Essential Thrombocythemia Group Research Articles

Polistemi Vera ve Esansiyel Trombositemi Tanısı Alan Hastalarda Elektrokardiyografik ve Ekokardiyografik Bulgularının Değerlendirilmesi

Background/Aims: Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative diseases. Along with the increase in myeloproliferative cell lines, they can cause various clinical outcomes such as arterial and venous thrombosis, pulmonary hypertension, and myocardial infarction. This study was designed to evaluate the cardiac effects of these diseases via electrocardiography and echocardiography. Methods: A total of 50 patients with a diagnosis of PV, 50 patients with a diagnosis of ET, and 50 healthy individuals as a control group were included in this study. Data on patient demographics were recorded in all subjects. All patients and control subjects had electrocardiography (ECG) recordings and routine transthoracic echocardiographic examination. Pulse wave velocity (PWV) was assessed with a Holter blood pressure device. Results: In total, 50 PV patients, 50 ET patients, and 50 control group were included in the study. The demographic characteristics of the PV, ET and control groups were similar. The PR interval was significantly shorter in control subjects than in PV and ET patients (p:0.007, p:0.024). Although the measured values were within normal limits, diastolic posterior wall thickness was significantly lower in the control group compared to PV and ET patients (p:0.019, p:0.009). PWV was significantly higher in ET patients compared to the control group (p:0.012). Conclusion: In this study, evaluating the effects of PV and ET on electrocardiography and transthoracic echocardiography; ECG parameters used to predict ventricular arrhythmias (QT, QTc, Tp-Te, Tp-Te/QT) and Pulmonary Artery Pressure showed no significant change, in opposition to existing literature. Nonetheless, similar to previous publications, PV and ET were found to negatively affect the diastolic function parameters on transthoracic echocardiography. While the aortic stiffness was significantly higher in ET patients compared to the control group, no significant difference was noted between PV patients and control subjects in terms of aortic stiffness.

Easily Applicable Predictive Score for Differential Diagnosis of Prefibrotic Primary Myelofibrosis from Essential Thrombocythemia.

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (prePMF) initially have a similar phenotypic presentation with thrombocytosis. The aim of our study was to determine significant clinical-laboratory parameters at presentation to differentiate prePMF from ET as well as to develop and validate a predictive diagnostic prePMF model. This retrospective study included 464 patients divided into ET (289 pts) and prePMF (175 pts) groups. The model was built using data from a development cohort (229 pts; 143 ET, 86 prePMF), which was then tested in an internal validation cohort (235 pts; 146 ET, 89 prePMF). The most important prePMF predictors in the multivariate logistic model were age ≥ 60 years (RR = 2.2), splenomegaly (RR = 13.2), and increased lactat-dehidrogenase (RR = 2.8). Risk scores were assigned according to derived relative risk (RR) for age ≥ 60 years (1 point), splenomegaly (2 points), and increased lactat-dehidrogenase (1 point). Positive predictive value (PPV) for pre-PMF diagnosis with a score of ≥points was 69.8%, while for a score of ≥3 it was 88.2%. Diagnostic performance had similar values in the validation cohort. In MPN patients with thrombocytosis at presentation, the application of the new model enables differentiation of pre-PMF from ET, which is clinically relevant considering that these diseases have different prognoses and treatments.

FTIR- based serum structure analysis in molecular diagnostics of essential thrombocythemia disease.

Essential thrombocythemia (ET) reflects the transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis remains obscure. Nevertheless, tyrosine kinase, especially Janus kinase 2 (JAK2), has been implicated in myeloproliferative disorders other than chronic myeloid leukaemia. FTIR analysis was performed on the blood serum of 86 patients and 45 healthy volunteers as control with FTIR spectra-based machine learning methods and chemometrics. Thus, the study aimed to determine biomolecular changes and separation of ET and healthy control groups illustration by applying chemometrics and ML techniques to spectral data. The FTIR-based results showed that in ET disease with JAK2 mutation, there are alterations in functional groups associated with lipids, proteins and nucleic acids significantly. Moreover, in ET patients the lower amount of proteins with simultaneously higher amount of lipids was noted in comparison with the control one. Furthermore, the SVM-DA model showed 100% accuracy in calibration sets in both spectral regions and 100.0% and 96.43% accuracy in prediction sets for the 800-1800cm-1 and 2700-3000cm-1 spectral regions, respectively. While changes in the dynamic spectra showed that CH2 bending, amide II and CO vibrations could be used as a spectroscopy marker of ET. Finally, it was found a positive correlation between FTIR peaks and first bone marrow fibrosis degree, as well as the absence of JAK2 V617F mutation. The findings of this study contribute to a better understanding of the molecular pathogenesis of ET and identifying biomolecular changes and may have implications for early diagnosis and treatment of this disease.

Efficacy and safety of peginterferon-α2b for treatment of myeloproliterative neoplasms

To evaluate the clinical efficacy and adverse reactions of peginterferon-α2b for treatment of chronic myeloproliferative neoplasms (MPN). We retrospectively analyzed the data of 107 patients with MPN, including 95 with essential thrombocythemia (ET) and 12 with polycythemia vera (PV), who all received peginterferon-α2b treatment for at least 12 months. The clnical and follow-up data of the patients were analyzed to evaluate the efficacy and adverse reactions of the treatment. After receiving peginterferon- α2b treatment, both ET and PV patients achieved high hematological remission rates, and the total remission rates did not differ significantly between the two groups (86% vs 78%, P>0.05). In the overall patients, the spleen index decreased by 13.5% (95%CI: 8.5%-18.5%) after the treatment. The patients with hematological remission showed a significantly greater reduction of the total symptom score than those without hematological remission (P < 0.01). The median percentage of JAK2V617F allele load of PV patients decreased from 67.23% (49.6%-84.86%) at baseline to 19.7% (0.57%-74.6%) after the treatment, and that of JAK2V617F-positive ET patients decreased from 48.97% (0.45%-74.24%) at baseline to 22.1% (0.33%-65.42%) after the treatment. Mild adverse reactions (grade 1-2) were observed in both ET and PV groups without significant differences between them. The overall incidence of thrombotic events during the treatment was 2.8% in these patients, and no serious adverse reactions were observed. For patients with chronic myelodysplasia, peginterferon-α2b treatment can achieve a high peripheral blood cell remission rate and maintain a long-term stable state with good effect in relieving symptoms such as splenomegaly. Peginterferon- α2b treatment caused only mild adverse reactions, which can be tolerated by most of the patients.

