Essential Thrombocythemia Patients Research Articles

Real-world study of children and young adults with myeloproliferative neoplasms: identifying risks and unmet needs

AbstractMyeloproliferative neoplasms (MPNs) are uncommon in children/young adults. Here, we present data on unselected patients diagnosed before 25 years of age included from 38 centers in 15 countries. Sequential patients were included. We identified 444 patients, with median follow-up 9.7 years (0-47.8). Forty-nine (11.1%) had a history of thrombosis at diagnosis, 49 new thrombotic events were recorded (1.16% patient per year [pt/y]), perihepatic vein thromboses were most frequent (47.6% venous events), and logistic regression identified JAK2V617F mutation (P = .016) and hyperviscosity symptoms (visual disturbances, dizziness, vertigo, headache) as risk factors (P = .040). New hemorrhagic events occurred in 44 patients (9.9%, 1.04% pt/y). Disease transformation occurred in 48 patients (10.9%, 1.13% pt/y), usually to myelofibrosis (7.5%) with splenomegaly as a novel risk factor for transformation in essential thrombocythemia (ET) (P= .000) in logistical regression. Eight deaths (1.8%) were recorded, 3 after allogeneic stem cell transplantation. Concerning conventional risk scores: International Prognostic Score for Essential Thrombocythemia-Thrombosis and new International Prognostic Score for Essential Thrombocythemia-Thrombosis differentiated ET patients in terms of thrombotic risk. Both scores identified high-risk patients with the same median thrombosis-free survival of 28.5 years. No contemporary scores were able to predict survival for young ET or polycythemia vera patients. Our data represents the largest real-world study of MPN patients age < 25 years at diagnosis. Rates of thrombotic events and transformation were higher than expected compared with the previous literature. Our study provides new and reliable information as a basis for prospective studies, trials, and development of harmonized international guidelines for the specific management of young patients with MPN.

Longitudinal trends of peripheral blood counts in polycythaemia vera and essential thrombocythemia patients in the UK.

There is sparse evidence of how well haematological targets are met in practice for essential thrombocythemia (ET) and polycythaemia vera (PV) patients. Patient data was collected between 2008 and 2020 from two UK NHS Trusts for ET and PV patients. Longitudinal changes in peripheral blood counts, including the proportion of patients meeting peripheral blood count remission, was modelled. Relative risk of cardiovascular‐related events for patients achieving remission within 3‐months was estimated. A total of 620 ET and 429 PV patients were analysed. For high‐risk patients, haematological parameters decreased in the first months of observation then stabilised within normal reference ranges until year 5. Total time spent in peripheral blood count remission was 39.2% for ET and 29.1% for PV. A lower proportion of ET patients reached target platelet counts (48.3%) compared to WBC (79.1%), whilst PV patients were less likely to reach target haematocrit levels (56.9%) compared to platelets (77.3%) or WBC (74.6%). There was no statistically significant association between reaching target blood counts within 3‐months and cardiovascular risk. Complete haematological remission remains a challenging target in managing PV and ET, however this study was unable to show statistically‐significant evidence that this was associated with increased risk of cardiovascular events.

Hematological relevance of JAK2 V617F and calreticulin mutations in Tunisian patients with essential thrombocythemia

BackgroundThe genetic investigation of essential thrombocythemia(ET) has highlighted the presence of driver mutations in ET. Janus kinase JAK2V617F and calreticulin(CALR) mutations are the most frequent driver mutations and have significantly improved the molecular diagnosis of ET. The impact of genetic heterogeneity on clinical features has not been fully elucidated.This is the first study which aimed to determine the frequency of JAK2V617F and CALR exon9 mutations in Tunisian ET patients and to establish the correlation between hematological characteristics and mutational status.MethodsThis study included Tunisian patients suspected with ET and was conducted between September 2017 and March 2021. Genomic DNA of patients was isolated from peripheral blood samples. JAK2V617F was detected by AS‐PCR and CALR mutations were detected by PCR/direct sequencing. Clinical and hematological characteristics were also analyzed.ResultsTwo hundred and fifty ET patients were enrolled in this study. JAK2V617F mutation was found in 166/250 (66.4%) of patients, whereas CALR mutations were detected in 27/84 (32.1%) patients without JAK2V617F. Compared with JAK2V617F‐positive patients, those with CALR mutations showed lower hemoglobin level and lower leucocytes count (p = 0.007 and p = 0.004,respectively). CALR type 2 was the most frequent mutation of CALR detected in 55.55% of CALR mutated. Six of seven patients with thrombotic events harbored JAK2V617F mutation.ConclusionThe prevalence of driver mutations JAK2V617F or CALR mutations was 77.2% in Tunisian ET patients. Moreover, patients with JAK2 V617F mutation had a higher risk of thrombosis. The mutational status is necessary to improve the diagnosis and contribute to the therapeutic decisions.

