treatments with JAK2 inhibitors are effective not only in MPN patients (PMF and polycythemia vera, PV) harboring JAK2 mutations but also in PMF patients harboring mutations in CALR3. We have previously reported that erythroid cells from PV and PMF patients express abnormal activity of the glucocorticoid receptor (GR), a nuclear receptor whose transcriptional activity plays an important role in the regulation of stress erythropoiesis4,5. Since GR is one of the numerous proteins regulated by CALR6, we hypothesized that in human erythroid cells CALR regulates GR functions and that this regulation is disrupted both by CALR and JAK2 mutations in MPN. In this study we tested this hypothesis by determining whether GR and CALR are associated in normal erythroid cells and whether this association is impaired in those from MPN patients. First, biochemical studies determined that human erythroblasts (Erys) expanded ex-vivo from normal stem cell sources [cord blood (CB) and adult blood (AB)] and from MPN patients contain similar levels of CALR and GR. Analyses of cell fractions indicated that in normal Erys, CALR was constitutively localized in the cytoplasm while GR was detected either in the cytoplasm or in the nucleus, depending on the growth factor (the glucocorticoid receptor agonist dexamethasone, erythropoietin or stem cell factor) to which they had been exposed. Second, robust levels of CALR and GR expression were also detected by confocal microscopy. In addition, this analyses revealed that in Erys expanded from normal sources CALR and GR are co-localized in the cytoplasm and that the cytoplasmic association between the two proteins is increased by growth factor deprivation and further enhanced by stimulation with growth factors that activate the JAK2/STAT5 signaling (dexamethasone and/or erythropoietin) while it is inhibited by stimulation with factors that do not use this pathway (stem cell factor). By contrast, in Erys expanded from MPN carrying either CALR or JAK2 mutations, CALR and GR are not associated and remain not associated when the cells are exposed to dexamethasone or erythropoietin. However, in Erys from JAK2V17F-positive MPN patients, association between CALR and GR in the cytoplasm is restored by exposing the cells to the JAK2 inhibitor ruxolitinib. These results suggest that CALR/GR association is a downstream event induced by the JAK2/STAT5 pathway and identify for the first time that CALR functions are impaired in erythroid cells from MPN patients carrying JAK2 mutations.