Efficacy and safety outcomes in Japanese patients with low-risk polycythemia vera treated with ropeginterferon alfa-2b.

Polycythemia vera (PV) is a Philadelphia chromosome-negative myeloproliferative neoplasm characterized by clonal erythrocytosis. A phase 2 study reported that ropeginterferon alfa-2b is a well-tolerated and effective treatment for PV in Japanese patients. This post hoc analysis of the phase 2 data further evaluated outcomes in patients at low risk of thrombosis (low-risk PV). Among 20 patients with low-risk PV, 60.0% (12/20) and 85.0% (17/20) achieved < 45% hematocrit by weeks 24 and 52, respectively. The proportion of responders with complete hematologic response (CHR) was 60.0% (12/20) at week 52, and the median time to response was 11.9 months. The mean JAK2 V617F allele burden decreased from 75.8% at baseline to 53.7% at week 52. No patient experienced thrombosis or bleeding episodes. All patients experienced treatment-emergent adverse events (TEAEs) related to ropeginterferon alfa-2b, but no grade ≥ 3 TEAEs or deaths related to ropeginterferon alfa-2b occurred, and no new safety concerns arose. This analysis indicated that ropeginterferon alfa-2b may be an effective treatment option for Japanese patients with low-risk PV.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

[Recent findings and advances in treatment of myeloproliferative neoplasms].

Many novel agents have been developed for BCR::ABL1-negaive myeloproliferative neoplasms (MPN), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Some of these agents not only achieve hematologic complete response, reduce spleen size, and alleviate constitutional symptoms, but also induce molecular response, which means that they reduce the allele burden of driver gene mutations. These agents also prevent and alleviate fibrosis in bone marrow, which reduces the incidence of thrombotic events and disease progression and might improve prognosis. This article discusses the latest findings and promising treatments, including ongoing clinical trials, in PV, ET, and PMF.

Mathematical modelling of stem and progenitor cell dynamics during ruxolitinib treatment of patients with myeloproliferative neoplasms.

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms are a group of slowly progressing haematological malignancies primarily characterised by an overproduction of myeloid blood cells. Patients are treated with various drugs, including the JAK1/2 inhibitor ruxolitinib. Mathematical modelling can help propose and test hypotheses of how the treatment works. Materials and methods: We present an extension of the Cancitis model, which describes the development of myeloproliferative neoplasms and their interactions with inflammation, that explicitly models progenitor cells and can account for treatment with ruxolitinib through effects on the malignant stem cell response to cytokine signalling and the death rate of malignant progenitor cells. The model has been fitted to individual patients' data for the JAK2 V617F variant allele frequency from the COMFORT-II and RESPONSE studies for patients who had substantial reductions (20 percentage points or 90% of the baseline value) in their JAK2 V617F variant allele frequency (n = 24 in total). Results: The model fits very well to the patient data with an average root mean square error of 0.0249 (2.49%) when allowing ruxolitinib treatment to affect both malignant stem and progenitor cells. This average root mean square error is much lower than if allowing ruxolitinib treatment to affect only malignant stem or only malignant progenitor cells (average root mean square errors of 0.138 (13.8%) and 0.0874 (8.74%), respectively). Discussion: Systematic simulation studies and fitting of the model to the patient data suggest that an initial reduction of the malignant cell burden followed by a monotonic increase can be recapitulated by the model assuming that ruxolitinib affects only the death rate of malignant progenitor cells. For patients exhibiting a long-term reduction of the malignant cells, the model predicts that ruxolitinib also affects stem cell parameters, such as the malignant stem cells' response to cytokine signalling.

Case Report and Literature Review of Acute Spontaneous Intraspinal Epidural Hematoma (SSEH) Secondary to Myeloproliferative Disease.

Background: Spontaneous spinal epidural hematoma (SSEH) presenting in the context of JAK2 V617F-positive myeloproliferative neoplasms is a rare condition, characterized by the compression of the spinal cord leading to various symptoms. The etiology, pathogenesis, and optimal treatment strategies for this condition remain undetermined. The occurrence of spontaneous spinal epidural hematoma (SSEH) in the context of JAK2 V617F-positive myeloproliferative neoplasms (MPNs) represents a rare manifestation. Magnetic Resonance Imaging (MRI) plays a crucial role in the definitive diagnosis of this condition. With a good understanding of the pathogenic characteristics and clinical presentations of this disease, a diagnosis can be reasonably made, even in the absence of MRI, based on physical examinations indicating the affected area. Once diagnosed, immediate surgery is recommended to attempt the restoration of spinal cord function. Postoperatively, the use of hydroxyurea has proven effective in disease control. Case Presentation: We report a case of a 65-year-old male patient who presented with progressive lumbar back pain and bilateral lower limb paralysis lasting for 36 hours. CT imaging revealed an intraspinal lesion at the L1-3 level, and genetic testing confirmed the presence of the JAK2V617F mutation. Following surgery, there was a significant recovery of sensory and motor function in the lower limbs. At one-year follow-up, the patient demonstrated good functional status, and blood tests indicated a platelet count within the normal range. Conclusion: The presented case adds to the existing literature on SSEH by highlighting the association with myeloproliferative neoplasms (MPNs), as evidenced by the JAK2V617F mutation. MPNs constitute a group of hematologic malignancies, and the association with SSEH is a rare occurrence. The exact interplay between MPNs and SSEH warrants further investigation, as the underlying mechanisms linking these conditions remain elusive. The case also underscores the importance of a multidisciplinary approach, involving hematologists and neurosurgeons, in the comprehensive management of such complex cases.