Allelic burden of Janus kinase 2 in a 6-month course of therapy for myeloproliferative neoplasms.

Janus kinase 2 (JAK2) V617F gene mutation is an important marker for the diagnosis of Philadelphia negative Myeloproliferative neoplasms (MPN) which is subdivided into Polycythemia Vera (PV), Primary Myelofibrosis (PMF), and Essential Thrombocythemia (ET). The aim here is to investigate the JAK2 allele burden of the patients diagnosed with the subgroups of MPN and to demonstrate the alterations of hematological parameters and spleen size between diagnosis and 6 months of treatment. A total of 107 patients with the diagnosis of MPN and negative Philadelphia chromosome, 51 males and 56 females with a mean age of 59,74 ± 16,41 years, were included in the study. Diagnosis of MPN was based on the World Health Organization (WHO) criteria. Subgroups of MPN distributed as 49,5% ET, 46,7% PV, and 3,8% PMF. Findings such as the age of the patients, JAK-2 allele burden, and laboratory findings of splenomegaly were examined at the time of diagnosis, 3rd month, and 6th month. JAK2 allele burden and spleen size were re-evaluated in 6th month. Our study confirmed the findings of high Hb, HCT, and RBC but low platelet values in PV patients with high JAK2 allele burden with respect to other groups, a positive correlation between JAK2 allele burden and LDH. A novel finding of our study is, that there is not any reducing effect of the phlebotomy on JAK2 allele burden in PV patients whether they receive phlebotomy or not. Evaluation of the spleen size alteration during 6 months within the subgroups demonstrated a decrease in PV and ET groups whereas no statistically significant difference was found in the PMF group.

Restless Legs Syndrome (RLS) in Patients (pts) with Polycythemia Vera (PV): Is Iron Deficiency (ID) the Only Culprit?

Background: RLS is a sensorimotor disorder that causes discomfort in the legs and the urge to move them. In its etiology, there are many conditions including ID. PV is a myeloproliferative neoplasia (MPN), and in pts with PV, ID may develop during the course of the disease. In one study, 29.6% of PV pts had RLS, which was thought to be associated with phlebotomy and ID (Med Oncol. 2010;27(1):105-7). Essential thrombocythemia (ET) is another MPN, in which ID is not typically observed. In our study, we sought to evaluate the frequency of RLS in PV and to search for possible factors other than ID that might have a role in the etiology of RLS among MPN pts. Methods: Previously diagnosed pts with PV and ET, who admitted to our clinic between August and December 2021 were included. All PV pts were evaluated for the study regardless of their iron status. ET pts with ID were not included, since ET cases formed the non-ID MPN control group. Pts with a transferrin saturation below 20% and ferritin level below 20 µg/L were considered as cases with ID. Pts with comorbidities that may lead to RLS generation were excluded. Fasting plasma glucose, HbA1c values, urea, creatinine, thyroid function tests, vitamin B12, and magnesium values were measured. According to these parameters, pts with biochemical disorders that may affect the development of RLS were also excluded (Figure 1). In accordance with the PV and ET groups, pts with ID and healthy subjects (HS) were included as the positive and negative controls, respectively. Demographic characteristics, treatments, complete blood count (CBC) parameters, iron studies, and biochemical tests of the subjects were recorded. Neurological examination was performed in all cases, electromyography (EMG) was taken and evaluated for polyneuropathy. All subjects were questioned according to the RLS diagnostic criteria, and all subjects underwent Pittsburgh Sleep Quality Index (PSQI) tests. Results: In the final analysis, 27 PV, 23 ET, and 22 ID pts and 23 HS were included (Figure 1). Pts in the ID group were significantly younger than other groups (p=0.025) (Table 1). The median follow-up duration of the ET pts was significantly higher than those of others (p=0.011). Other demographic features were comparable between groups (Table 1). No significant pathology was detected in the neurological examination in all subjects. RLS was detected in 25.9%, 34.8%, and 45.5% of PV, ET, and ID pts, respectively. While there was no difference between these groups in terms of the presence of RLS, RLS was not detected in HS. In pts with PV 66% had ID, and there were no significant differences in terms of CBC parameters, iron studies, biochemical parameters, and the JAK2V617F mutational status in PV and ET pts with and without RLS. In addition, polyneuropathy was detected in one ET patient by EMG and RLS was not detected in this patient. There was no significant difference between these groups regarding median PSQI scores, but the median PSQI score was found to be significantly higher in pts with RLS (p=0.001) (Table 1). When we investigated the relationship between cytoreductive treatments and RLS in PV and ET pts, we found that the frequency of RLS was higher in pts receiving interferon-alfa (IFN-a) and anagrelide (Table 2). In pts with PV and ET, IFN-a and anagrelide usage, and magnesium (Mg) levels were found to have an impact on RLS in univariate analysis, but only Mg levels was associated with RLS in multivariate analysis (Table 3). Conclusion: In our study, the high prevalence of RLS in pts with ID and the absence of RLS among HS both indicate that our RLS questionnaire results are reliable. Although not all PV pts had ID, the prevalence of RLS was higher when compared to HS as was described previously (Med Oncol. 2010;27(1):105-7). In addition, although not significantly different, the frequency of RLS was higher in ET pts than that observed in PV cases. These results suggest that, RLS may occur during the course of the disease in MPN pts, due to factors independent of ID and other known RLS triggering conditions. In light of all these, further studies are needed to elucidate the etiology of RLS developing in pts with PV and ET. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Features and Mutational Landscape of Patients Referred for Suspected Essential Thrombocytosis: A Descriptive Study Using a 'Real-World’ Database

