JAK2V617F Burden Research Articles

Somatic Variants Acquired Later in Life Associated with Thoracic Aortic Aneurysms: JAK2 V617F.

The JAK2 V617F somatic variant is a well-known driver of myeloproliferative neoplasms (MPN) associated with an increased risk for athero-thrombotic cardiovascular disease. Recent studies have demonstrated its role in the development of thoracic aortic aneurysm (TAA). However, limited clinical information and level of JAK2 V617F burden have been provided for a comprehensive evaluation of potential confounders. A retrospective genotype-first study was conducted to identify carriers of the JAK2 V617F variant from an internal exome sequencing database in Yale DNA Diagnostics Lab. Additionally, the overall incidence of somatic variants in the JAK2 gene across various tissue types in the healthy population was carried out based on reanalysis of SomaMutDB and data from the UK Biobank (UKBB) cohort to compare our dataset to the population prevalence of the variant. In our database of 12,439 exomes, 594 (4.8%) were found to have a thoracic aortic aneurysm (TAA), and 12 (0.049%) were found to have a JAK2 V617F variant. Among the 12 JAK2 V617F variant carriers, five had a TAA (42%), among whom four had an ascending TAA and one had a descending TAA, with a variant allele fraction ranging from 11.2% to 20%. Among these five patients, 60% were female, and average age at diagnosis was 70 (49-79). The mean ascending aneurysm size was 5.05 cm (range 4.6-5.5 cm), and four patients had undergone surgical aortic replacement or repair. UKBB data revealed a positive correlation between the JAK2 V617F somatic variant and aortic valve disease (effect size 0.0086, p = 0.85) and TAA (effect size = 0.004, p = 0.92), although not statistically significant. An unexpectedly high prevalence of TAA in our dataset (5/594, 0.84%) is greater than the prevalence reported before for the general population, supporting its association with TAA. JAK2 V617F may contribute a meaningful proportion of otherwise unexplained aneurysm patients. Additionally, it may imply a potential JAK2-specific disease mechanism in the developmental of TAA, which suggests a possible target of therapy that warrants further investigation.

Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

Exploratory Study on Impact of Comorbid Sleep Apnea Treatment in Patients with Polycythemia Vera and Essential Thrombocythemia

Background: Systemic inflammation has been implicated in the progression of myeloproliferative neoplasms (MPNs), polycythemia vera (PV) and essential thrombocythemia (ET). Obstructive sleep apnea (OSA) is a highly prevalent disorder that can lead to TNF-driven systemic inflammation. OSA leads to oxidative stress and NF-kB driven inflammatory gene expression through the mechanistic pathways of intermittent hypoxia and autonomic activation. We therefore sought to understand the prevalence of OSA in patients with MPNs and the feasibility of understanding the impact of treating comorbid sleep apnea in patients with PV and ET. Methods: Patients with PV and ET diagnosed using the 2008 or 2016 WHO criteria underwent STOP-BANG questionnaires to evaluate for the likelihood of OSA. Based on the finding of 3 or more positive responses on STOP-BANG questionnaire (PLoS One 2015;10:e0143697), patients underwent further evaluation with polysomnography or home sleep apnea test to diagnose OSA at an American Academy of Sleep Medicine-certified sleep laboratory (Figure 1). Patients with apnea-hypopnea index (AHI) of 5 or more events per hour and Eastern Cooperative Oncology Group scale score of 0-2 underwent continuous positive airway pressure therapy (CPAP). Effects of CPAP therapy on MPN-symptom assessment form total symptom scores (MPN-SAF TSS), JAK2V617F, MPL and CALR allelic burden, and exploratory endpoints of changes in inflammatory and thrombotic markers were ascertained (Figure 1). Comparisons with observational cohorts were planned to ascertain changes in the above endpoints at 0-, 3- and 6-months intervals (Figure 1). Based on a 40% reduction in allele burden (SD of 25%) and correlation of 0.5 between paired measurements, it was estimated that using a paired t-test with 27 evaluable patients will provide a 95% power to detect a 20% change in JAK2V617F burden from 40% to 20%. Similarly, assuming a 50% correlation (SD of 16) between pre- and post-therapy MPN-SAF TSS scores, 27 evaluable patients will provide a 80% power to detect a change of 10 at the two-sided 0.025 significance level. Results: Screening for OSA in patients with ET and PV revealed a high prevalence of OSA risk based on STOP-BANG scores. 26/29 males (mean age 63.6±11.8 years; BMI 29±3.3) and 16/26 females (mean age 61.5±12.8 years; BMI 28.2±6.5) had STOP-BANG scores of 3 or more. 8 patients with PV and ET have so far been enrolled into this study. 6/8 patients (age ranges from 41-74 years; 4 females and 2 males) with OSA (AHI 5-56 events/hr; 4% oxygen desaturation index 4.7-30/hr) underwent CPAP therapy. 1/6 patients in the CPAP arm did not tolerate CPAP and was excluded. 5/6 patients completed study procedures as did 2 additional patients without OSA in the observational study arm. Of 5/6 patients undertaking CPAP, 3/5 had the JAK2V617F mutations whereas 2/5 had the CALR mutation. JAK2V617F allele burdens at baseline in 2/5 patients were 80.7% and 4% at baseline and reduced to 73% and 1% at 6 months respectively whereas the third patient had undetectable JAK2V617F burdens throughout the study. All except one patient experienced improvement in MPN-SAF TSS scores after 6 months of CPAP therapy. While all 5 patients treated with CPAP showed improvements in C-reactive protein levels, analysis of NFkB1, TNF and IL-6 transcript levels showed mixed results. Similar mixed results were found with hypoxia-inducible factor (HIF) target gene expression ( SLC2A1, VEGF-A, EDN1) and markers of iron turnover (% transferrin saturation, transferrin, iron and ferritin levels) despite reduction in erythropoietin values in 4/5 patients with CPAP therapy at 6 months. Conclusions: Given the significant likelihood of OSA in patients with PV and ET (as based on STOP-BANG scores of 3 or more), there is need to evaluate and treat for OSA. Preliminary results show abnormalities in inflammatory markers with reductions in C-reactive protein and erythropoietin levels following CPAP therapy even though mixed results were noted in inflammatory and HIF-target gene transcripts, and markers of iron turnover. Larger studies and longer follow-up are required to understand the impact of OSA on not only the thrombotic and inflammatory milieu in patients with MPNs, but also the benefit of treating on prevention of disease progression and thrombotic manifestations common in MPNs. Funding: Huntsman Interventional Trial Grant CTO #HCI-17-HEM 28.

Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

Association of Myelofibrosis Phenotypes with Clinical Manifestations, Molecular Profiles, and Treatments.

Myelofibrosis (MF) presents an array of clinical manifestations and molecular profiles. The two distinct phenotypes- myeloproliferative and myelodepletive or cytopenic- are situated at the two poles of the disease spectrum and are largely defined by different degrees of cytopenias, splenomegaly, and distinct molecular profiles. The myeloproliferative phenotype is characterized by normal/higher peripheral blood counts or mildly decreased hemoglobin, progressive splenomegaly, and constitutional symptoms. The myeloproliferative phenotype is typically associated with secondary MF, higher JAK2 V617F burden, fewer mutations, and superior overall survival (OS). The myelodepletive phenotype is usually associated with primary MF, ≥2 cytopenias, modest splenomegaly, lower JAK2 V617F burden, higher fibrosis, greater genomic complexity, and inferior OS. Cytopenias are associated with mutations in epigenetic regulators/splicing factors, clonal evolution, disease progression, and shorter OS. Clinical variables, in conjunction with the molecular profiles, inform integrated prognostication and disease management. Ruxolitinib/fedratinib and pacritinib/momelotinib may be more suitable to treat patients with the myeloproliferative and myelodepletive phenotypes, respectively. Appreciation of MF heterogeneity and two distinct phenotypes, the different clinical manifestations and molecular profiles associated with each phenotype alongside the growing treatment expertise, the development of non-myelosuppressive JAK inhibitors, and integrated prognostication are leading to a new era in patient management. Physicians can increasingly tailor personalized treatments that will address the unique unmet needs of MF patients, including those presenting with the myelodepletive phenotype, to elicit optimal outcomes and extended OS across the disease spectrum.

Using a Humanized Animal Model to Determine the Minimum JAK2V617F Mutant Allele Burden Required to Generate MPN Pathologies

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases driven by somatic mutations acquired in hematopoietic stem cells (HSCs) and characterized by the deviant, unregulated proliferation of one or more myeloid lineages. Chronic MPN can present as polycythemia vera (PV; excess erythrocytes), essential thrombocythemia (ET; excess platelets), or myelofibrosis (MF; bone marrow fibrosis). More than 300,000 patients are currently living with an MPN diagnosis in the United States. The JAK2V617F variant is an acquired somatic mutation recurrently found in MPN patients and is used as a diagnostic marker along with increased hematocrit, increased hemoglobin, and adverse thrombotic events. In MPN patients, the frequency of JAK2V617F mutant cells varies widely but is known to induce an MPN phenotype with both high (>50%) and very low (<5%) variant allelic frequency (VAF). The reason for this is currently unknown. Most JAK2-mutant animal models model a VAF which is much higher than the lower end of detectable VAFs found in MPN patients. This results in a very limited understanding of what the minimal mutant cell burden required for disease manifestation is. The understanding of genotype/phenotype relationships in patients with low JAK2V617F VAF chronic MPN is paramount to our understanding of this disease and its hematological progression. To this end, we have designed a clinically relevant, humanized mouse model wherein we employ CRISPR and homology directed repair (HDR) via a single stranded oligo donor nucleotide sequence (ssODN) to knock-in the JAK2V617Fmutation at the endogenous locus of human cells at low VAF. In addition, a synonymous JAK2V617V mutation was introduced via CRISPR/ssODN in parallel cells to serve as a control tracked by VAF alongside the VAF of JAK2V617F in their corresponding cohorts. The respective ssODNs were nucleofected into either umbilical cord blood (UCB) derived CD34+ cells or healthy adult bone marrow (BM) derived CD34+ cells to investigate the possibility that developmental age is a variable in low JAK2V617F VAF inducing a clinical MPN phenotype. After nucleofection, 20,000 CD34+ cells were intra-tibially transplanted into sub-lethally irradiated (250 rads) NSGS mice. The low cell input was utilized to limit engraftment and initial disease burden of mutant cells. Effect was measured by mutant allele burden by droplet-digital PCR (ddPCR) analysis, flow cytometry at selected timepoints to track human cell engraftment, spleen weight, and complete blood count (CBC) analysis including hematocrit, hemoglobin, white blood cells, and platelets. At 4 months post-transplant, peripheral blood hCD45+ engraftment across all cohorts averaged less than 3%. Average VAF of circulating hCD45+ cells increased in both JAK2V617F cohorts 5 months post-transplant compared to day 0 post nucleofection. Notably, overall JAK2V617F VAF in each sample remained <5% when adjusted to include both human and mouse derived peripheral blood cells. Remarkably, both hematocrit (p= .037 in UCB cohorts and p=.0096 in BM cohorts; Figure 1A) and hemoglobin (p=.046 in UCB cohorts and p=.0026 in BM cohorts; Figure 1B) were significantly elevated in both JAK2V617F cohorts compared to their respective JAK2V617V controls, while white blood cell and platelet counts remained unchanged within normal ranges. In addition, spleen weights were significantly increased in both JAK2V617F cohorts compared to their respective controls. These data demonstrate a successful clinical model of MPN which induces a classical MPN phenotype where a low VAF of JAK2V617F corresponds with a significant increase of hematocrit, hemoglobin, and spleen weight in a humanized mouse model compared to relevant controls. In addition, we demonstrate that while engraftment due to low hCD34+ cell input remains low after 4 months, the VAF of JAK2V617F within the hCD45+ sub- population increases. This successfully enables the overall VAF contributing to the MPN phenotype to remain at clinically relevant lower levels compared to other models with much higher VAF contributions. We believe this model to be of high clinical relevance into the investigation of how low JAK2V617F burden relates to disease development and progression in MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1036: VOLUMETRIC SPLENOMEGALY IN PATIENTS WITH POLYCYTHEMIA VERA

