JAK2 V617F, MPL, and CALR Mutations in Korean Patients with Essential Thrombocythemia and Primary Myelofibrosis

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

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B Cell Immune Profiles in Essential Thrombocythemia with Calr Mutations: Clinical and Molecular Correlates

Blood ◽

10.1182/blood.v126.23.1610.1610 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1610-1610

Author(s):

Yu-Cheng Chang ◽

Ken-Hong Lim ◽

Huan-Chau Lin ◽

Yi-Hao Chiang ◽

Ling Huang ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Healthy Adults ◽

Tlr4 Expression ◽

Reactive Thrombocytosis ◽

Membrane Bound ◽

Calr Mutations ◽

Activated B Cells

Abstract Introduction: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood. We have reported that activated B cells are increased in patients with essential thrombocythemia, and can facilitate platelet production mediated by cytokines, such as interleukin-1beta (IL-1β) and interleukin-6 (IL-6) regardless JAK2 V617F mutational status (Thromb Haemost. 2014, 112: 537). Recently, Calreticulin (CALR) mutations were discovered in JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. Although CALR mutations may be associated with activated JAK-STAT signaling pathway, its exact molecular pathogenesis remains elusive in MPN. Interestingly, in vitro study has shown that CALR is capable of driving B cells activation through the toll-like receptor 4 (TLR4) pathway (J Immunol.2010; 185: 4561). Here we sought to evaluate the association between CALR mutations and B cell immune profiles in ET patients. Methods: Fifty-four patients diagnosed with ET based on the 2008 WHO classification were enrolled into this study. CALR mutations were screened by high-resolution melting analysis and nucleotide sequencing. JAK2 V617F and MPL mutations were screened by allele-specific PCR and nucleotide sequencing, respectively. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) and CALR levels, B cells TLR4 expression and intracellular levels of IL-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF and plasma CALR concentrations were measured by ELISA. Forty-eight healthy adults and 17 patients with reactive thrombocytosis were used for comparison. The association between clinical, laboratory and molecular characteristics were studied. Statistical significance was defined as a two-sided p value <0.05 and SPSS version 22.0 (IBM, New York, USA) was used for all analyses. Results: In this series, 19 (35.2%) patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2 V617F mutations. Compared to JAK2 V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Among all ET patients, CALR mutations correlated with significantly lower serum BAFF level (median 1.6 ng/mL, p =0.049) and higher fraction of B cells with TLR4 expression (median 11.3%, p=0.021). Compared to healthy adults, patients with ET had statistically significant higher serum BAFF concentrations and higher mBAFF levels on both granulocytes and monocytes, and higher fraction of B cells with TLR4 expression and higher fractions of B cells with intracellular IL-1β and IL-6 expression irrespective of their genotypes. ET patients with both JAK2 and CALR mutations had statistically higher number of CD69-positive and CD86-positive activated B cells when compared with healthy adults. Among the three mutational groups of ET patients, there were no significant differences in granulocytes/monocytes mBAFF, in the fraction of B cells with intracellular IL-1β or IL-6 expression, and the numbers of CD80-positive and CD86-positive activated B cells. Granulocyte membrane-bound CALR levels were highest in patients with reactive thrombocytosis. Plasma CALR concentrations were highest in patients with reactive thrombocytosis (mean +/- SE: 9.04 +/- 0.59) and lowest in CALR -mutated ET patients (5.35 +/- 0.90, p <0.001). Conclusions: Activation of B cells is universally present in ET. Both granulocyte membrane-bound CALR levels and plasma CALR concentrations were lower in CALR-mutated ET patients suggesting that CALR may not play a major role in the activation of B cells in these patients. Disclosures No relevant conflicts of interest to declare.

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STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

Cancers ◽

10.3390/cancers12041021 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1021 ◽

Cited By ~ 3

Author(s):

Emir Hadzijusufovic ◽

Alexandra Keller ◽

Daniela Berger ◽

Georg Greiner ◽

Bettina Wingelhofer ◽

...