Background: Essential Thrombocythemia (ET) is a myeloproliferative neoplasm characterized by persistent thrombocytosis and often associated with mutations in JAK2, CALR, or MPL. Next Generation Sequencing (NGS) has become widely used in patients with suspected hematologic malignancies but its role in the investigation of unselected patients referred for thrombocytosis remains to be defined. This project describes the characteristics and mutational landscape of patients referred for isolated thrombocytosis with the aim of developing a clinical prediction rule for ET to help guide diagnosis and improve utilization of genomic testing. Methods: All adult patients referred to the Hematology Service at London Health Sciences Centre, Ontario, Canada, between January 1, 2018, and May 31, 2021 were reviewed for thrombocytosis (≥ 450 x 109 cells /L). NGS testing was performed using the Oncomine Myeloid Research Assay (Thermofisher Scientific, MA, USA). Patients were classified into two groups: Clinical ET and secondary thrombocytosis. Clinical ET was defined as any patient meeting the World Health Organization (WHO) 2016 criteria for ET or any patient meeting all criteria but lacking a bone marrow biopsy. Results: 446 consecutive patients were evaluated for thrombocytosis with NGS testing. 102 patients were excluded due to meeting criteria for other WHO defined conditions such as chronic myeloid leukemia, polycythemia vera, other myeloproliferative neoplasms and/or myelodysplastic syndromes. In the remaining, 342 patients, the overall yield of molecular testing was 188 (55.0%). Mutations in JAK2, CALR, MPL were identified in 84.7% of patients with clinical ET (JAK2 in 48.9%, CAL-R type 1 or 2 in 27.6%, and MPL in 8.5%), with triple negative making up the remaining 8.5%. There was no statistically significant difference between CBC parameters, ferritin, ESR, and CRP between clinical ET and secondary thrombocytosis groups. In contrast, clinical factors such as previous history of arterial thrombosis were highly predictive of ET, whilst active malignancy, chronic inflammatory disease and post splenectomy and iron deficiency were all associated with secondary thrombocytosis. Conclusion: Overall yield of molecular testing for this population of patients referred for thrombocytosis was high (55.0%), suggesting appropriate use of NGS testing, but the identification of clinical factors predicting secondary causes might suggest further improvement in utilization. In our cohort, CBC parameters did not readily distinguish between ET and secondary thrombocytosis. Clinical factors such as smoking, active malignancy, chronic inflammatory disease and post splenectomy and iron deficiency are associated with secondary thrombocytosis. These results may help inform development of a clinical prediction rule to assist in determining the pre-test probability of ET in patients referred for thrombocytosis. #co-first authors Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Von Willebrand factor activity levels are influenced by driver mutation status in polycythemia vera and essential thrombocythemia patients with well-controlled platelet counts.

von Willebrand factor ristocetin cofactor (vWF activity) and platelet count (PLT) are negatively correlated in patients with polycythemia vera (PV) and essential thrombocythemia (ET). However, vWF activity does not always normalize upon controlling PLT in those patients. To address this issue, we investigated the correlation between vWF activity and PLT in PV and ET patients. The negative correlation between vWF activity and PLT was stronger in calreticulin mutation-positive (CALR+) ET than in Janus kinase 2 mutation-positive (JAK2+) PV or ET groups. When PLT were maintained at a certain level (<600 × 109 /L), low vWF activity (<50%) was more frequently observed in JAK2+ PV patients than in JAK2+ ET (p=.013) or CALR+ ET (p=.013) groups, and in PV and ET patients with ≥50% JAK2+ allele burden than in those with allele burden <50% (p=.015). High vWF activity (>150%) was more frequent in the JAK2+ ET group than in the CALR+ ET group (p=.005), and often associated with vasomotor symptoms (p=.002). This study suggests that some patients with JAK2+ PV or ET have vWF activity outside the standard range even with well-controlled PLT, and that the measurement of vWF activity is useful for assessing the risk of thrombosis and hemorrhage.

P997: PROTEOMIC ANALYSIS ON PLATELETS OF ESSENTIAL THROMBOCYTHEMIA PATIENTS UNDERSCORES THE ROLE OF MITOCHONDRIA IN JAK2 V617F PLATELET REACTIVITY AND FUNCTION

Background: Essential thrombocythemia (ET) is a heterogeneous disease subdivided into five genetic groups according to WHO, based on the common MPN driver mutations (JAK2 V617F, MPL W515K/L, and CALR Type I&II). Current treatment strategies include anti-platelet (e.g. acetylsalicylic acid (ASA)) or cytoreductive agents (e.g. hydrea (HU)) and target mainly complications related to platelet dysfunction (i.e. hemorrhage/thrombosis). Platelet activation is an energy demanding process fueled mainly by a dynamic equilibrium between mitochondria oxidative phosphorylation and glycolysis1. Importantly, altering the platelet catabolic response to activation has been shown to prevent thrombus formation2. JAK2 V617F platelets have been reported to be more activated than CALR mutated platelets3, as well as JAK2 mutated patients have a greater thrombotic risk in comparison to other ET groups. However, the cause of this phenotype is not completely understood. Aims: In this study we aimed to analyze the proteome of ET platelets and to characterize their functional properties according to the mutational background and treatment regimens. Methods: 22 healthy donors (HD) and 67 ET patients have been included in the study. Most of the patients were treated with low dose aspirin (ASA), anagrelide (ANA) or hydrea (HU) or a combination of these. Specifically, 35 out of the 67 ET platelet samples and 8 out of 22 HD were subjected to label-free quantitation mass spectrometry (LFQ-MS). All HD and ET platelets were also analyzed for surface marker expression, degranulation and aggregation capacity by flow cytometry. Results: Hierarchical clustering of the mass spectrometry data revealed different proteomic profiles between HD and ET mutational groups and specifically between JAK2 V617F and CALR I and CALR Type II treated and/or untreated platelets. In general, HD platelets were more enriched for proteins related to platelet activation, and degranulation as compared to ET platelets and clustered next to JAK2 V617F ASA-treated platelet samples (Figure 1). In particular, the JAK2 V617F ASA-treated platelets presented significant enrichment in platelet activation metabolic and mitochondrial proteins in contrast to the CALR Type I and Type II ET platelets. Of note, no peptides corresponding to the mutant CALR were detected using label-free MS methods. Surface marker expression was variable among ET patients. However, CD49B and CD36 surface markers were the most affected among the JAK2V617F and CALR Type I and II platelets and their levels (mean fluorescence intensity-MFI-) were inversely correlated. With regards to platelet aggregation, JAK2 V617F platelets presented similar or lower levels when compared to HD platelets, in line with their proteomic profiles. Differences in aggregation and degranulation levels were also observed between CALR Type I and Type II platelets specifically in the untreated condition. Image:Summary/Conclusion: ET platelets are functional but they show different capacities to respond upon various stimuli and treatment regimens. JAK2 V61F platelets are more activated than any other ET group according to their proteome profile, which is not fully reflected by the functional assays. Mitochondrial activity arises as an important factor in the control of platelet reactivity and it could be critical in disease management and treatment strategies, specifically for the JAK2 V617F patients.