PB2039: CLINICAL FEATURES AND NEXT-GENERATION SEQUENCING LANDSCAPE OF ESSENTIAL THROM-BOCYTHEMIA，PREFIBROTIC PRIMARY MYELOFIBROSIS, AND OVERT FIBROTIC PRIMARY MYELOFIBROSIS: A CHINESE RETROSPECTIVE STUDY

Background: Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate enti-ty in the 2016 revised classification of MPN differed from essential thrombocythemia (ET) or overt fibrotic primary myelofibrosis (overt PMF), it has been a subject of de-bate among experts due to its indefinite diagnosis. Aims: We conducted a retrospective study of patients with myeloproliferative neoplasms (MPNs) in China, including 56 ET pa-tients, 19 pre-PMF patients, and 43 overt PMF patients. Methods: We conducted a retrospective study of patients with BCR-ABL-negative MPN from October 2014 to May 2021 in the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Patients who were diagnosed with ET, pre-PMF or overt PMF according to the 2016 WHO revised Classification of MPN were included. Patients with incomplete information from laboratory tests and medical records were excluded. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University School of Medicine in line with the principles of the Declaration of Helsinki.Statistical analyses were performed using SPSS statistics 26.0 and GraphPad Prism 8.0 software. Results: Pre-PMF patients exhibited higher leukocyte counts[14.2(6.0-28.1)×109/L vs 9.6(4.0-55.0)×109/L, P=0.003], LDH values[307(233-479)U/L vs 241(129-1182)U/L, P<0.001], onset ages[67(32-76)years vs 50(16-79) years, P=0.006], a higher frequen-cy of splenomegaly(47.4% vs 16.7%, P=0.018) and hypertension(57.9% vs 23.2%, P=0.005) than ET patients. On the other hand, pre-PMF patients had higher platelet counts[960(500-2245)×109/L vs 633(102-1720)×109/L, P=0.017], haemoglobin levels[152(115-174)g/L vs 119(71-200)g/L, P=0.003], lower LDH val-ues[307(233-479)U/L vs 439(134-8100)U/L, P=0.007] and a lower frequency of splenomegaly(47.4% vs 75.6%, P=0.031) than overt PMF patients. Next-generation sequencing landscape was performed in 50 patients, revealed the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients (60% vs 10% vs 15.79%, P=0.033), and WT1 was more often overexpressed (WT1/ABL1 copies≥1.0%) in patients with overt PMF than in those with ET or pre-PMF(54.55% vs 16.67% vs 17.65%, P=0.009). In terms of outcome, male sex, along with symptoms including MPN10, anaemia (haemoglobin<120g/L), thrombocytopenia (platelet count<100×109/L), leucocytosis (leukocyte counts>13×109/L), high LDH value (>350U/L), splenomegaly, WT1 overexpres-sion(WT1/ABL1 copies≥1.0%), KMT2A, ASXL1 and TP53 mutations, indicated a poor prognosis for PMF patients. The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China. *Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. E-mail: [email protected] Image:Summary/Conclusion: In summary, the results of this study indicated that comprehensive evaluation of BM features, clinical phenotypes, haematologic parame-ters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

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.