Background: At present, palpable splenomegaly is relatively uncommon in patients with polycythemia vera (PV), possibly because of early diagnosis facilitated by a lowering of the diagnostic thresholds for hemoglobin and hematocrit levels as well as the wide application of driver gene mutation tests. Radiological volumetric analysis of spleen size is now readily available. However, non-palpable splenomegaly in patients with PV has seldom been addressed. Aims: In this retrospective study, we evaluated non-palpable, volumetric splenomegaly defined based on age- and body surface area (BSA)–matched criteria in patients with PV diagnosed according to the 2016 World Health Organization diagnostic criteria. Methods: Patients with PV who underwent abdominal computed tomography (CT) and who had palpable splenomegaly at diagnosis from January 1991 to December 2020 at Chungnam National University Hospital were enrolled in the study. The spleen volume of each patient was determined by volumetric analysis of abdominal CT and adjusted for the patient’s age and BSA. And then the degree of splenomegaly was classified as follows: no splenomegaly, spleen volume less than the mean plus 2 SD of the reference volume based on both age and BSA; overt volumetric splenomegaly, spleen volume greater than the mean plus 3 SD of the reference volume based on both age and BSA; borderline volumetric splenomegaly, a spleen volume between no splenomegaly and overt splenomegaly; or palpable splenomegaly, spleen palpable below the left costal margin. Results: Of the 87 PV patients enrolled in the study, 15 (17.2%) had no splenomegaly, whereas 17 (19.5%), 45 (51.7%), and 10 (11.5%) had borderline volumetric, overt volumetric, and palpable splenomegaly, respectively. Spleen volume was significantly positively correlated with lactate dehydrogenase level (r = 0.227, P = 0.042) and tended to be positively related to white blood cell count (r = 0.209, P = 0.055) and monocyte count (r = 0.210, P = 0.059) at diagnosis. Spleen volume was not correlated with hemoglobin level, platelet count, or JAK2V617F burden at diagnosis. The degree of splenomegaly did not affect the cumulative incidence of thrombotic vascular events (10-year incidence: 7.7%, 0%, 22.3%, and 50.7%, respectively, P = 0.414). By contrast, splenomegaly tended to adversely affect myelofibrotic transformation (10-year cumulative incidence: 0%, 0%, 7.1%, and 30.3%, respectively, P = 0.062). Moreover, the cumulative incidence of myelofibrotic transformation was significantly higher in patients with overt volumetric or palpable splenomegaly than those with no or borderline volumetric splenomegaly (10-year incidence: 0% vs. 10.3%, respectively; 15-year incidence: 0% vs. 26.3%, respectively, P = 0.020). Overall survival (OS) differed among patients with different degrees of splenomegaly (15-year OS: 100%, 78.6%, 71.7%, and 51.9%, respectively, P = 0.021). The degree of splenomegaly was independent risk factor for both myelofibrotic transformation (hazard ratio [HR]: 7.75; 95% confidence interval [CI]: 1.87-21.10; P = 0.005) and OS (HR: 6.70; 95% CI: 1.89-23.72; P = 0.003). Summary/Conclusion: The degree of splenomegaly, including volumetric splenomegaly, based on age- and BSA-matched reference spleen volumes at diagnosis reflects disease progression in PV patients. Therefore, volumetric splenomegaly should be evaluated at the time of PV diagnosis and taken into consideration when predicting the prognosis of patients with this disorder.

Thrombosis in Patients with Post-Polycythemia Vera Myelofibrosis: Incidence and Risk Factors