Keyword(s):

Stem Cells ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Signaling Cascades ◽

Nuclear Compartment ◽

Downstream Signaling ◽

Direct Targeting ◽

Calr Mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

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Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0054 ◽

2019 ◽

Vol 44 (4) ◽

pp. 492-498

Author(s):

Gonca Gulbay ◽

Elif Yesilada ◽

Mehmet Ali Erkurt ◽

Harika Gozukara Bag ◽

Irfan Kuku ◽

...

Keyword(s):

Blood Cell ◽

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Hematological Parameters ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

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A Phase I Study of XL019, a Selective JAK2 Inhibitor, in Patients with Primary Myelofibrosis, Post-Polycythemia Vera, or Post-Essential Thrombocythemia Myelofibrosis

Blood ◽

10.1182/blood.v112.11.98.98 ◽

2008 ◽

Vol 112 (11) ◽

pp. 98-98 ◽

Cited By ~ 25

Author(s):

Neil P. Shah ◽

Patrycja Olszynski ◽

Lubomir Sokol ◽

Srdan Verstovsek ◽

Ronald Hoffman ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Reversible Inhibitor ◽

Hematologic Toxicity ◽

Low Grade ◽

Costal Margin ◽

Spleen Size ◽

Activating Mutation

Abstract JAK2 V617F has been identified as a constitutive activating mutation in approximately half of patients with myelofibrosis (MF). MF, a myeloproliferative disorder comprised of primary myelofibrosis and the clinically indistinguishable entities of post-polycythemia vera or post essential thrombocythemia MF, has been reported to have a median survival of 4 years [Dupriez et al. (1996) Blood88:1013–18]. No effective therapies exist for patients with MF. XL019 is a potent, highly selective and reversible inhibitor of JAK2 which may have utility in treating MF, by ameliorating hepato-splenomegaly, constitutional symptoms, and progressive anemia. The objectives of this phase 1 study include safety evaluation, preliminary assessments of efficacy using International Working Group (IWG) response criteria for MF, and evaluation of pharmacokinetic and pharmacodynamic endpoints. Pharmacodynamic evaluations include quantitative PCR for peripheral blood JAK2 V617F allele burden and erythropoietin-independent colony formation. In addition, plasma and fixed blood samples are being collected to evaluate changes in protein biomarkers and JAK2 signaling pathways. To date, XL019 has been studied in 21 patients over multiple dose levels ranging from doses of 25 mg to 300 mg using different schedules of administration (3 weeks on, 1 week off; QD; and QMWF). Median age was 64 years (range, 47–87 years) and 16 patients (76%) carried the JAK2V617F mutation. Additionally, one patient had a MPLW515F mutation in the absence of a JAK2 mutation. No treatment-related hematologic adverse events (i.e. thrombocytopenia, anemia, neutropenia) have been observed to date. Reversible low-grade peripheral neuropathy (PNP) was observed in 7/9 patients treated at daily doses of ≥100 mg (Grade 1: 5 patients; Grade 2: 2 patients). XL019 doses below 100 mg using 2 different dosing schedules are currently being evaluated. To date, XL019 has resulted in reductions in splenomegaly and leukocytosis, stabilization of hemoglobin counts, improvements in blast counts, and resolution or improvement in generalized constitutional symptoms. The median spleen size in 15 patients measured below the costal margin by palpation was 14cm (range, 3–26cm). Three of 15 patients with palpable splenomegaly at baseline were JAK2 V617F mutation negative and did not experience spleen size reduction. Twelve of 12 (100%) evaluable patients with an activating mutation (JAK2 V617F: 11 patients; MPLW515F: 1 patient) experienced reduction in spleen size and 5 (42%) had a ≥50% decline from baseline. Ten of 11 patients with JAK2V617F activating mutations and baseline constitutional symptoms, reported improvements in generalized constitutional symptoms which include pruritus and fatigue. No significant non-hematologic or hematologic toxicity has been observed at the current dose level. On 25 mg dosing schedules, no signs of PNP have been observed with a follow-up period of up to 4 months. Overall, XL019 has demonstrated encouraging clinical activity and is generally well tolerated.