Clinical Characteristics of Patients with JAK2 Gene Mutation Myeloproliferative Neoplasms

To investigate the relationship between JAK2 gene mutation and clinical indicators in patients with myeloproliferative neoplasms (MPN). 122 MPN patients in the Department of Hematology, Xiyuan Hospital, China Academy of Chinese Medical Sciences from September 2017 to January 2020 were retrospectively analyzed. The relationship between JAK2 gene mutation and sex, age, peripheral blood cell count, splenomegaly, and thrombosis and bleeding events were analyzed. In 122 patients with MPN, the patients with polycythemia vera (PV) accounted for 36 (29.5%), the patients with essential thrombocythemia (ET) accounted for 56 (45.9%), the patients with myelofibrosis (MF) accounted for 30 (24.6%). The JAK2 gene mutation rate in MPN patients was 64.6% (79/122), and the JAK2 gene mutation rate in PV, ET and MF groups were 77.7% (28/36), 60.7% (34/56) and 56.7% (17/30), the JAK2 gene mutation rate of the patients in PV group was statistically significant as compared with those in the ET group (P＜0.05). The hemoglobin (Hb) count of the patients in JAK2 gene mutation group was higher than those in wild-type group ［(150.0±39.6)g/L vs (129.4±38.9)g/L, P＜0.05］; the white blood cell (WBC) count of the patients in JAK2 gene mutation group was higher than those in the wild type group ［(9.5±4.7)×109/L vs (8.4±46.9)×109/L, P＜0.05］. As for the patients in PV group, the platelet count of the patients in JAK2 gene mutation group was higher than those in the wild type group ［(370.2±113.1)×109/L vs (264.8±63.9)×109/L, P＜0.05］. The incidence of splenomegaly in MPN patients was 35.2% (43/122), and the incidence of splenomegaly in MF patients was 63.3% (19/30), and the incidence of splenomegaly in the patients in JAK2 gene mutation group in MF group (82.4%, 12/17) was significantly higher than those in the wild-type group (38.5%, 5/13) (P<0.05). The mutation rate of JAK2 gene in MPN patients is higher, and the mutation rate of JAK2 gene in PV patients is higher than that in ET and MF patients; JAK2 gene mutations in MPN patients are related to hemogram index; the incidence of splenomegaly is the highest in MF patients, and splenomegaly is related to the occurrence of JAK2 gene mutations in MF patients.

METABOLIC PROFILE OF BONE MARROW PLASMA IN MYELOPROLIFERATIVE NEOPLASMS

Bioactive lipids have been related to several biological functions such as immune system regulation, angiogenesis, cell survival, and proliferation. They are categorized into eight families: fatty acyls (fatty acids and eicosanoids), glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, polyketides, and saccharolipids. The deregulation of bioactive lipids secretion and production has been shown to contribute to cancer pathogenesis. To quantify eicosanoids levels in bone marrow plasma from myeloproliferative neoplasms patients and healthy subjects. Nine polycythemia vera (PV), 8 primary myelofibrosis (PMF) and 8 essential thrombocythemia (ET) patients at diagnosis and 8 healthy subjects (CT) were investigated. CT group was composed by 4 males and 4 females with a median age of 43 years (y). The PV group was composed by 5 males and 4 females, median age of 61 y; ET group was composed of 1 male and 7 females, median age of 58, and, PMF group was composed by 3 female and 5 males, median age of 68 y. Bone marrow plasma samples were analyzed by high performance liquid chromatography followed by mass spectroscopy. The metabolomics analyzes were performed by using the mass spectral data acquired with negative electrospray ionization and the full scan of the mass/charge ratio (m/z) from 100 to 1500. The results showed that 7346 bioactive lipids compounds were detected in bone marrow plasma from patients and controls. Among all the analyzed compounds we found that 129 of them overlap between all comparisons, as 241 are specific to PMF, 873 to PV and 374 to ET. The predictive analysis of activity of metabolic pathways showed that for PMF and ET, the main deregulated metabolic pathways are bile acid biosynthesis and fatty acid oxidation, while for in PV the deregulated pathways are: arginine and proline metabolism, biopterin metabolism, glutamate metabolism, Glycosphingolipid biosynthesis, Glyoxylate and Dicarboxylate Metabolism, Pyruvate Metabolism, Phytanic acid peroxisomal oxidation, Sphingolipid metabolism, Vitamin E metabolism, Polyunsaturated fatty acid biosynthesis and Xenobiotics metabolism. These data suggest that the bone marrow microenvironment from MPN patients is deregulated, culminating in the deregulation of bioactive lipids and several metabolic pathways. Each disease category presents a metabolic signature, which could be used as biomarker for diagnosis and prognosis and therapeutic target. CAPES, CNPq and FAPESP (2018-01756-5; 2018/18714-7). Metabolomic analysis; Polycythemia vera; Essential thrombocythemia; Primary myelofibrosis.