▪Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV). Post PV myelofibrosis (MF) is the advanced phase in the natural progression of PV. To explore the risk factors for thrombosis in post-PV MF, clinical characteristics, laboratory characteristics, the incidence of thrombosis and survival were retrospectively analyzed in a cohort of 163 Chinese patients with post-PV MF. The Kaplan-Meier method and multivariate Cox analysis were used to identify the risk factors and a risk model for thrombosis was established. Among the 163 patients, the median follow-up duration was 6 (1-18) years. During follow-up, 84 (51.5%) patients developed thrombosis, 11 (6.7%) patients progressed to acute leukemia, and 35 (21.5%) patients died (20% of whom died due to thrombosis). The 5-year, 10-year, and 15-year thrombosis-free survival (TFS) rates were 59.8%, 28.2%, and 9%, respectively. The TFS time of the post-PV MF patients was significantly lower than that of the age- and sex-matched PV patients (P<0.001). The incidence of venous thrombosis was significantly higher after the diagnosis of post-PV MF than before or at the time of the diagnosis, and the proportions of patients with JAK 2 V617F allele burden ≥75% or absolute monocyte count ≥1.5×10 9/L was significantly higher in the venous thrombosis group than in the group without venous thrombosis (P<0.05). Multivariate analysis showed that palpable splenomegaly (P=0.008, HR=3.284, 95% CI [1.373,7.855]), age ≥60 years (P=0.048, HR=1.604, 95% CI [1.004,2.56]), and a history of thrombosis (P<0.001, HR=2.767, 95% CI [1.735, 4.412]) were risk factors for thrombosis in post-PV MF patients, then a risk model for thrombosis was established according to these data. The median TFS durations in patients in the extremely high-risk group, high-risk group, intermediate-risk group, and low-risk group were 2 years, 4 years, 9 years, and 13 years, respectively. In summary, post-PV MF patients have a higher incidence of thrombosis. Reducing the volume of the spleen and the allele burden of JAK2 V617F is critical to prevent thrombosis in post-PV MF patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Polycythemia Vera Patients Respond Better to Ropeginterferon Alfa-2b Than HU/BAT Irrespective of Pretreatment or Mutational Status; Results from 5 Years' Treatment in a Randomized, Controlled Setting in the PROUD-PV/Continuation-PV Trials

▪Introduction:Ropeginterferon alfa-2b (BESREMi®), a novel pegylated interferon with an extended half-life, was approved in Europe for treatment of patients with PV based on results from the phase 3 PROUD-PV and CONTINUATION-PV trials. Ropeginterferon alfa-2b treatment is recommended in hydroxyurea (HU) naïve patients as well as in those who have previously received HU. Therefore, treatment response was analyzed by prior HU treatment status, and the influence of baseline JAK2V617F allele burden and additional mutations - which may increase over time during non-disease modifying treatment - was explored.Methods:In PROUD-PV, patients aged ≥18 years, diagnosed with PV according to WHO 2008 criteria, and either cytoreduction-naïve or HU-pre-treated (for <3 years, without intolerance or resistance) were randomized 1:1 to receive ropeg or HU at individualized doses. After 12 months' treatment, patients could roll over into CONTINUATION-PV and patients in the HU arm were permitted to switch to best available treatment (BAT). After 5 years' treatment, complete hematologic response (CHR) and molecular response defined by modified ELN criteria were assessed in patients enrolled in the extension study CONTINUATION-PV (N=171). Sub-group analyses were performed by prior HU treatment, JAK2V617F allele burden category (≤50% or>50%), and in patients with available data (N=159), by the presence of non-driver mutations (TruSight™ Myeloid Panel, Illumina) or chromosomal aberrations (Affymetrix SNP6.0 array) at baseline.Results:After 5 years of treatment with ropeginterferon alfa-2b, high rates of CHR were sustained in both HU-naïve and HU pre-treated patients (53.1% and 61.3%, respectively), whereas in the control arm, the CHR rate was lower among HU pre-treated patients (36.0% compared to 48.0% for HU-naïve patients). Molecular response rates at 5 years in HU naïve and pre-treated patients were 71.4% and 64.5% respectively in the ropeginterferon alfa-2b arm and 26.0% versus 12.5% respectively in the control arm. Rates of adverse events (AEs), treatment-related AEs, serious AEs, and AEs leading to discontinuation were similar between the subgroups regardless of HU pre-treatment.Similar CHR rates were observed at 5 years irrespective of baseline JAK2V617F allele burden category (ropeginterferon alfa-2b arm: 57.1% versus 53.1% for allele burden ≤50% or >50%, respectively; control: 46.9% versus 38.5%, respectively). The molecular response rate in the ropeginterferon alfa-2b arm was higher among patients with baseline allele burden >50% (84.4% versus 61.3% for allele burden ≤50%); in the control arm there was no difference in molecular response rates between the allele burden subgroups (23.1% versus 20.8%, respectively). Of interest, the presence of non-driver mutations or chromosomal aberrations at baseline had no apparent influence on molecular response rates to ropeginterferon alfa-2b (64.5% compared with 70.7% in patients without these genetic abnormalities).Conclusion:High hematologic and molecular response rates in both HU-pretreated and HU-naïve patients and in those with more advanced JAK2V617F burden suggest that ropeginterferon alfa-2b is also a suitable treatment option in patients switching from HU. DisclosuresGisslinger: AOP Orphan Pharmaceuticals GmbH: Other: Personal fees, Research Funding; Novartis: Other: Personal fees, Research Funding; PharmaEssentia: Other: Personal fees; MyeloPro Diagnostics and Research: Other: Personal fees; Janssen-Cilag: Other: Personal fees; Roche: Other: Personal fees; Celgene: Other: Personal fees. Klade: AOP Orphan Pharmaceuticals GmbH: Current Employment. Pylypenko: Communal nonprofit enterprise “Cherkasy regional oncology dispensary of Cherkasy oblast council: Current Employment. Mayer: AOP Orphan Pharmaceuticals GmbH: Research Funding. Krejcy: AOP Orphan Pharmaceuticals GmbH: Current Employment. Empson: AOP Orphan Pharmaceuticals GmbH: Current Employment. Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board. Kralovics: AOP Orphan Pharmaceuticals GmbH: Other: Personal fees; PharmaEssentia: Other: Personal fees; Qiagen: Other: Personal fees; Novartis: Other: Personal fees; MyeloPro Diagnostics and Research: Current holder of individual stocks in a privately-held company. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; Taiho Oncology, Inc.: Research Funding.