Myeloproliferative Neoplasm Symptom Assessment Total Symptom Score (MPN-SAF TSS) in Chronic Myeloproliferative Neoplasms, Related with Genetic Burden, and Thrombosis.

Myeloproliferative Neoplasm Symptom Assessment Total Symptom Score (MPN-SAF TSS) is a surrogate marker for symptom evaluation in Chronic Myeloproliferative Neoplasms. There is not enough data to show the relationship between MPN-SAF TSS, JAK2 mutation allele burden, and thrombosis. In this retrospective analysis, we aimed to determine the genetic burdens, clinical features, and relationship with MPN-SAF TSS in MPN patients. One hundred thirty JAK2V617F positive MPN were included in our study. We have calculated MPN-SAF TSS and compared it with clinical features. Patients with higher JAK2V617F mutation allele burden had higher MPN-SAF TSS (p-value 0,008). Patients with thrombosis had higher MPN-SAF TSS scores than patients without thrombosis (p-value 0.003). The mean MPN-SAF TSS was higher in primary myelofibrosis (PMF) patients compared to PV and ET patients. Thrombosis was associated with increased symptom severity in several domains, including fatigue, abdominal discomfort, inactivity, night sweats, pruritus, weight loss, and early satiety. Additionally, an increase in JAK2 allele burden was observed with higher symptom scores. The MPN-SAF TSS proved to be a reliable tool for assessing symptom burden in Turkish MPN patients. Furthermore, a significant association between thrombosis occurrence and symptom severity suggests that thrombotic events may contribute to symptom development. Notably, increasing JAK2 allele burden was correlated with more severe symptoms, highlighting its potential role in predicting disease burden. This study emphasizes the importance of symptom assessment in MPN patients and supports the incorporation of MPN-SAF TSS in routine clinical practice to enhance patient care and management.

JAK-2 V617F Mutation in Endothelial Cells of Patients with Atherosclerotic Carotid Disease.

Objective: It has been shown that clonal mutations occur in hematopoietic stem cells with advancing age and increase the risk of death due to atherosclerotic vascular diseases, just like in myeloproliferative neoplasms. It is known that endothelial cells (EC) and hematopoietic stem cells develop from a common stem cell called hemangioblast in early embryonic period. However, the presence of hemangioblast in the postnatal period is controversial. In this study, JAK2 gene variants was examined in patients with atherosclerotic carotid disease and without any hematological malignancy. Materials and Methods: Ten consecutive patients (8 men and 2 women) with symptomatic atherosclerotic carotid stenosis were included in this study. EC (CD31+CD45-) were separated from tissue samples taken by carotid endarterectomy. JAK2 variants was examined in EC, peripheral blood mononuclear cells and oral epithelial cells of the patients with next generation sequencing. Results: The median age of the patients was 74 (58-80) and the median BMI was 24,44 (18,42-30,85) kg/m2. Smoking history was present in 50%, hypertension in 80%, diabetes in 70%, and ischemic heart disease in 70% of the patients. JAK2V617F mutation was detected in peripheral blood mononuclear cells in three out of 10 patients, two of them also had JAK2V617F mutation in their EC. JAK2V617F mutation was not found in oral epithelial cells in any of the patients. Conclusion: In this study, for the first time in the literature, we showed that JAK2V617F mutation was found somatically in both peripheral blood cells and EC in patients with atherosclerosis. This finding may support that EC and hematopoietic cells originate from a common clone or that the somatic mutation can be transmitted to EC by other mechanisms. Examining the molecular and functional changes caused by JAK2V617F mutation in EC may help open a new avenue for treating atherosclerosis.