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Sequential Analysis By Next Generation Sequencing of 18 Genes in Polycythemia Vera and Essential Thrombocythemia Reveals a Association Between Mutational Status and Clinical Outcome after 3-Year Follow-up

Blood ◽

10.1182/blood.v126.23.2808.2808 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2808-2808

Author(s):

Damien Luque Paz ◽

Aurelie Chauveau ◽

Caroline Buors ◽

Jean-Christophe Ianotto ◽

Francoise Boyer ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Poor Prognostic Factor ◽

Disease Evolution ◽

Allele Burden ◽

V617f Mutation

Abstract Introduction Myeloproliferative neoplasms (MPN) are molecularly characterized by driver mutations of JAK2, MPL or CALR. Other somatic mutations may occur in epigenetic modifiers or oncogenes. Some of them have been shown to confer a poor prognosis in primary myelofibrosis, but their impact is less known in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). In this study, we investigated the mutational profile using NGS technology in 50 JAK2 V617F positive cases of MPN (27 PV and 23 ET) collected at the time of diagnosis and after a 3 year follow-up (3y). Patients and Methods All patients were JAK2 V617F positive and already included in the prospective cohort JAKSUIVI. All exons of JAK2, MPL, LNK, CBL, NRAS, NF1, TET2, ASXL1, IDH1 and 2, DNMT3A, SUZ12, EZH2, SF3B1, SRSF2, TP53, IKZF1 and SETBP1 were covered by an AmpliseqTM custom design and sequenced on a PGM instrument (Life Technologies). CALR exon 9 mutations were screened using fragment analysis. Hotspots that mutated recurrently in MPN with no sequencing NGS coverage were screened by Sanger sequencing and HRM. A somatic validation was performed for some mutations using DNA derived from the nails. The increase of a mutation between diagnosis and follow-up has been defined as a relative increase of twenty percent of the allele burden. An aggravation of the disease at 3y was defined by the presence of at least one of the following criteria: leukocytosis >12G/L or immature granulocytes >2% or erythroblasts >1%; anemia or thrombocytopenia not related to treatment toxicity; development or progressive splenomegaly; thrombocytosis on cytoreductive therapy; inadequate control of the patient's condition using the treatment (defined by at least one treatment change for reasons other than an adverse event). Results As expected, the JAK2 V617F mutation was found in all patients with the use of NGS. In addition, we found 27 other mutations in 10 genes out of the 18 genes studied by NGS (mean 0.54 mutations per patient). Overall, 29 of 50 patients had only the JAK2 V617F mutation and no other mutation in any of the genes analysed. No CALR mutation was detected. Nine mutations that were not previously described in myeloid malignancies were found. The genes involved in the epigenetic regulation were those most frequently mutated: TET2, ASXL1, IDH1, IDH2 and DNMT3A. In particular, TET2 mutations were the most frequent and occurred in 20% of cases. There was no difference in the number or in the presence of mutations between PV and ET. At 3y, 4 mutations appeared in 4 patients and 15 out of 50 patients (9 PV and 6 ET) were affected by an allele burden increase of at least one mutation. At 3y, 24/50 patients suffered an aggravation of the disease as defined by the primary outcome criterion (16 PV and 8 ET). The presence of a mutation (JAK2 V617Fomitted) at the time of the diagnosis was significantly associated with the aggravation of the disease (p=0.025). Retaining only mutations with an allele burden greater than 20%, the association with disease aggravation is more significant (p=0.011). Moreover, a mutation of ASXL1, IDH1/2 or SRSF2, which is a poor prognostic factor in primary myelofibrosis, was found in 8 patients, all having presented an aggravation of their disease (p=0.001). Only 4 patients had more than one somatic mutation other than JAK2 V617F and all of them also had an aggravation at 3y (p=0.046). In this cohort, appearance of a mutation at 3y was not associated with the course of the disease. Conversely, the increase of allele burden of at least one mutation was associated with an aggravation (p=0.019). Discussion and conclusion Despite the short follow-up and the limited number of patients, this study suggests that the presence of additional mutations at the time of the diagnosis in PV and TE is correlated to a poorer disease evolution. The increase of mutation allele burden, which reflects clonal evolution, also seems to be associated with the course of the disease. These results argue for a clinical interest in large mutation screening by NGS at the time of the diagnosis and during follow-up in ET and PV. Disclosures Ugo: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: ASH travel.