Poor Responsiveness to Low-Dose Aspirin Contributes to Persistent In Vivo Platelet Activation in Polycythemia Vera

▪Introduction . Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by peripheral erythrocytosis, increased thrombotic risk affecting morbidity and mortality. Low-dose aspirin once-daily (od) is currently recommended for cardiovascular prevention in PV. However, it has been recently suggested that aspirin should be used at 100 mg twice-daily (bid) in PV patients at high cardiovascular risk (JAK2V617F allelic burden, age >60 years) on the assumption of a shorter lasting antiplatelet effect, similarly to essential thrombocythemia. However, pharmacological and clinical data supporting the need for a bid ASA regimen in PV are missing.Methods . We studied the platelet responsiveness to standard aspirin (100 mg od) in PV patients, assessed at the end of the 24-h dosing interval. Patients were diagnosed according to the WHO 2008 criteria, were all on od low-dose aspirin and underwent a run-in phase (7-10 days) where aspirin tablets were always ingested at breakfast. Routine hematochemistry, genotype and allelic burden, clinical characteristics and history, medications were recorded. Serum thromboxane B2 (sTXB2) was measured as ex vivo biomarker of aspirin pharmacodynamics, urinary 11-deidro-TXB2 (TXM) as in vivo index of platelet activation, the major urinary prostacyclin metabolite, 2,3 dinor 6-keto-PGF1α (PGIM), the urinary isoprostane 8-iso-PGF2α and plasma salicylic acid esterase activity.Results . Forty-nine patients (67±10 years, 16 females) on aspirin for ≥1 month gave their informed consent; aspirin compliance and non-steroidal anti-inflammatory drug intake were checked by phone calls and patient's interview on the day of the visit. Nine patients were on phlebotomy only, while the remaining patients were on phlebotomy and hydroxyurea to keep the hematocrit <45%. Average hematologic values were: hematocrit 43.8±3%; erythrocytes 5.3±1.3x106/µL; leukocytes 9.1±4.3x103/µL, platelets 359±165x103/µL, immature platelets 11.8±7.4x103/µL. The median sTXB2 value was 8.6 [2-50] ng/ml, urinary TXM 509 [205-3428] pg/mg creatinine, 8-iso-PGF2α 768 [234-2104] pg/mg creatinine, PGIM 172 [120-275] pg/mg creatinine, plasma esterase activity 53 [47-60] µmol/L salycilic acid/min. Overall, sTXB2 significantly (p<0.01) correlated with immature platelets (rho=0.46), erythrocyte count (rho=0.41), leukocytes (rho=0.39). Patients were then divided according to sTXB2 tertiles, and patients in the lowest tertile showed sTXB2 levels within the expected range (3.4 [2.4-4.2] ng/ml, Figure). The patients in the upper tertile of sTXB2 levels (19.8 [15.3-40.9] ng/ml) were characterized by significantly (p<0.05) higher erythrocyte counts, total platelet counts, and immature platelets (p=0.05), while age, JAK2V617F allelic burden, leukocytes, hematocrit, hemoglobin, and sex distribution were comparable across tertiles. Urinary TXM significantly (p<0.05) correlated with sTXB2 (rho=0.39 and Figure), disease duration (rho=0.33), and leukocytes (rho=0.33). By multivariable analysis, only sTXB2 (p=0.009) positively predicted urinary TXM excretion. Urinary PGIM correlated with 8-iso-PGF2α excretion but not with TXM. Plasma esterase activity correlated with hemoglobin levels (rho=0.33, p=0.04), but not with the hematocrit, erythrocyte count, urinary TXM or sTXB2. JAK2V617F burden was significantly associated with polymorphonucleate count (rho=0.65), hemoglobin (rho=-0.49), and male sex (rho=0.41) while it was unrelated to previous thrombosis, sTXB2, urinary TXM and PGIM. By multivariable analysis JAK2V617F allelic burden predicted leukocyte counts only.Conclusions . A fraction of PV patients characterized by high erythrocyte, platelet and immature platelet count have a reduced response to a standard regimen of low-dose aspirin, which may contribute to persistently enhanced in vivo platelet activation. An improved antiplatelet regimen should be tested in these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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

Sex determines the presentation and outcomes in MPN and is related to sex-specific differences in the mutational burden

AbstractThe factors underlying the variable presentation and clinical course of myeloproliferative neoplasms (MPNs) remain unclear. The aim of this study was to evaluate the independent effect of sex on MPN presentation and outcomes. A total of 815 patients with essential thrombocytosis, polycythemia vera, or primary myelofibrosis were evaluated between 2005 and 2019, and the association of sex with presenting phenotype, JAK2 V617F burden, progression, and survival was examined. Men presented more often with primary myelofibrosis vs essential thrombocytosis (relative risk, 3.2; P < .001) and polycythemia vera (relative risk, 2.1; P < .001), had higher rates of transformation to secondary myelofibrosis (hazard ratio [HR], 1.55; P = .013) and acute myeloid leukemia (HR, 3.67; P < .001), and worse survival (HR, 1.63; P = .001) independent of age, phenotype at diagnosis, and MPN-specific mutation. Men had higher JAK2 V617F allele burdens in their CD34+ cells (P = .001), acquired more somatic mutations (P = .012) apart from the MPN-specific mutations, and had an increased frequency of 1 (odds ratio, 2.35; P = .017) and 2 (odds ratio, 20.20; P = .011) high-risk mutations independent of age, phenotype, and driver mutation. Male sex is an independent predictor of poor outcomes in MPNs. This seems to be due to an increased risk of non–MPN-specific somatic mutations, particularly high-risk mutations, rather than MPN-specific mutation allele frequency. Conversely, disease progression in female subjects is more dependent on JAK2 mutation allele burden than on acquisition of other somatic mutations. Sex should be considered in prognostic models and when evaluating therapeutic strategies in MPNs.