PB2053: PRIMARY ARTERIAL HYPERTENSION IN MYELOPROLIFERATIVE NEOPLASMS: A SYSTEMATIC REVIEW

Background: Patients diagnosed with myeloproliferative neoplasms (MPNs) are prone to experience thrombotic events and often experience a high burden of cardiovascular comorbidities. Primary arterial hypertension (HTN) remains one of the most common comorbidities in the elderly. However, the impact of HTN on MPNs is unclear. Aims: Report on the occurrence of HTN in MPNs. Describe its epidemiology, influence on development of thrombosis, HTN treatment strategies. Methods: Systematic search in PubMed/Medline, Web of Science and SCOPUS, from the inception of these databases to retrieve relevant English-written articles. Eligibility criteria: 1. Confirmed MPNs diagnosis, 2. Confirmed HTN diagnosis, 3. Studies reported on the epidemiology of HTN in MPNs, impact of HTN on the development of thrombosis or on the therapeutic options used to treat HTN in MPNs. We excluded studies investigating secondary HTN, e.g., drug-induced HTN. Results: The systematic search resulted in 590 potentially relevant manuscripts. After removal of duplicates and screening of abstracts/titles (n=503), 87 manuscripts were selected for full-text review. After applying the eligibility criteria and full-text evaluation, 25 articles were excluded and the remaining 62 were entered into the qualitative/quantitative analysis. HTN emerged as the most common comorbidity in MPNs, affecting 41-63% of primary myelofibrosis (PMF), 40-64% of essential thrombocythemia (ET), 46-90% of polycythemia vera (PV) cases. In PV, the presence of HTN was associated with decreased serum free epinephrine and aldosterone levels, reduced retinal microperfusion, elevated kidney arteries resistance, JAK2-V617F mutation and high-risk PV. In PMF, it was linked with high uric acid concentrations and elevated risk of thrombosis (OR=1.96). PV subjects with HTN were non-dippers, suffered from systolic/diastolic dysfunction as assessed by cardiac ultrasound, displayed elevated cell-free hemoglobin and nitrite/nitrate ratio, and an increased risk of thrombosis (HR=1.77). HTN in ET was associated with overall thrombosis, especially arterial thrombosis (HR=1.91-3.8). The cardio-IPSET score was superior in predicting thrombosis in ET patients with HTN. Individuals with MPNs and HTN exhibited signs of carotid artery injury, end-organ damage, increased albumin-to-creatinine ratio, P-selectin concentrations and were prone to develop kidney dysfunction. Moreover, stroke was more common in MPNs with concomitant HTN (HR=4.24) and HTN was a predictor of ischemic stroke recurrence in MPNs. The risk of thrombosis in MPNs was higher if HTN was also associated with other cardiovascular risk factors, i.e., dyslipidemia, diabetes or smoking. The risk of cerebral venous sinus thrombosis was lower in MPNs with co-occurring HTN. In terms of treatment options for patients with HTN and MPNs, angiotensin-converting enzyme inhibitors (ACEI), e.g., lisinopril, and calcium-channel blockers (CCB - nifedipine for hypertensive emergencies) have been successfully used. The recommendation to avoid the use of diuretics in patients with elevated blood viscosity, e.g., PV, remains unaltered. Summary/Conclusion: Our findings suggest that HTN is the most common comorbidity in MPNs and is associated with development of thrombosis, end-organ damage, i.e., retinal and kidney dysfunction. Integration of HTN in the current scores employed to predict the development of thrombosis and stratify MPNs subjects in warranted. Screening for HTN in MPNs is urgently needed. Accurate management strategies based on the use ACEI and CCB need further exploration in MPNs with associated HTN.