Quantification of JAK2V617F mutation load by droplet digital PCR can aid in diagnosis of myeloproliferative neoplasms.

This study developed a method for quantifying the JAK2V617F mutation load in patients with myeloproliferative neoplasm (MPN) using droplet digital PCR (ddPCR), which provides a new laboratory method for diagnosing polycythemia vera (PV), essential thrombocythemia (ET), and pre-primary myelofibrosis (pre-PMF). Patients with MPN who had JAK2V617F mutations from March 2013 to August 2019 were enrolled in this study. JAK2V617F mutation loads were quantified using ddPCR technology. The study examined 225 patients, including 135 with ET, 58 with PV, and 32 with PMF. JAK2V617F mutation loads significantly differed (P<.001) between the ET and PV groups and between the ET and PMF groups. Bone marrow biopsies were reclassified in accordance with the 2016 World Health Organization diagnostic criteria, which revealed 132 patients with MPN: 62 with ET, 35 with PV, 17 with pre-PMF, and 18 with overt-PMF. JAK2V617F mutation loads significantly differed (P<.001) between the ET and PV groups and between the ET and pre-PMF groups. The cutoff value between the ET and pre-PMF groups was 49.9. JAK2V617F mutation loads provide an additional basis for diagnosis of ET, PV, and PMF, particularly regarding differentiation between ET and pre-PMF.

Organ Stiffness in the Work-Up of Myelofibrosis and Philadelphia-Negative Chronic Myeloproliferative Neoplasms.

To define the role of spleen stiffness (SS) and liver stiffness (LS) in myelofibrosis and other Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), we studied, by ultrasonography (US) and elastography (ES), 70 consecutive patients with myelofibrosis (MF) (no.43), essential thrombocythemia (ET) (no.10), and polycythemia vera (PV) (no.17). Overall, the median SS was not different between patients with MF and PV (p = 0.9); however, both MF and PV groups had significantly higher SS than the ET group (p = 0.011 and p = 0.035, respectively) and healthy controls (p < 0.0001 and p = 0.002, respectively). In patients with MF, SS values above 40 kPa were significantly associated with worse progression-free survival (PFS) (p = 0.012; HR = 3.2). SS also correlated with the extension of bone marrow fibrosis (BMF) (p < 0.0001). SS was higher in advanced fibrotic stages MF-2, MF-3 (W.H.O. criteria) than in pre-fibrotic/early fibrotic stages (MF-0, MF-1) (p < 0.0001) and PFS was significantly different in the two cohorts, with values of 63% and 85%, respectively (p = 0.038; HR = 2.61). LS significantly differed between the patient cohort with MF and healthy controls (p = 0.001), but not between the patient cohorts with ET and PV and healthy controls (p = 0.999 and p = 0.101, respectively). We can conclude that organ stiffness adds valuable information to the clinical work-up of MPNs and could be employed to define patients at a higher risk of progression.

Eryptosis in polycythemia vera and essential thrombocythemia*

Aim: Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia–negative myeloproliferative neoplasms with documented apoptosis impairment at the level of hematopoietic stem cell. However, so far no study has evaluated apoptosis of circulating blood neoplastic cells, including the suicidal death of erythrocytes – eryptosis. Material/Methods: Erythrocytes from 61 patients (24 PV and 37 ET) naïve to and treated with hydroxyurea (HU) and 13 healthy individuals were analysed using flow cytometry to quantify phosphatidylserine (PS) externalization from Annexin-V-binding, calpain activity from 7-amino-4-chloromethylcoumarin (CMAC)-fluorescence, cell volume from forward scattered light (FSC) and cell shape from side scattered light (SSC). Results: Significantly increased levels of calpain activity and PS exposure were observed in both ET and PV naïve patients, indicating enhanced eryptosis. Among HU-treated patients, a significant increase in calpain activity in the ET group and a decrease in the PV group were observed compared to patients without cytoreductive therapy. Among PV patients, FSC was substantially higher in the HU-treated group than in the naïve group, whereas no significant differences were found between HU-treated and HU-naïve groups of ET patients. Conclusions: The enhanced eryptosis in ET and PV patients may be a form of systemic compensation of the pathological bone marrow overproduction of erythrocytes. HU, the basic cytoreductive drug used in ET and PV, may affect eryptosis in PV and ET in different ways depending on disease. The JAK2V617F mutation was not observed to have any effect on eryptosis in ET.

Increased von Willebrand factor levels in polycythemia vera and phenotypic differences with essential thrombocythemia

BackgroundAcquired von Willebrand factor (VWF) deficiency was described in Philadelphia‐negative myeloproliferative neoplasms, especially in essential thrombocythemia (ET). VWF phenotype in contemporary patients with polycythemia vera (PV) remains less explored. ObjectivesTo characterize the VWF phenotype in PV and to compare VWF phenotype in PV with matched healthy subjects and ET patients. Patients/MethodsWe studied 48 PV patients, treated according to current recommendations (hematocrit ≤ 45%, on low‐dose aspirin prophylaxis); 48 healthy and 41 subjects with ET, all sex, age, and blood group matched. We measured VWF antigen, activity, multimeric pattern, ADAMTS‐13, and factor VIII (FVIII) antigen. ResultsIn patients with PV, VWF antigen and activity were significantly higher than in healthy subjects (antigen: 119[96‐137] vs 93[79‐107] IU/dL; activity: 114[95‐128] vs 90[79‐107] IU/dL, respectively, medians and interquartile, P < 0.01), with normal multimeric distribution. ADAMTS‐13 levels were similar between patients with PV and healthy subjects. FVIII levels were higher in PV than in healthy subjects (141[119‐169] versus 98[88‐123] IU/dL, respectively, P < 0.01). By multivariable analysis, JAK2‐p.V617F allelic burden, erythrocyte count, and male sex significantly predicted VWF antigen and activity levels. As compared to patients with ET, patients with PV showed similar VWF antigen levels but approximately 40% higher activity (79[49‐104] vs 112[93‐125] IU/dL, respectively, P < 0.01). ConclusionsPatients with PV show increased VWF and FVIII levels, predicted by JAK2‐p.V617F burden and erythrocyte count. At variance with ET, acquired VWF defect was not observed in PV. High VWF/FVIII levels may sustain the thrombotic diathesis of PV and may be investigated as biomarkers for risk stratification.