PB2206 HEMOSTATIC MARKERS OF HYPERCOAGULABILITY IN PRIMARY MYELOFIBROSIS PATIENTS, RELATION WITH JAK2V617F ALLELE BURDEN

Background:Thrombotic complications contribute significantly to morbidity and mortality in Ph‐negative myeloproliferative neoplasms, including primary myelofibrosis (PMF). The majority of PMF patients have JAK2V617F mutation that is associated with increased thrombotic risk, CALR gene mutations are also common in PMF. Clonal myeloproliferation in PMF followed by secondary inflammation with pathologic cytokine production induce endothelium, leukocytes and platelets activation resulting in coagulation activation. Natural anticoagulants protein C (PC) and protein S (PS) deficiencies with activated protein C resistance development are supposed among the crucial procoagulant features in Polycythemia Vera and Essential Thrombocythemia, while the data on hemostatic abnormalities in PMF and their relationship with JAK2V617F burden is scarce.Aims:To evaluate the hemostatic parameters in PMF patients, predisposing high thrombosis risk, and their relationship with JAK2V617F presence and allele burden.Methods:The study included 36 PMF patients (age 30‐86 years, median 60 years). Somatic mutations (JAK2V617F and CALR) presence and JAK2V617F allele burden were assessed using allele specific polymerase‐chain reaction. The control group consisted of 68 healthy persons (30‐73 years, median 46 years). Coagulation assays (factors VIII, V, von Willebrand (VWF), antithrombin and PC activities, fibrinogen concentration, VWF antigen and free PS level) were performed by standard clotting, chromogenic and immunoturbidimetric techniques. STATISTICA 6.0 was used for data analysis. The results were presented as median with 95% confidence interval (CI). Mann‐Whitney and Spearman rank tests were applied, the differences considered statistically significant with p &lt; 0.05(∗).Results:23 (63.9%) PMF patients in the study were JAK2V617F‐positive, 8 (22.2%) had mutation in CALR gene. JAK2V617F allele burden data was available in 13 PMF patients with median 8.1% (95%CI 3.4‐43.8%). The results of hemostatic parameters in PMF patients and controls are shown in Table 1.Unexpectedly there were no significant differences in studied parameters between PMF patients with JAK2V617F and CALR mutations. However, a statistically significant inverse correlation was revealed between JAK2V617F allele burden and PC activity (r = ‐0.68301, p = 0.01).imageSummary/Conclusion:PC and, especially, PS activities were decreased in PMF, predisposing hypercoagulation. The increased FVIII activity and fibrinogen in PMF patients confirm the hypercoagulability and also reflect high inflammation level, while VWF:Ag increase suggests the endothelium damage and dysfunction, enhancing prothrombotic state in PMF. An inverse relationship between JAK2V617F allele burden and PC activity can serve as one of the mechanisms contributing to the higher thrombosis incidence depending on JAK2V617F burden.

PB2217 EPIDEMIOLOGICAL, CLINICAL, MOLECULAR AND GENETIC CHARACTERISTICS OF PRIMARY MYELOFIBROSIS IN THE RUSSIAN FEDERATION

Background:To date studies have not been conducted to assess the epidemiological characteristics of Ph‐negative myeloproliferative neoplasms, in particular primary myelofibrosis (PMF) in the Russia.Aims:Creation of the Russian registry of primary myelofibrosis to assess the epidemiological clinical molecular characteristics of the PMF.Methods:In 2017 initiated a multicenter prospective observational cohort study involving 14 research centers from 11 regions of Russia. The study included 188 patients: 177 (94%) with PMF, 10 patients with post‐PV MF and 1 patient with post‐ET MF. Men: women ratio – 1: 1,3.Results:The incidence of PMF in various regions of the Russia ranges from 0.05 to 2.12 per 100000 (Table 1, 2). Among patients with PMF, post‐PV MF and post‐ET MF, 44% (83) are patients in the age group 60‐74 years, 28% (53) in the age group 45‐59 years, 17% (32) ‐ patients aged 18‐44 years, 11% (20) ‐ in the age group 75‐90 years. Constitutional symptoms were recorded in the majority of patients: fatigue ‐ 78% (147), lethargy ‐ 65 (122), headache ‐ 40% (75), discomfort in the stomach ‐ 38% (72), discomfort in the left hypochondrium ‐ 38% (71), night sweats ‐ 32% (61), feeling of early satiety ‐ 30% (56), weight loss ‐ 26% (48), bone pain ‐ 23% (43), sweating ‐ 15% (28), skin itching ‐ 11% (20), fever ‐ 6% (12). No symptoms mediated by the disease ‐ 5% (10). Splenomegaly at the time of diagnosis was recorded in 76% (143 patients). Molecular analysis performed at 94% of patients (176): JAKV617F mutation detected in 122 patients, JAK2exon12 ‐ 1, CALR ‐ 34, MPL ‐ 7, triple negative variant ‐ 13. Allelic burden of JAK2V617F defined in 90 patients with PMF, median ‐ 34% (1 ‐ 100%). Data on allelic burden JAK2V617F was in 7 patients with post‐PV MF, the median was 57% (3 ‐ 88%). Histological examination of bone marrow performed by 91% (171 patients). Histochemical studies with an assessment of the degree of reticulin fibrosis ‐ 81% (153). Confirmation of the diagnosis according to the full set of criteria in patients with PMF registered in 54% of patients. The distribution of patients by risk groups in accordance with the IPSS prognostic scale: low ‐ 26%, intermediate‐1 ‐ 33%, intermediate‐1 ‐ 23%, high ‐ 18%. The first line of therapy: hydroxyurea ‐ 58%, ruxolitinib ‐ 14%, interferon‐alpha ‐ 10%, glucocorticosteroids ‐ 5%, erythropoietin 3%, anagrelide ‐ 1%, other therapy ‐ 9%. Blood transfusions performed in 70% of patients, thromboconcentrate transfusions ‐ 6%. Concomitant therapy is given to 63% (119 patients): acetylsalicylic acid ‐ 78, allopurinol ‐ 49, erythropoietin ‐ 11, clopidogrel ‐ 6, NSAID ‐ 3, sulodexide ‐ 3, tranexamic acid ‐ 1, etamzilat ‐ 1, other (dipyridamole, iron preparations, folic acid, vitamin B12) ‐ 30. The efficacy of therapy assessed for 52 patients: complete clinical and hematological remission ‐ 23% (12), partial clinical and hematological remission ‐ 48% (25), stabilization of the disease ‐ 29% (15). 5 deaths reported. The 2‐year overall survival rate was 97% (Figure 1).Summary/Conclusion:In general the registry of patients with PMF allows integrating information into a common system and obtaining information characterizing the population of patients with this disease in Russia.image