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Clinical Relevance of Molecular Markers in Taiwan Chinese Patients with Primary Myelofibrosis: Endogenous Erythroid Colony Growth Is the Most Important Predictor of Outcomes

Blood ◽

10.1182/blood.v126.23.4083.4083 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4083-4083

Author(s):

Ming-Chung Kuo ◽

Tzung-Chih Tang ◽

Tung-Liang Lin ◽

Po-Nan Wang ◽

Po Dunn ◽

...

Keyword(s):

Molecular Markers ◽

Clinical Relevance ◽

Triple Negative ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Important Predictor ◽

Over Expression ◽

Allele Burden ◽

Calr Mutations

Abstract Background and purpose: The clinical features and molecular markers of primary myelofibrosis (PMF) in Asian population have rarely been reported. We examined the clinical relevance of molecular markers in a large cohort of PMF patients in Taiwan. Methods: Bone marrow or blood samples at initial diagnosis from 145 patients consecutively diagnosed with PMF based on WHO criteria in Chang Gung Memorial Hospital-Linkou, Taiwan, were examined. EEC assay was performed in a serum free culture system. PRV-1 mRNA expression in granulocytes was measured by real-time RQ-PCR TaqMan assay. Pyrosequencing was used to detect JAK2 V617Fand its allele burden as well as 46/1 rs12343867 genotype in granulocytes. Mutational analysis of MPL (exon 10) was performed by PCR assay followed by direct sequencing. CALR (exon 9) mutations were screened by GeneScan analysis followed by sequencing for those with length changes. Ten of 20 patients progressed to secondary AML (sAML) had matched paired diagnosis and sAML samples available for comparative analysis. Results: Of the 145 patients with PMF, the median age was 64 years, 76 were male, IPSS low risk 25, Int I 23, Int II 41, and high risk 56 patients. In a median follow-up of 35.8 months (range 1.1 to 275.5 months), 20 patients progressed to sAML, 88 patients died with a median overall survival (OS) of 67.4 months. JAK2 V617F was detected in 52% (74/143) patients, CALR mutations in 30% (41/135) (type1 n=29; type 2 n=5; and others n=7), MPL mutations in 4% (5/141) (n=2/2/1 for W515L/K/A), and 11.0% of PMF patients were triple-negative. The incidence of 46/1 haplotype in 112 patients analyzed was TT 32 %, CT 36 %, and CC 32 %; C-allele frequency was significantly higher in PMF compared with 50 normal subjects (50% vs. 24%; P< 0.0001).EEC growth was detected in 48.9% (45/92) of patients examined. PRV-1 over-expression was present in 40% (28/70) of patients. Of the 10 matched paired PMF/sAML samples, 6 patients had CALR mutations with similar allele burden at both phases of disease whereas sAML evolved from a non-JAK2 V617F clone in one of the 3 patients carrying JAK2 V617F at diagnosis. Patients with EEC growth or PRV-1 over-expression were significantly associated with younger age, higher WBC and platelet counts. EEC-positive patients had higher Hb level and lower circulating blasts. JAK2 V617F was closely associated with higher WBC and platelet counts whereas patients with CALR mutations had lower WBC counts. None of these molecular markers had a correlation with constitutional symptom, IPSS, occurrence of thrombosis or risk of sAML transformation. EEC growth conferred a favorable leukemia-free survival (LFS) (P =0.019) and OS (P =0.013) compared with those without EEC. PRV-1 over-expression was associated with better OS (P =0.036). JAK2 V617F and MPL mutations did not influence LFS and OS. Allele burden of JAK2 V617F had no impact on outcomes. CALR mutations were associated with a favorable OS compared with mutation-negative patients (P =0.034). There were no difference in outcomes between type 1 and type 2 mutations of CALR. Patients with triple-negative mutations had a significantly inferior OS (P =0.020). CT genotype (46/1) was associated with shorter LFS (P =0.026). EEC growth was strongly associated with PRV-1 over-expression and JAK2 V617F mutation, whereas EEC formation and CALR mutations were mutually exclusive. In multivariate analysis, EEC growth was the most important predictor for LFS (HR 0.058; 95% CI: 0.005-0.676, P =0.023) and OS (HR 0.21; 95% CI 0.076-0.581, P =0.003) among the molecular markers; CALR mutations also held favorable OS (HR 0.245; 95% CI 0.085-0.709, P =0.009). Conclusions: Approximately 90% of PMF patients in Taiwan had JAK2 V617F, CALR, or MPL mutations, half were associated with C-allele genotype, 78% had EEC growth and /or PRV-1 over-expression. EEC growth was the most important independent factor for predicting better outcomes and CALR mutations also conferred a favorable OS. (Grant support: NSC96-2314-B-182-003, CMRPG330303, OMRPG3C0021, and MOHW103-TD-B-111-09) Disclosures No relevant conflicts of interest to declare.