PB2218 MONITORING OF BIOCHEMICAL PARAMETERS AND THROMBOEMBOLIC COMPLICATIONS IN PATIENTS WITH PHILADELPHIA NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

Background:Myeloproliferative neoplasms (MPN) are the group of clonal, malignant hematopoietic stem cell disorders, characterized by the proliferation of one or more blood lines with normal or nearly normal maturing in the bone marrow and in extramedullar hematopoietic organs. MPNs are acquired prothrombogenic conditions. It is thought that the mechanisms that lead to thrombosis in MPN are the following: increased blood cell; abnormal platelet function and the phenomenon of spontaneous aggregation. In patients with MPN there is increased activity of the coagulation system due to the resistance to the anticoagulant function of thrombomodulin.Aims:The aim of this study is to monitor certain biochemical parameters and thromboembolic complications in patients with chronic Philadelphia negative myeloproliferative neoplasms. We have simultaneously monitored the presence of JAK2V617F mutation in those patients, as well as its correlation with biochemical parameters.Methods:Over a five‐year period, we monitored 129 patients of both sexes, aged between 30 and 82 years, being diagnosed with Ph‐negative myeloproliferative neoplasm. Patients were classified into the following groups: 1. Group with polycythemia vera (PV) (51); 2.Group with essential thrombocythemia (ET) (28); 3.Group with primary myelofibrosis (PMF) (21); 4. Group with myeloproliferative neoplasm unclassified (MPNs) (29). Among biochemical parameters, we monitored the levels of lactate dehydrogenase, urate, cholesterol, C‐reactive protein, total proteins and albumins. Each group of patients was divided into two subgroups: A) patients with JAK2V617F‐positive mutation; B) patients with JAK2V617F‐negative mutation. We used methods of clinical, laboratory, ultrasound and CT scans.Results:JAK2V617F mutation was statistically more significantly present in patients with PV (about 81%). In patients with ET it was noticed 58%, in patients with PMF about 38%, and in group of patients with MPNs about 48%. The highest percentage of thrombotic complications (arterial and venous) was found in the group of patients with ET, which was statistically more significant relative to PV. Thrombotic complications were in both groups more frequent in percentage with JAK2V617F positive patients, but without statistical significance. At the same time, LDH and cholesterol values statistically significantly higher in the PV and ET group of patients, who had some of the thromboembolic complications. The percentage of LDH and cholesterol values were higher in the group of patients who were JAK2V617F positive, but without statistical significance. CRP values were also higher in percentage in the group of patients with thrombotic complications who were JAK2V617F positive, but without statistical significance. Urate values were elevated in all patient groups but without statistical significance in relation to the occurrence of thromboembolic complications.Summary/Conclusion:The level of LDH, cholesterol, and CRP in patients with Philadelphia negative myeloproliferative neoplasms could be a significant indication of possible thromboembolic complications, particularly in correlation with the presence of JAK2V617F mutations. Those parameters could also have a significant role in defining the risk factors for the incidence of thromboembolic complications in those patients. Further monitoring is required and larger number of subjects.

S1610 MASKED POLYCYTHEMIA VERA: ANALYSIS OF A SINGLE CENTER SERIES OF 2480 RED CELL MASSES.