P1724: QUALITY OF LIFE AND UNMET NEEDS AMONG PH-NEGATIVE MYELOPROLIFERATIVE NEOPLASM PATIENTS: RESULTS OF THE NATIONWIDE SURVEY

Background: Patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), including myelofibrosis (MF), polycythemia vera (PV) and essential thrombocythemia (ET), experience troublesome symptoms leading to impaired functioning (physical and psychosocial) and quality of life (QoL). Understanding symptom burden, QoL and unmet patient needs across disease subgroups is of value to aid optimal treatment and rehabilitation decision-making for patients with different MPNs. It is also worthy to explore perceptions on the impact of the disease and its treatment among MPN patients and hematologists to ensure patient-centered care. Aims: The national survey MPN-QoL-2020 was aimed to evaluate QoL and symptom burden in patients with MPN, as well as to examine the perceptions of patients and physicians about the impact of MPN and its treatment in a real world setting in Russia. Methods: A survey of patients with MPN and their treating physicians was conducted from September to December 2020. This survey included in- or out-patients aged ≥18 years with a confirmed diagnosis of MF, PV, or ET. All the patients completed the HM-PRO, a hematological malignancy (HM) specific patient-reported outcomes (PRO) measure, the MPN10 symptom assessment tool, and a patient’s survey checklist. The HM-PRO consists of two scales: Part A measuring the ‘impact on patients’ QoL’; Part B measuring ‘signs and symptoms’ experienced by the patients. Part A has 4 domains: physical behaviour (PB), social well-being (SW), emotional behaviour (EB) and eating and drinking habits (ED). Physicians completed a physician’s survey checklist which included a record for each patient. Both paper and electronic versions of the survey forms were available. ANOVA and χ2 test were applied to examine the differences between groups. Results: 1100 patients with MPNs (MF, n=355, PV, n= 408 and ET, n=337; mean age = 58±14 yrs; female = 61%) and 100 hematologists (mean age = 42±12 yrs, female = 85%) from 37 medical centers completed the survey. The HM-PRO Parts A & B total scores were significantly worse (higher scores) in MF and PV patients than in ET (Table 1). Impact on PB and EB was higher in MF and PV as compared to ET: PB = 28.6 and 21.4 vs 14.3; EB = 31.8 and 27.3 vs 22.7, respectively. Impact on ED was higher in MF as compared to PV and ET. SF was less impaired and similar across different MPNs. Among MF there were more patients with moderate/large effect on QoL as compared to PV and ET – 40% vs 34% and 28% (p=0.003). The vast majority of MPN patients experienced symptoms (95%); more than 80% had fatigue and inactivity. The MPN10 Total Symptom Score was the highest in MF, intermediate in PV and the lowest in ET: 24 vs 20 vs 14 (p<0.001). Notably, 29% MF, 34% PV and 44% ET patients did not attribute their symptoms to MPN. Physicians and patients had a discordant perspective of the most bothersome symptoms. There was agreement between MF patients and their physicians about the impact of the disease on QoL and symptom burden. For PV and ET, physicians underestimated patient’s concerns; the discrepancies were pronounced for QoL impact (PV, p=0.028; ET, p<0.001) and symptom burden (PV, p=0.018; ET, p<0.001). Across MPNs, 41.5% MF, 37.8% PV and 35.2% ET agreed that there were areas of patient-physician relationship needed to be improved. Image:Summary/Conclusion: The findings of this nationwide survey demonstrate differences in the impact on QoL and symptom burden across MPNs, identify the areas of different perspective between patients and physicians about MPN and its treatment as well as highlight the unmet needs among patients with MPN.

Clinical features and next-generation sequencing landscape of essential thrombocythemia, prefibrotic primary myelofibrosis, and overt fibrotic primary myelofibrosis: a Chinese monocentric retrospective study.

Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate entity in the 2016 revised classification of MPN differed from essential thrombocythemia (ET) or overt fibrotic primary myelofibrosis (overt PMF), it has been a subject of debate among experts due to its indefinite diagnosis. We retrospectively reviewed the clinical parameters, haematologic information, and genetic mutations of patients who were diagnosed withmyeloproliferative neoplasms (MPNs)according to the WHO 2016 criteria in China, including 56 ET patients, 19 pre-PMF patients, and 43 overt PMF patients. Pre-PMF patients exhibited higher leukocyte counts [14.2(6.0-28.1) × 109/L vs 9.6(4.0-55.0) × 109/L, P = 0.003], LDH values [307(233-479)U/L vs 241(129-1182)U/L, P < 0.001], onset ages [67(32-76) years vs 50(16-79) years, P = 0.006], a higher frequency of splenomegaly(47.4% vs 16.7%, P = 0.018) and hypertension (57.9 vs 23.2%, P = 0.005) than ET patients. On the other hand, pre-PMF patients had higher platelet counts [960(500-2245) × 109/L vs 633(102-1720) × 109/L, P = 0.017], haemoglobin levels [152(115-174)g/L vs 119(71-200)g/L, P = 0.003], lower LDH values [307(233-479)U/L vs 439(134-8100)U/L, P = 0.007] and a lower frequency of splenomegaly(47.4 vs 75.6%, P = 0.031) than overt PMF patients. Next-generation sequencing landscape was performed in 50 patients, revealed the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients (60 vs 10 vs 15.79%, P = 0.033), and WT1 was more often overexpressed (WT1/ABL1 copies ≥ 1.0%) in patients with overt PMF than in those with ET or pre-PMF(54.55 vs 16.67 vs 17.65%, P = 0.009). In terms of outcome, male sex, along with symptoms including MPN10, anaemia (haemoglobin < 120g/L), thrombocytopenia (platelet count < 100 × 109/L), leucocytosis (leukocyte counts > 13 × 109/L), high LDH value (> 350U/L), splenomegaly, WT1 overexpression(WT1/ABL1 copies ≥ 1.0%), KMT2A, ASXL1 and TP53 mutations, indicated a poor prognosis for PMF patients. The results of this study indicated that a comprehensive evaluation of BM features, clinical phenotypes, haematologic parameters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

Cytoreductive treatment and association with platelet function and maturity in patients with essential thrombocythaemia.

SummaryPatients with essential thrombocythaemia (ET) have an increased risk of thromboembolic events, which may differ according to different cytoreductive drugs. We investigated the effect of cytoreductive treatment on platelet function and turnover in ET patients. Blood samples were obtained at 1 and 24 h after aspirin intake. Platelet function was evaluated by platelet aggregation and flow cytometry. Platelet turnover was assessed by immature platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV). A total of 47 ET patients were included and grouped into 21 patients not receiving cytoreductive treatment, 15 patients receiving hydroxycarbamide and 11 patients receiving pegylated interferon alpha (peg‐IFN). Patients receiving peg‐IFN had significantly higher IPF and MPV than the other ET groups. Patients not receiving cytoreductive treatment had significantly higher platelet aggregation 24 h after aspirin intake than the other ET groups (p‐values from 0.03 to 0.0002). Patients receiving hydroxycarbamide had significantly higher expression of platelet granule makers, P‐selectin and CD63, than patients receiving peg‐IFN (p‐values ≤0.003). Cytoreduction provides more consistent platelet inhibition compared with no cytoreductive treatment. Moreover, peg‐IFN provides superior inhibition of platelet activation markers than hydroxycarbamide, which in part may explain differences in risk of thromboembolic events in ET patients.

Analysis of Differential Proteins Related to Platelet Activation in Patients with Essential Thrombocythemia Based on Label-Free Quantitative Technology

To analysis the specific protein markers of essential thrombocythemia (ET) based on proteomics technology, to explore and verify the differential protein related to platelet activation. Blood samples were obtained from ET patients and healthy people and a certain protein mass spectrometry was detected using label-free quantitative technology. The proteins relative abundance increased or down-regulated by 1.3 times in the disease group compared with the control group, and the protein abundance in the two groups t test P＜0.05 were defined as differential proteins. Bioinformatics analysis of the differential proteins was performed using GO and KEGG. The difference in the average protein abundance between the two groups was analyzed by t test and P＜0.05 was considered statistically significant. Differential proteins were selected for verification by parallel reaction monitoring (PRM) technology. A total of 140 differential proteins were found, of which 72 were up-regulated and 68 were down-regulated. KEGG enrichment showed that the differential protein expression was related to the platelet activation pathway. The differential proteins related to platelet activation were GPV, COL1A2, GP1bα, COL1A1 and GPVI. Among them, the expressions of GPV, GP1bα and GPVI were up-regulated, and the expressions of COL1A2 and COL1A1 were down-regulated. PRM verification of COL1A1, GP1bα, GPVI and GPV was consistent with LFP proteomics testing. Differential proteins in ET patients are related to platelet activation pathway activation.Differential proteins such as GPV, GPVI, COL1A1 and GP1bα can be used as new targets related to ET platelet activation.

RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS.

Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2mutation in the CALR gene. Allele-specific primers designed for detecting type 2mutation (5-bp insertion; c.1154_1155ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. The type 2mutation (K385fs*47ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2mutation (ins 5) of the CALR gene in patients with MPNs.

Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

Objective:The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. Methods:The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F–negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. Results:28.3% of JAK2 V617F–negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. Conclusion:Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

Low-risk polycythemia vera and essential thrombocythemia: management considerations and future directions.

Thrombotic events are a distinctive feature of the myeloproliferative neoplasms (MPNs) polycythemia vera (PV) and essential thrombocythemia (ET). Patients with these MPNs may also experience a poor quality of life secondary to symptom burden, as well as progression of disease to acute leukemia or myelofibrosis. Over the years, various risk stratification methods have evolved in order to attempt to predict thrombotic risk, which is the largest contributor of morbidity and mortality in these patients. More than half of PV and ET patients are low- or intermediate-risk disease status at the time of diagnosis. While therapeutic development is presently focused on high-risk patients, there is a paucity of therapies, outside of aspirin and therapeutic phlebotomy, which can reduce the thrombotic risk or delay disease progression in low-risk patients. In this review, we first describe the various complications that patients with PV and ET experience, and then detail our evolving understanding of risk stratification in these diseases. We then highlight the available evidence on the management of low-risk PV and ET and include a description of novel therapies currently under investigation in this space. We conclude with recommendations for future directions to advance our understanding and improve the treatment of low-risk PV and ET.

Analysis of factors associated with the development of myelofibrosis in polycythemia vera and essential thrombocythemia patients: a single-center experience

Analysis of factors associated with the development of myelofibrosis in polycythemia vera and essential thrombocythemia patients: a single-center experience

PEAR1 Genetic Variants in Essential Thrombocythemia: The Prevalence and Association with Hematological Parameters and ET Mutations

Objective: Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm characterized by the expansion of the megakaryocytic/platelet line. Given the undeniable role of genetic variations in the pathogenesis of ET, as well as the proven effects of PEAR1 SNPs on platelet function, the innovative purpose of this study is to investigate the prevalence of PEAR1 variants (rs12041331 and rs12566888)and their relationship to hematological parameters and ET-related mutations. Materials and Methods: We studied 105 ET patients and analyzed ET patients’ mutational profiles, including JAK2 V617F mutation (detected by Allele-specific PCR), CALR, and MPL mutations (both through PCR amplification). Two SNPs of the PEAR1 gene were assessed through ARMS-PCR, and the Sanger method was used for the validation of ARMS-PCR amplification. Results: The prevalence of rs12041331 and rs12566888 in ET patients were 43.9% and 38.5%, respectively, and rs12041331 was significantly associated with increased platelet counts (P-Value: 0.02). As expected, the incidence of thrombotic events in JAK2+ patients was high and significantly associated with JAK2 mutation (P-Value: 0.02). The prevalence of thrombotic events was also high in patients with the rs12041331 variant. Besides, a significant relationship was also found between the rs12041331 and CALR mutation (P-Value: 0.03). Conclusions: In recent years, the footprint of the PEAR1 variant’s effect on platelet aggregation led to evaluating these variants in ET patients. Finally, the significant relationship between the rs12041331 variant and increased platelet count and CALR mutation announced that the idea of this study could be pursued and challenged in the future.