SLAMF7high CD16negative Monocytes Increase in Peripheral Blood of Patients with Myelofibrosis in Correlation with JAK2V617F Mutation

IntroductionMyelofibrosis (MF) occurrence can be attributed to various pathogenic mechanisms. A recent study showed that the neoplastic clone of fibrocytes (spindle shaped, fibroblast-like blood cells derived from monocyte lineage) was essential in primary MF pathogenesis; moreover, serum amyloid P (PRM-151), which suppresses fibrocyte differentiation, markedly improved survival and MF in a murine xenograft model (J Exp Med 2016; 213: 1723). Using a romiplostim-induced murine MF model, direct induction of fibrocyte differentiation using TPO receptor activation, leading to MF progression, was demonstrated (Leukemia 2017; 31: 2709).Using DNA microarray analysis, we previously showed that compared with macrophages, human fibrocytes highly express SLAMF7 (J Immunol 2015; 195: 4341). Elotuzumab (Elo) is an anti-SLAMF7 antibody recently developed for treating multiple myeloma. We showed that Elo inhibits human fibrocyte differentiation in vitro and relieves MF using the mouse model in vivo, identifying Elo as a potential therapeutic agent (manuscript in preparation). Additionally, monocytes with high SLAMF7 expression and negative CD16 expression were significantly elevated in the peripheral blood (PB) of MF patients compared with that of healthy controls (HCs). We hypothesized that this monocyte subset reflects fibrocyte activation and is the target of Elo.For the clinical study of Elo, we evaluated SLAMF7high CD16− monocyte percentage in PB of HCs, myeloproliferative neoplasm (MPN) patients with or without MF, and non-MPN patients with MF in a cross-sectional manner.MethodsSix HCs, 12 non-MPN patients with MF, and 44 MPN patients (18 without and 26 with MF) were enrolled; their blood samples were collected. All MPN patients underwent bone marrow biopsy in 2016-2018 and were diagnosed by the 2016 WHO classification and diagnostic criteria. Non-MPN patients underwent bone marrow biopsy in 2015-2018, and MF was confirmed over MF-1 according to the European Consensus Criteria.SLAMF7high CD16- monocyte percentage in PB monocytes of HCs and patients was calculated using flow cytometry. Further, all patients were tested for JAK2V617F, CALR, or MPL genetic mutations. The allele burden of JAK2V617F was measured. Moreover, fibrocytes were differentiated from PB mononuclear cells of patients with genetic mutation and were verified for genetic mutation in their fibrocytes.ResultsPatients were classified in three groups (MPN without MF, MPN with MF, and non-MPN with MF). The median percentage of SLAMF7high CD16− monocytes in PB of HCs, MPN patients without MF, MPN patients with MF, and non-MPN patients with MF were 1.66%, 2.48%, 27.4%, and 19.8%, respectively. Compared with HCs and MPN patients without MF, SLAMF7high CD16− monocyte percentage of MPN and non-MPN patients with MF significantly increased (p < 0.05 and p < 0.01, respectively) (Figure A).MPN patients with MF harboring JAK2V617F (n = 17) had a significantly higher SLAMF7high CD16− monocyte percentage than those without MF harboring JAK2V617F (n = 8) (median: 43.70% vs. 7.00%, p < 0.001). While a similar trend was observed in patients not harboring JAK2V617F (median: 1.15% vs. 3.26%, p = 0.05), the contrast between patients with and without MF patients was significant for those with JAK2V617F (Figure B). More than 25% of SLAMF7high CD16− monocytes in PB indicates MF in MPN patients harboring JAK2V617F (sensitivity: 82.4%, specificity: 87.5%).In addition, the JAK2V617F allele burden of fibrocytes was strongly correlated with the SLAMF7high CD16− monocyte percentage in PB (p = 0.0003) (Figure C). In all patients, fibrocytes and PB harbored the same genetic mutation.ConclusionThe percentage of SLAMF7high CD16− monocytes in PB elevated in MF patients regardless of MPN, whereas it was not elevated in MPN patients without MF. In MPN patients, those with MF harboring JAK2V617F presented a high SLAMF7high CD16-monocyte percentage in PB, positively correlating with the JAK2V617F allele burden of fibrocytes. In conclusion, SLAMF7high CD16- monocyte levels are closely related with MF and neoplastic clones of fibrocytes harboring JAK2V617F, indicating fibrocyte activation and representing a non-invasive marker of MF progression and a potential target for Elo treatment. [Display omitted] DisclosuresUsuki:Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Novartis: Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau.