Background:Recently, masked polycythemia vera (mPV) was defined in the WHO 2016 Myeloproliferative Neoplasms (NMP) classification as a new entity: harboring JAK2V617F mutation, combining features of both Essential Thrombocythemia (ET) (isolated thrombocytosis) and Polycythemia vera (PV). It is impossible to distinguish ET from mPV based on hemoglobin or hematocrit values and bone marrow biopsy is required to discriminate between the two.Aims:We retrospectively analyzed the red cell masses (RCM) made in our institution to determine whether this test could help differentiating these entities.Methods:We analyzed 2480 consecutive patients in whom a RCM was performed in our center for suspected polycythemia during the last 7 years. Among the JAK2V617F positive patients, the characteristics of the following groups of patients were studied: ”PV“ (JAK2V617F mutation, RCM &gt; 125%, hemoglobin greater than 18.5/16.5 g/dL (male/female)); ”mPV“ (JAK2V617F mutation, RCM &gt; 125% and hemoglobin 16.5–18.5/15–16.5 g/dL); ”JAK2V617F‐positive ET with hemoglobin level similar to mPV“ (JAK2V617F mutation, VG &lt;125%, hemoglobin 16.5–18.5/15–16.5 g/dL).Results:In this cohort, 37.3% of the 2480 patients had a RCM &gt; 125% confirming polycythemia. Patients with lower hemoglobin levels (between 15–16.5 g/dL in 56 women and 16.5–18.5 g/dL in 112 men) could be classified according to the RCM level between mPV (n = 113) when RCM was &gt; 125% or JAK2V617F‐positive ET (n = 55) when RCM was &lt;125% of predicted value. Mean platelet counts were significantly lower in PV patients than in mPV and ET patients, while no difference was observed between mPV and ET patients. Mean platelet counts were 460 G/L (+/‐ 222 G/L) in PV, 560 G/L (+/‐ 245 G/L) in mPV and 563 G/L (+/‐ 252 G/L) in ET (p PV vs mPV = 0.0014, p PV vs ET = 0.0075 and p mPV vs ET = NS). Mean serum Erythropoietin levels were comparable in the mPV and PV groups and lower than in the ET group (PV: 3.41 +/−2.3 mIU/mL, mPV: 3.42 +/−2.2 mIU/mL and ET: 6.22 +/−4.09 mIU/mL, p PV vs mPV = NS, p mPV vs ET = 0.0003 and p PV vs ET = 0.0002). JAK2V617F allelic burden was significantly different between the three groups. Mean mutant allelic ratios were 46% +/‐ 22%, 31% +/‐ 23% and 19.9% +/‐ 20% in PV, mPV and ET, respectively (p PV vs mPV &lt;0.0001, p mPV vs ET = 0.0094 and p PV vs ET &lt; 0.0001). Finally, the proportion of patients with hemodilution was significantly higher in the mPV group with plasma volume &gt; 110% in 35% of mPV, 14% of PV, and 2% of ET patients respectively (p mPV vs. PV &lt; 0.001, p mPV vs ET &lt; 0.001 and p PV vs. ET = NS). This difference between PV and mPV patients could not be attributed to an increased spleen volume as the proportion of patients with splenomegaly was 19% in PV, 19% in mPV and 5.7% in ET patients (p PV vs mPV = NS, p mPV vs ET = 0.02 and p PV vs ET = 0.02).Summary/Conclusion:This study demonstrates that mPV can be distinguished from PV and ET based on RCM measurement even when bone marrow biopsy is not performed. Our findings may explain why masked PV presents some characteristics of ET (thrombocytosis) and PV (low EPO, increased RCM, relative microcytosis), but is a distinct entity (hemodilution even in the absence of splenomegaly, intermediate JAK2 mutant allelic burden between ET and PV). These results also suggest that mPV probably needs to be managed like PV, since these patients present a true erythrocytosis.

Clinical Analysis of 208 Patiets with BCR/ABL Negative Myeloproliferative Neoplasms

To analyze the incidence, hemogram, genetics, clinical manifestations, therapeutic efficacy and outcome of patients with myeloproliferative neoplasms(MPN) so as to provide much more therapeutic basis for clinically studying the pathogenesis, diagnosis, and treatment as well as evaluating the prognosis of MPN patients. The clinical data and related laboratory test results in 208 cases of BCR/ABL fusion gene regative MPN were collected and analyzed retrospectively. The MPN could occur at any age, but the highest incidence was observed in patients aged 40-79. Among 208 patients with MPN, the patients with essential thrombocythemia(ET) accounted for 48.56%(101/208), the patients with polycythemia vera(PV) accounted for 25.96%(54/208), and the patients with primary myelofibrosis(PMF) accounted for 25.48(53/208). The clinical manifestation of MPN varied, the first manifestations was no-specific, onset of disease presented slow. The JAK2V617F gene mutation existed in 130 out of 208 patients with MPN, total mutation rate was 62.5%;JAK2V617F mutation rate in PV patients was 81.5%(44/54), while that in ET and PMF patients was 58.4%(59/101) and 50.9%(27/53) respectively, the detected rate of this mutation in PV patients was significantly higher than that in ET and PMF patients (P<0.05), while there was no significant difference between ET and PMF patients(P>0.05). In PV group, the WBC count of JAK2V617F positive patients was significantly enhanced (P<0.01), while there were no statistical differences of hemoglobin level and platelet count (P>0.05); in ET and PMF groups, the JAK2V617F positive patients had a higher WBC count and hemoglobin level(P<0.05), while the difference of platelet count was no significant(P>0.05). The most common vascular event in patients with MPN was ischemic cerebrovascular disease. The JAK2V617F mutation related with risk of thrombosis (OR=2.222, 95% CI=1.101 to 4.486). The difference in the incidence of vascular event between ET and PV patients was no statistically significant (P>0.05), but the incidence of vascular event in ET and PV patients was higher than that in PMF patients(P<0.05). The disease conversion much more easily happened in JAK2V617F positive patients. After treatment, the MPN could be controlled, yet the maintained treatment is needed. The MPN can occur almost at any age, but more commonly occures in middle-aged and elderly persons. The onset of MPN varies, the clinical manifestation was similar, a high detected rate of JAK2V617F mutation is observed in MPN patients and relates closely with onset of MPN; moreover, JAK2V617F mutation rate relates with type of MPN. The MPN patients with JAK2V617F mutation have higher WBC count and higher incidence of thrombosis. After treatment, the MPN can be better controlled, and need maintenance treatment. So as to avoid the reccurence of disease, control the complications and obtain the longterm survival.

