scholarly journals Procoagulant Platelet Subpopulation As a Novel Predictive Biomarker of Poor Outcomes in Essential Thrombocythemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3622-3622
Author(s):  
Christine Shu Mei Lee ◽  
Kristen L Estabrooks ◽  
Kaitao Lai ◽  
Agnibesh Dey ◽  
Jonathan Cheng ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Risk Stratification ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Healthy Controls ◽  
Event Analysis ◽  
Time To Event ◽  
Mutation Status ◽  
Poor Outcomes

Abstract Background: Patients with essential thrombocythemia (ET) remain at risk of significant thrombotic complications, despite use of validated risk stratification and treatment pathways. Procoagulant platelets, a platelet subpopulation which supports thrombin generation, are elevated in prothrombotic conditions. We hypothesize that ET patients exhibit high procoagulant platelet potential and the procoagulant platelet subset may be a biomarker for risk of poor disease outcomes, enabling identification of patients for therapeutic escalation. Methods: 70 ET patients, 20 polycythemia vera (PV) patients and 32 age-matched healthy controls were recruited at Concord Hospital, Australia and Singapore General Hospital (development cohort). Outcome data including thrombosis, death and transformation to myelofibrosis and acute leukemia were prospectively collected. Procoagulant platelets were measured by uptake of a tripeptide trivalent arsenical (GSAO) combined with P-selectin expression by flow cytometry, with or without stimulation with increasing thrombin concentrations (0.5-5 U/mL) ± 10 µg/mL collagen, in whole blood samples. a Flow cytometry data were analyzed using both standard and a multiparameter clustering approach (FlowSOM) with FlowJo software. A hypergate algorithm based on fluorescence intensity of six parameters was developed using R programming language to enumerate a novel GSAO+ platelet subpopulation in each patient flow cytometry dataset. Time to event analysis was performed on cohorts defined by receiver operating characteristics (ROC) analysis identified cut-off for the subpopulation, with clinician adjudication of outcomes blinded to biomarker data. 25 consecutive ET patients were subsequently recruited from Concord Hospital outpatient clinics in a prospective validation study for an independent time to event analysis. Results: ET (n=70) and PV (n=20) patients in the development cohort demonstrated marked increased GSAO+/P-selectin+ procoagulant response to thrombin±collagen compared to healthy controls (n=32), segregating according to JAK2 V617F mutation status (Figure 1). Multivariate analysis (age, sex, thrombosis history, cardiovascular risk factors, hydroxyurea, platelet and leukocyte counts and mutation status) demonstrated that only male sex (p=0.013) and JAK2 V617F mutation (p=0.004) were independent predictors of thrombin response in ET. FlowSOM analysis identified a high GSAO+ platelet subpopulation significantly overexpressed in ET patients with thrombosis history (p=0.001). A ROC curve to evaluate the high GSAO+ platelet subpopulation as a predictor of disease transformation and/or death yielded a higher predictive value than the total GSAO+/P-selectin+ population (AUC of 0.9022, p=0.0081, Figure 2A). Prospective follow up with time to event analysis (development cohort) showed a ROC derived cut-off of 2.09% GSAO+ subpopulation at time of recruitment (100% sensitivity, 73.9% specificity) was predictive of death and/or disease transformation (p=0.0006, Figure 2B) or new thrombotic events (p=0.0026, Figure 2C) in patients already on primary or secondary prevention therapy. In the validation cohort, time to event analysis (n=25, median time to census 20 months, range 2.7-30) demonstrated a hazard ratio of 7.3 (95% CI: 1.4 to 37.4, p=0.02, Figure 2D) for new thrombotic events with GSAO+ subpopulation of >2.09%, independent of potential covariates (mutation status, IPSET score, historical thrombosis, anti-platelet therapy). In 3 of 3 patients in which biomarker bloods were collected at clinical review for thrombosis prior to initiation of therapy, the GSAO+ subpopulation was markedly increased and then reduced following disease modification therapy. Conclusion: This study, including validation in a small prospective cohort with pre-specified outcomes, identifies a high GSAO+ platelet flow cytometry subpopulation as a potential novel risk stratification marker for transformation and/or recurrent thrombotic events in ET. The biomarker can be collected at multiple timepoints during a patient's clinical course for dynamic risk assessment and early data suggests utility in monitoring response to therapy. Larger multisite studies to evaluate the hypergate performance as a biomarker for poor outcomes in patients with ET appear warranted. aPasalic et al. J Thromb Haemost. 2018 Jun;16(6):1198-1210. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5228-5228
Author(s):  
Kohtaro Toyama ◽  
Norifumi Tsukamoto ◽  
Akio Saito ◽  
Hirotaka Nakahashi ◽  
Yoko Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Mononuclear Cells ◽  
Rna Extraction ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Mutation Status ◽  
V617f Mutation

Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p < 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

scholarly journals Impact of JAK2(V617F) mutation status on treatment response to anagrelide in essential thrombocythemia: an observational, hypothesis-generating study

2015 ◽  
pp. 2687
Author(s):  
Alessandro M Vannucchi ◽  
Nicola Cascavilla ◽  
Valerio De Stefano ◽  
Alessandro Pancrazzi ◽  
Alessandra Iurlo ◽  
...  
Keyword(s):  
Treatment Response ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mutation Status ◽  
V617f Mutation

Tissue Factor and Soluble Markers of Platelet and Endothelial Activation in Essential Thrombocythemia: Relationship with Thrombosis and JAK2 V617F Mutation Status.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2707-2707
Author(s):  
Francisco Cervantes ◽  
Eduardo Arellano-Rodrigo ◽  
Alberto Alvarez-Larran ◽  
Juan-Carlos Reverter ◽  
Neus Villamor ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Essential Thrombocythemia ◽  
Plasma Concentrations ◽  
Jak2 V617f ◽  
Endothelial Activation ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Mutation Status ◽  
Significant Difference ◽  
V617f Mutation

Abstract There is increasing evidence that platelet and leukocyte activation plays an important role in the thrombotic complications of patients with essential thrombocythemia (ET), but the relationship of both thrombosis occurrence and JAK2 V617F mutation status with the levels of circulating tissue factor (TF) and of soluble markers of platelet and endothelial activation is not known. In 53 ET patients (26 of whom had a previous history of thrombosis), platelet TF expression and plasma levels of TF, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer, and prothrombin fragment 1+2 (F1+2), measured by ELISA, were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 V617F mutation status. ET patients with thrombosis had significantly higher levels of sP-selectin than patients without thrombosis and the controls, whereas ET patients without thrombosis had significantly higher levels than the controls (99.8 ± 47.1 ng/mL versus 70.6 ± 37.8 ng/mL versus 32.4 ± 11.9 ng/mL; p= 0.0001 for all comparisons). The same applied to sCD40L levels (226.7 ± 104.7 pg/mL in patients with thrombosis, 186.4 ± 92.1 pg/mL in patients without thrombosis, and 81.3 ± 22.0 pg/mL in controls; p= 0.0001 for all comparisons). Circulating VWF:Ag and F1+2 levels were higher in ET patients than in controls, but no significant difference was observed between patients with and without thrombosis. No differences in TF platelet expression, TF and sTM plasma concentrations were found between patients and controls. A positive correlation was observed between sP-selectin and F1+2, a marker of thrombin generation (r= 0.378, p= 0.01). Patients with the JAK2 mutation (22 out of 52 assessable patients), as compared with those with the wild-type allele, had significantly higher levels of sP-selectin (p= 0.002), sCD40L (p= 0.03), TF (p= 0.016), VWF:Ag (p= 0.0001), and sTM (p= 0.032). These results support a role for soluble markers of platelet activation in the thrombosis of ET as well as their potential to identify ET patients at greater risk of thrombosis. The association between JAK2 mutation and increased levels of TF and soluble markers of platelet and endothelial activation would suggest that the mutation could promote an enhanced prethrombotic state in ET.

Activation of the tissue factor-dependent extrinsic pathway and its relation to JAK2 V617F mutation status in patients with essential thrombocythemia

2016 ◽  
Vol 27 (7) ◽  
pp. 817-821 ◽  
Author(s):  
Grażyna Gadomska ◽  
Katarzyna Stankowska ◽  
Joanna Boinska ◽  
Alicja Bartoszewska-Kubiak ◽  
Olga Haus ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Extrinsic Pathway ◽  
Mutation Status ◽  
V617f Mutation

Increased risk of heparin induced thrombocytopenia and thrombosis in patients with essential thrombocythemia carrying the homozygous JAK2 V617F mutation

2018 ◽  
Vol 47 (1) ◽  
pp. 155-156 ◽  
Author(s):  
Roberto Castelli ◽  
Paolo Gallipoli ◽  
Riccardo Schiavon ◽  
Thomas Teatini ◽  
Giorgio Lambertenghi Deliliers ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Heparin Induced Thrombocytopenia ◽  
Increased Risk ◽  
V617f Mutation

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

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.

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3676-3682 ◽  
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Daniela Pietra ◽  
Matteo G. Della Porta ◽  
Emanuela Boveri ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Gene Dosage ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Disease Category ◽  
Mutation Status ◽  
Cell Counts ◽  
Cd34 Cells ◽  
V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3503-3503
Author(s):  
Ruben A. Mesa ◽  
Ayalew Tefferi ◽  
Heather Powell ◽  
Terra Lasho ◽  
David Loegering ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Jak2 Mutation ◽  
Myeloid Metaplasia ◽  
Mutation Status ◽  
Stat3 Phosphorylation ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

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