Influential factors of thrombosis in patients with polycythemia vera

Polycythemia vera (PV) is a clonal myeloproliferative neoplasms (MPN) deriving from hematopoietic stem cells (HSC), with a median survival of about 14 years. Thrombosis is the major complication affecting the survival of PV patients as well as the most common cause of death in PV patients. For PV patients, thrombosis is the result of the combined effects of various factors, and its pathogenesis is very complicated. There are many factors affecting the incidence of thrombosis in PV patients, including age, history of thrombosis, count of blood cells, platelet activity, treatment modality, JAK2 V617F burden. This paper reviews the factors affecting thrombosis in PV patients. Key words: Polycythemia vera; Thrombosis; Influential factors

Influence of Blood Count, Cardiovascular Risks, Inherited Thrombophilia, and JAK2 V617F Burden Allele on Type of Thrombosis in Patients With Philadelphia Chromosome Negative Myeloproliferative Neoplasms

IntroductionThrombosis is the most common complication in Philadelphia chromosome negative (Ph−) myeloproliferative neoplasms patients. Patients and MethodsIn a cohort of 258 Ph− myeloproliferative neoplasm patients, the difference between patients with and without thrombosis was analyzed according to genetic thrombophilia factors, JAK2 V617F status and burden allele, blood count, cardiovascular risk factors and age. Patients were also divided in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) subgroups as well as by the type of thrombosis. ResultsAnalysis of cardiovascular risk factors regarding arterial thrombosis showed that PV patients with thrombosis had higher incidence of diabetes (P = .030), ET patients more often had hypertension (P = .003) and hyperlipidemia (P = .005), while PMF patients had hyperlipidemia (P = .046) and at least one cardiovascular risk factor (P = .044). Moreover, leukocytes > 18 × 109/L and V617F burden allele > 25.7% were statistically significantly different in PV patients (P = .019 and borderline significant at P = .055, respectively), while in ET patients leukocytes > 9.2 × 109/L (P < .001) and age at diagnosis of > 55 years were statistically significantly different (P = .002). PMF patients with V617F burden allele ≤ 34.8% were more prone to thrombosis (P = .032). When comparing patients with and without venous thrombosis, cutoff value of V617F burden allele > 90.4% was significant for PV patients with thrombosis (P = .036), as was > 56.7% for PMF patients with thrombosis (P = .046). Platelets ≤ 536 × 109/L and age at diagnosis > 54 years showed statistically significant difference for ET patients with thrombosis (P = .015 and P = .041, respectively). ConclusionOn the basis of our results, a new scoring system for thrombosis risk in PV could be made, while PMF prognostic model may be expanded for better recognition of potential thrombotic risk factors.

Gender Determines the Myeloproliferative Neoplasms Phenotype Independently of Age, Driver Mutation and JAK2V617F Burden

Gender Determines the Myeloproliferative Neoplasms Phenotype Independently of Age, Driver Mutation and JAK2V617F Burden

GATA1 Expression in BCR/ABL1-negative Myeloproliferative Neoplasms.

BackgroundThis study aimed to determine GATA1 expression levels to better characterize subgroups in BCR/ABL1-negative myeloproliferative neoplasms (MPNs).MethodsThis study enrolled 49 patients diagnosed as having BCR/ABL1-negative MPN on the basis of the 2016 World Health Organization classification : nine polycythemia vera (PV), 17 essential thrombocythemia (ET), 12 prefibrotic primary myelofibrosis (prePMF), and 11 overt primary myelofibrosis (PMF). Relevant clinical and laboratory data were retrieved from the medical records. The molecular analysis of CALR and MPL mutations and quantification of JAK2 V617F allele burden were performed. GATA1 expression was assessed by an immunohistochemical assay on bone marrow biopsy. GATA1 expression was analyzed serially in 18 patients.ResultsGATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of JAK2 V617F, but it decreased significantly in patients with overt fibrosis or leukemic transformation.ConclusionsOur results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.

Comparison of JAK2V617F -positive essential thrombocythaemia and early primary myelofibrosis: The impact of mutation burden and histology.

An accurate histological diagnosis may distinguish essential thrombocythaemia (ET) from early primary myelofibrosis (early-PMF), which is associated with worse outcome. Outcome of ET is also negatively affected by the presence of the JAK2V617F mutation. To investigate the impact of JAK2V617F mutation burden and histology on outcome, we collected 475 WHO-diagnosed ET (69.2%) or early-PMF JAK2V617F -positive patients followed in 4 Italian haematology centers. JAK2V617F allele burden was ≤50% in 90% and 87% of ET and early-PMF patients, respectively (P=.34). During follow-up, 32 (9.7%) ET and 18 (12.3%) early-PMF patients experienced 59 thrombotic events, and 27 patients (5.6%) and 6 (1.2%) patients evolved to myelofibrosis and acute leukemia, respectively. At last contact, 28 (5.8%) patients had died. In early-PMF compared to ET, the 10-year mortality rates (6.7% and 4.3%, P=.73), leukemic transformation rates (1.4% and 1.2%, P=.45), and thrombosis rates (16.7% and 12.2%, P=.12) were comparable. Only progression to overt myelofibrosis at 10years was significantly worse (11.4% and 1.5%, P=.004). In multivariate analysis, a higher (>50%) JAK2V617F burden was significantly correlated with fibrotic progression and histology. Considering JAK2V617F -positive disease, a higher (>50%) JAK2V617F burden and histological classification are independent prognostic risk factors for disease progression. These findings reinforce the need for standardized detection of this mutation.