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Function of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation

Blood ◽

10.1182/blood.v126.23.4079.4079 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4079-4079

Author(s):

Jun Yamanouchi ◽

Takaaki Hato ◽

Etsuko Matsubara ◽

Taichi Azuma ◽

Hiroshi Fujiwara ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Lower Risk ◽

Healthy Subjects ◽

Thrombotic Event ◽

Jak2 V617f ◽

Β1 Integrin ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Calr Mutations ◽

Calr Mutation

Abstract Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia and it has been turned out that CALR mutated patients have a lower risk of thrombosis than JAK2 V617F patients. However, the molecular mechanism for this differential risk remains obscure. It has been reported that CALR is a potential regulatory protein of integrin activation based on the interaction between CALR and a conserved sequence of GFFKR in the integrin α cytoplasmic tails. Recent studies suggest that calreticulin activates β1 integrin and modulates integrin-associated signaling. In this study, we examined if the mutant CALR proteins observed in patients with ET affect integrin αIIbβ3 activation which plays a crucial role on thrombus formation. We first identified mutations of JAK2, MPL, and CALR genes in 37 patients with WHO defined ET and explored clinical characteristics of patients with CALR mutation. The patients with JAK2 V617F were 22 (59%), MPL W515L was 1 (3%), and CALR mutations were 10 (27%). The two types of CALR mutations were found; deletion (52-bp deletion; c.1092_1143del) and insertion (5-bp insertion; c.1154_1155insTTGTC) mutations. The patients with CALR mutations had lower hemoglobin and leukocyte count compared with JAK2 V617F patients, but platelet count did not have a difference between the CALR and JAK2 mutation groups. Nine (41%) of 22 patients with JAK2V617F had a thrombotic event while 1 (10%) of 10 patients with CALR mutation did (p<0.05), suggesting that patients with CALR mutation had a lower risk of thrombosis than JAK2 V617F patients. Two patients with CALR mutations developed myelofibrosis while no patient with JAK2V617F did. One patient with CALR mutation developed acute myeloid leukemia, with persistence of the CALR mutation in his leukemic cells. To see if the CALR mutation affects functional status of αIIbβ3, we examined the binding of PAC1, a monoclonal antibody recognizing the active conformation of αIIbβ3, to platelets from 5 patients with CALR mutation and 12 patients with JAK2V617F in the presence or absence of ADP. Platelets from all the 5 patients with CALR mutation showed the same level of PAC1 binding as platelets from healthy subjects. Overexpression of recombinant CALR proteins in Chinese Hamster Ovary (CHO) cells expressing αIIbβ3 by transfection of a protein-expression vector containing wild-type, deletion, or insertion mutant CALR had no effect on PAC1 binding. We further examined adhesive function of CHO cells stably expressing αIIbβ3 and mutant or wild-type CALR to various concentrations of immobilized fibrinogen. Expression of wild-type or mutant CALR had no effect on αIIbβ3-mediated cell adhesion to fibrinogen. Moreover, each cell adherent to fibrinogen showed apparently the similar extent of spreading. On the other hand, platelets from 4 of 12 patients with JAK2V617F had an increase in PAC1 binding in the presence and absence of ADP compared with platelets from healthy subjects. All 4 (100%) of 4 patients with increased PAC1 binding had a thrombotic event while 4 (30%) of 13 patients with normal PAC1 binding did. Our study suggests no functional activation of integrin αIIbβ3 by CALR mutation, which is contrary to a recent finding that CALR activates β1 integrin. Nonetheless, our finding is rather in line with a clinical finding of a low risk for thrombosis in patients with CALR mutation and may provide the molecular basis for the differential thrombotic risk between the patient with CALR and JAK2 mutations. Disclosures Fujiwara: Celgene: Honoraria, Other: Travel, Acomodations, Expenses.