Validation and reliability of current guidelines for the treatment of essential thrombocythemia under real-world clinical settings in Japan

ABSTRACT Objective Current guidelines for essential thrombocythemia (ET) patients recommend different treatment approaches based on thrombosis risk stratification models. However, these recommendations may not be applicable to some patients under real clinical settings. Therefore, we carried out a retrospective real-world validation study. Methods Thrombosis-free survival (TFS) was compared between treatment naïve ET patients receiving different treatment approaches. ET patients were stratified by three representative risk models, the conventional, the International Prognostic Score for thrombosis in ET (IPSET-thrombosis), and revised IPSET-thrombosis. Treatment decisions were largely made by individual physicians, taking into account patient preferences and backgrounds. Results A total of 179 ET patients were included, and thrombotic events were observed in 26 patients. TFS was significantly longer in high-risk patients of all risk models receiving a combination of cytoreductive therapy (CRT) and antiplatelet therapy (APT) compared to CRT alone. Similar results were seen in intermediate-risk patients stratified by IPSET-thrombosis. In contrast, in very low- and low-risk patients of all risk models, TFS was not affected by addition of CRT, indicating that observation or APT alone is an appropriate treatment approach for these patients. Conclusion We demonstrate that current guidelines provide optimal treatment approaches for Japanese ET patients under real-world clinical settings.

Philadelphia-negative myeloproliferative neoplasms display alterations in monocyte subpopulations frequency and immunophenotype.

Philadelphia-negative myeloproliferative neoplasms (MPN) are clonal hematological diseases associated with driver mutations in JAK2, CALR, and MPL genes. Moreover, several evidence suggests that chronic inflammation and alterations in stromal and immune cells may contribute to MPN’s pathophysiology. We evaluated the frequency and the immunophenotype of peripheral blood monocyte subpopulations in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). Peripheral blood monocytes from PV (n = 16), ET (n = 16), and MF (n = 15) patients and healthy donors (n = 10) were isolated and submitted to immunophenotyping to determine the frequency of monocyte subpopulations and surface markers expression density. Plasma samples were used to measure the levels of soluble CD163, a biomarker of monocyte activity. PV, ET, and MF patients presented increased frequency of intermediate and non-classical monocytes and reduced frequency of classical monocytes compared to controls. Positivity for JAK2 mutation was significantly associated with the percentage of intermediate monocytes. PV, ET, and MF patients presented high-activated monocytes, evidenced by higher HLA-DR expression and increased soluble CD163 levels. The three MPN categories presented increased frequency of CD56+ aberrant monocytes, and PV and ET patients presented reduced frequency of CD80/86+ monocytes. Therefore, alterations in monocyte subpopulations frequency and surface markers expression pattern may contribute to oncoinflammation and may be associated with the pathophysiology of MPN.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12032-022-01825-6.

Correlation of the Number of TGFβF1-Expressing Atypical Megakaryocytes with the Degree of Bone Marrow Stroma Fibrosis and Osteosclerosis in Patients with Essential Thrombocythemia and Different Stages of Primary Myelofibrosis

Background. As morphological pattern of bone marrow (BM) biopsy samples at advanced stages of clonal evolution in essential thrombocythemia (ET) appears similar to that in the development of post-thrombocythemic myelofibrosis and primary myelofibrosis (PMF), the expression of fibro-genesis factors by atypical megakaryocytes (MKC) acquires increased interest. Aim. To study the expression of the transforming growth factor TGFβF1 by atypical MKC; to relate the number of TGFβF1-positive MKCs with the degree of BM stroma fibrosis and trabecular bone changes in patients with ET and different PMF stages. Materials &amp; Methods. BM biopsy samples of ET and PMF patients, obtained before cytoreductive therapy, were subjected to histochemical study with Gomori stain and Masson trichrome as well as to CD42b and TGFβF1 antibody immu-nohistochemical assays. The degree of myelofibrosis and osteosclerosis was estimated by semi-quantitative method in accordance with the European Consensus guidelines. The morphological characteristics of atypical MKC included the comparative evaluation of nuclear-cytoplasmic ratio. Results. The number of MKCs with high nuclear-cytoplasmic ratio was significantly higher in BM biopsy samples of patients with pre-fibrosis/early PMF (pre-PMF) stage and fibrosis stage of PMF (f-PMF) compared with BM biopsy samples of ET patients. The analysis of TGFβF1 expression showed different numbers of positive MKCs in the study groups. The matching of the number of TGFβF1-positive MKCs with the degree of myelofibrosis and osteosclerosis, with no regard to nosologic entities, revealed significant moderate correlation between these features (r = 0.431, p = 0.001 and r = 0.499, p = 0.001, respectively). In 55 % of pre-PMF patients’ BM biopsy samples, histochemical study with Masson trichrome stain visualized minimal immature osteoid deposits on bone trabeculae. Similar changes were also identified in f-PMF patients’ BM biopsy samples, whereas the ET patients’ samples featured none of them. Conclusion. The results of the study prove that the pathological clone of MKC with TGFβF1 expression affects myelofibrosis and osteosclerosis processes whose manifestation in BM biopsy samples is associated with the number of TGFβF1-expressing atypical MKCs.