Excess Mortality in Polycythemia Vera and Essential Thrombocythemia

Background and objective. An important proportion of patients with polycythemia vera (PV) and essential thrombocythemia (ET) are diagnosed in the seventh and eighth decades of life. Because of the chronic course of PV and ET and the advanced age of patients, many will actually die from age-related ailments instead of the myeloproliferative neoplasm, so the disease's impact on life-expectancy remains largely unknown. This registry‐based study was aimed at investigating the excess mortality attributable to PV and ET in a large series of patients diagnosed and managed according to modern criteria.Methods. We queried the databases of the Spanish Group for Chronic Myeloproliferative Neoplasms (GEMFIN) to retrieve patients diagnosed with PV or ET after 1980. Criteria for diagnosis and treatment modalities were the internationally recommended ones in each period.Excess mortality was defined as the complementary of the ratio between the actuarial survival observed in the cohort of patients and the expected survival derived from the general Spanish population matched to the patients by age, sex, and calendar year at diagnosis, and was estimated by the Dickman's method (Stat Med 2004;23:51). Spanish life-tables stratified by age, sex, and calendar year were obtained from the Human Mortality Database (www.mortality.org).Results. A total of 3,268 patients were included in the study (PV: 1,731; ET: 1,537). Median age (interquartile range) and sex distribution in the PV and ET groups were 67 (55-75) years, 48% females, and 62 (48-74) years, 61% females, respectively. At the study closing date, 270 (16%) patients with PV and 202 (13%) with ET had died. Estimated median survival was 20.2 years for PV patients and 23.8 years for ET patients. Transformation into acute myeloid leukemia/myelodysplastic syndrome was diagnosed in 76 (4.4%) patients with PV and 63 (4.1%) with ET. Three hundred and seventy-one patients (170 ET, 201 PV) had one or more episodes of thrombosis over the course of the disease.No significant excess mortality attributable to ET was detected at 20 years from diagnosis (figure 1a). In PV, mortality did not significantly deviate from matched general population until 18 years from diagnosis (figure 1b). In both diseases, excess mortality significantly increased after the first recorded thrombotic event (figure 1c).Conclusion. From the population viewpoint, ET and PV do not carry an increased excess mortality until late in the second follow-up decade or later. Nevertheless, appearance of a thrombotic event significantly increases the subsequent excess mortality. [Display omitted] DisclosuresHernandez Boluda:Incyte: Consultancy; Novartis: Consultancy. Ferrer Marin:Novartis: Consultancy, Research Funding; Incyte: Consultancy. Gómez-Casares:Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Speakers Bureau. Cervantes:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hospital Clinic Barcelona: Employment.

Risk Factors for Secondary Cancer in a Case-Control Study on 1,259 Patients with Myeloproliferative Neoplasms

INTRODUCTIONThe incidence of secondary cancer (SC) in patients with myeloproliferative neoplasms (MPN) is high and comparable to that of thrombosis. However, the identification of patient subgroups that might be at increased susceptibility of developing SC has not been systematically addressed. We report here the results of an international case-control study (MPN-K) aimed at comparing the frequency of exposure to possible causes of SC in patients with classical MPN, polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF).METHODSThis European Leukaemia Network (ELN) study reports MPN patients from 28 sites of 5 European countries and Israel, diagnosed in the period from 2000 to 2016. Cases were MPN patients with concomitant diagnosis of a non-myeloid SC (n=15) or its presentation during the course of the disease (n=412). Controls were MPN patients cancer-free, matched to the paired case for sex, age (±5 years), date of MPN diagnosis (±5 years), and MPN disease duration (±6 years). A multivariable conditional logistic regression model was used to estimate the effect of selected variables on total SC risk and in different types of SC.RESULTSAmong 1,259 MPN patients, there were 427 cases and 832 matched controls. Cases presented melanoma (n=20; 4.7%), non-melanoma skin cancer (n=69; 16.2% - basal/squamous cell carcinoma), non-skin solid cancer (n=290; 67.9%) including breast, ovary/uterus, colorectal, upper gastrointestinal, liver/pancreas, lung, prostate/urinary, other and lymphoproliferative diseases (n=48; 11.2%) including multiple myeloma, chronic lymphocytic leukemia, low and high grade B- and T-lymphoma.At diagnosis, there were slightly more patients with PV among SC cases (n= 152; 35.6%) than controls (n=256; 30.8%), while conversely there were slightly less ET patients among cases (n=196; 45.9%) than controls (n=426; 51.2%). Cases and controls presented similar proportion of MF diagnosis (n=79 cases, 18.5% and 150 controls, 18.0%). Driver mutations (JAK2 V617, EXON-12, CALR, MPL), non-driver mutations and abnormal karyotype were equally represented in cases and controls. Other variables such as cardiovascular risk factors, exposure to cancerogens, family history of cancer and chronic inflammatory diseases were reported with similar frequency in cases and controls.After MPN diagnosis, exposure to first and other lines of treatments until the index event, with Phlebotomy (n=193; 15.3%), Hydroxyurea (n=814; 64.7%), Anagrelide (n=14; 1.1%), Interferon (n=30; 2.4%), Pipobroman (n=8; 0.6%), Busulphan (n=13; 1.0%), Ruxolitinib (n=11; 0.9%), was similar in the two groups except for aspirin that was used less frequently (p=0.043) in cases (n=320; 74.9%) compared to controls (n=664; 79.9%). In particular, the lower use of aspirin was circumscribed to non-skin solid tumors. A multivariable analysis was carried out in all patients and stratified by different type of tumors (Table). In non-skin solid cancers, the time to exposure of the MPN disease > 5 years (OR=2.95; 95% CI 1.54-5.66, p=0.001) and the PV phenotype (OR=2.40, 95% CI 1.15-5.01, p=0.020) were more burdened by the incidence of events than the reference ET group. No difference in SC risk was found for MF patients compared to patients with ET.Interestingly, the independent protective role of aspirin retained its statistical significance only in non-skin SC. In non-melanoma skin cancer, multivariable analysis revealed that the presence of JAK2 mutation was less associated with SC (OR=0.32, 95% CI 0.13-0.81, p=0.016) and confirmed that exposure to HU and other cytotoxic agents was associated with a significantly higher risk of SC (OR=6.00, 95% CI 1.23-29.28, p=0.027 and OR=9.80, 95% CI 1.24-77.78, p=0.031, respectively). This finding was not seen in non-skin SC and in lymphoma.CONCLUSIONThe considered clinical and biological features, at MPN diagnosis, were not different in cases with SC and controls. During the course of the disease, three factors significantly and independently affected the risk of SC in these MPN patients: 1) patients with PV had a 77% higher risk than those with ET, 2) patients with MPN duration of more than 5 years had a twice higher risk than those with lower duration, 3) for the first time, we documented that in non-skin solid cancers, aspirin treatment reduced SC risk of 38%. Exposure to HU and other cytoreductive drugs was confirmed as a risk factor for non-melanoma skin cancer. [Display omitted] DisclosuresPalandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marchetti:Gilead: Consultancy; takeda: Speakers Bureau; amgen: Speakers Bureau; janssen: Speakers Bureau. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.