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TET2 Mutations in Ph-Negative Myeloproliferative Neoplasms: Identification of Three Novel Mutations and Relationship with Clinical and Laboratory Findings

BioMed Research International ◽

10.1155/2013/929840 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Andrea Patriarca ◽

Donatella Colaizzo ◽

Gianluca Tiscia ◽

Raffaele Spadano ◽

Silvia Di Zacomo ◽

...

Keyword(s):

Multivariate Analysis ◽

Sequence Analysis ◽

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Coding Region ◽

Novel Mutations ◽

Laboratory Findings ◽

Mutational Frequency

High-throughput DNA sequence analysis was used to screen for TET2 mutations in peripheral blood derived DNA from 97 patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). Overall six mutations in the coding region of the gene were identified in 7 patients with an overall mutational frequency of 7.2%. In polycythemia vera patients (n=25) 2 mutations were identified (8%), and in those with essential thrombocythemia (n=55) 2 mutations (3.6%); in those with unclassifiable MPN (n=8) 3 mutations (37.5%). No primary myelofibrosis patients (n=6) harboured TET2 mutations. Three unreported mutations were identified (p.P177fs, p.C1298del, and p.P411del), the first two in patients with unclassifiable MPN, the last in a patient with essential thrombocythemia. On multivariate analysis the diagnosis of an unclassifiable MPN was significantly related to the presence of TET2 mutations (P=0.02; OR: 2.81; 95% CI 1.11–7.06). We conclude that TET2 mutations occur in both JAK2 V617F-positive and -negative MPNs and are more frequent in MPN-U patients. This could represent the biological link between the different classes of myeloid malignancies.

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Combination approach to diagnosis and treatment of an elderly patient with chronic Ph-negative myeloproliferative neoplasm and concomitant surgical pathology. Clinical observation

MD-Onco ◽

10.17650/2782-3202-2021-1-1-61-65 ◽

2021 ◽

Vol 1 (1) ◽

pp. 61-65

Author(s):

Yu. E. Ryabukhina ◽

P. A. Zeynalova ◽

O. I. Timofeeva ◽

F. M. Abbasbeyli ◽

T. V. Ponomarev ◽

...

Keyword(s):

Elderly Patient ◽

Essential Thrombocythemia ◽

Early Stage ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Left Femur ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms

Chronic myeloproliferative neoplasms (CMPN), Ph-negative, are of clonal nature, develop on the level of hematopoietic stem cell and are characterized by proliferation of one or more hematopoietic pathways. Currently, the group of Ph-negative CMPN includes essential thrombocythemia, primary myelofibrosis, polycythemia vera, myeloproliferative neoplasm unclassifiable.Identification of mutations in the Jak2 (V617F), CALR, and MPL genes extended understanding of biological features of Ph-negative CMPN and improved differential diagnosis of myeloid neoplasms. Nonetheless, clinical practice still encounters difficulties in clear separation between such disorders as primary myelofibrosis, early-stage and transformation of essential thrombocythemia into myelofibrosis with high thrombocytosis. Thrombocytosis is one of the main risk factors for thromboembolic complications, especially in elderly people.A clinical case of an elderly patient with fracture of the left femur developed in the context of Ph-negative CMPN (myelofibrosis) with high level of thrombocytosis is presented which in combination with enforced long-term immobilization and presence of additional risk created danger of thrombosis and hemorrhage during surgery and in the postoperative period.

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