Current Quality-of-Life Aspects in Patients with Classical Ph-Negative Myeloproliferative Neoplasms in the Russian Federation: Overview of the Outcomes of the National Observational Program MPN-QoL-2020

Background. The national observational program MPN-QoL-2020 was focused on quality of life (QoL) and symptoms in patients with classical Ph-negative myeloproliferative neoplasms (MPNs) in the Russian Federation, as well as on the perception of the disease and treatment from the patient's and physician's perspective. Aim. To evaluate QoL in patients with different MPNs using new standardized questionnaires, to assess the most common symptoms and their impact on QoL in patients with myelofibrosis (MF), polycythemia vera (PV) and essential throm-bocythemia (ET), and to characterize the perception of the disease and treatment concerns from patients' perspective and their treating physicians' perspective. Materials &amp; Methods. In total 1100 patients with MPNs (MF: n = 355, PV: n = 408, and ET: n = 337; mean age 58 ± 14 years; 61 % women) and 100 hematologists (mean age 42 ± 12 years; 85 % women) from 37 medical centers in 8 Federal districts of the Russian Federation participated in the study. All the patients filled out symptom assessment tool (MPN10), QoL questionnaire for patients with hematological nancies (HM-PRO) and patient's survey checklist; physicians filled out physician's survey checklist and patient record for each patient included in the study. Results. For the first time in Russia in a representative population of MPN patients in the real-world setting, QoL and symptom profiles in patients with different MPNs were characterized and symptom impact on the daily living of MPN patients was identified. MPN patients exhibited QoL impairment: noticeable detriments in physical and emotional functioning, as well as in eating and drinking regimen were found, social functioning was less impaired. More than one third of MPN patients had significant QoL impairment. The vast majority of patients experienced fatigue: 92.6 % MF patients, 83.7 % PI patients, and 82 % ET patients. Symptom prevalence severity differed across different MPNs. Top disease-related symptoms to be resolved were identified from patient's and physician's perspective. Discrepancies in the attitudes of MPN patients and their treating physicians to various aspects regarding the disease and its treatment were found as well as issues needed to be improved in the patient-physician communication were identified. Conclusion. The results of national research program MPN-QoL-2020 allowed to identify the areas of QoL impairment and symptom burden in MPN patients in Russia, to verify areas of concern related to the disease and its treatment in patients with different MPNs, as well as to highlight the unmet needs in this patients' population in our country. The outcomes of the study may contribute to establishing recommendations for improving/maintaining QoL in patients with MPNs and to developing measures aimed to raise awareness of this patients' population about the disease and its treatment.

Aortic Valve and Arch Replacements in a High-Risk Patient with Essential Thrombocythemia

Reports of the clinical results of cardiac surgery in patients with Essential Thrombocythemia (ET) are rare. In addition, cases of aortic arch aneurysm in ET have not been previously reported. We present a successful aortic valve and arch replacements with the frozen elephant trunk technique using hypothermic circulatory arrest in an ET patient with grade 3 aortic regurgitation and arch aneurysms. Reducing platelet count via the administration of anagrelide and the careful balancing of antiplatelets and anticoagulants is essential in determining the outcome of surgery.