Risk factors for ischemic stroke in patients with Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-negative MPNs) are linked with various complications, notably ischemic stroke. The study aims to identify risk factors for ischemic stroke in Ph-negative MPNs patients. METHODS: Patients were categorized into two groups based on whether they had experienced ischemic stroke. Subsequently, an analysis of demographics, biochemical makers, and genetic mutations (JAK2V617F and CALR mutations), was conducted to identify potential associations with an elevated risk of ischemic stroke in individuals with Ph-negative MPNs. RESULTS: A total of 185 patients diagnosed with Ph-negative MPNs participated in the study, including 82 with essential thrombocythemia (ET), 78 with polycythemia vera (PV), and 25 with primary myelofibrosis (PMF). Among these, 57 patients (30.8 %) had a history of ischemic stroke. Independent risk factors associated with ischemic stroke in Ph-negative MPNs patients included hypertension (OR = 5.076) and smoking (OR = 5.426). Among ET patients, smoking (OR = 4.114) and an elevated percentage of neutrophils (OR = 1.080) were both positively correlated with ischemic stroke incidence. For PV patients, hypertension (OR = 4.647), smoking (OR = 6.065), and an increased percentage of lymphocytes (OR = 1.039) were independently associated with ischemic stroke. Regardless of the presence of the JAK2V617F mutation, hypertension was the sole positively and independently associated risk factor for ischemic stroke. The odds ratios for patients with the JAK2V617F mutation was 3.103, while for those without the mutation, it was 11.25. CONCLUSIONS: Hypertension was a more substantial factor associated with an increased incidence of ischemic stroke in Ph-negative MPNs patients.

Acute upper gastrointestinal bleeding due to portal hypertension in a patient with primary myelofibrosis: A case report.

BACKGROUND: Acute upper gastrointestinal bleeding is a common medical emergency that has a 10% hospital mortality rate. According to the etiology, this disease can be divided into acute varicose veins and nonvaricose veins. Bleeding from esophageal varices is a life-threatening complication of portal hypertension. Portal hypertension is a clinical syndrome defined as a portal venous pressure that exceeds 10 mmHg. Cirrhosis is the most common cause of portal hypertension, and thrombosis of the portal system not associated with liver cirrhosis is the second most common cause of portal hypertension in the Western world. Primary myeloproliferative disorders are the main cause of portal venous thrombosis, and somatic mutations in the Janus kinase 2 gene (JAK2 V617F) can be found in approximately 90% of polycythemia vera, 50% of essential thrombocyrosis and 50% of primary myelofibrosis. CASE SUMMARY: We present a rare case of primary myelofibrosis with gastrointestinal bleeding as the primary manifestation that presented as portal-superior-splenic mesenteric vein thrombosis. Peripheral blood tests revealed the presence of the JAK2 V617F mutation. Bone marrow biopsy ultimately confirmed the diagnosis of myelofibrosis (MF-2 grade). CONCLUSION: In patients with acute esophageal variceal bleeding due to portal hypertension and vein thrombosis without cirrhosis, the possibility of myeloproliferative neoplasms should be considered, and the JAK2 mutation test should be performed.

Two Novel Genetic Variants Involved in the Oxygen Sensing Pathway in JAK2-unmutated Erythrocytosis.

JAK2-unmutated erythrocytosis or non-polycythemia vera erythrocytosis is a rare condition comprising both acquired and hereditary forms. Although acquired erythrocytosis has been well-studied, hereditary erythrocytosis remains poorly studied. Genetic alterations associated with hereditary erythrocytosis include mutations in erythropoietin receptor and erythropoietin (EPO), altered oxygen affinity mutations, and variants associated with the oxygen-sensing pathway. We established a molecular diagnostic approach based on these genes and retrospectively evaluated. Peripheral blood from 56 erythrocytosis patients, lacking JAK2 mutation, were screened for oxygen-sensing pathway abnormalities. Two novel mutations were identified in the EGLN1 gene: NM_022051.2:c.712G > C (p.Gly238Arg) and NM_022051.2:c.122A > C (p.Tyr41Ser) in two patients separately. Notably, both reported heterozygous mutations were absent in the population database. Predictions using multiple computer software indicated that these two missense mutations were harmful and induced a highly conserved amino acid change in EGLN1. Patients with the two mutations exhibited normal serum EPO levels and high hemoglobin and hematocrit levels. Additionally, three other variants of genes were identified in the oxygen-sensing pathway, including endothelial PAS domain protein 1 (EPAS1) rs184760160(2/56), and EGLN1 rs186996510(2/56), rs555121182(2/56). These variants were categorized as benign or likely benign. Our findings provide a framework for etiological research and highlight the importance of screening for genetic mutations associated with erythrocytosis in clinical practice.

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Adrenal infarction with latent myelodysplastic/myeloproliferative neoplasm, unclassifiable with JAK2V617F mutation.

Key Clinical Message: Hematopoietic neoplasms can cause adrenal infarction. In cases of thrombosis occurring at uncommon sites, it is necessary to consider evaluating for the JAK2V617F mutation, even in the absence of notable abnormalities in blood counts. Abstract: Adrenal infarction, a rare ailment, has been sporadically linked to hematopoietic neoplasms. A 46-year-old male encountered left adrenal infarction, which coincided with a progressive rise in platelet counts. Subsequent diagnosis revealed myelodysplastic/myeloproliferative neoplasm-unclassifiable, featuring a JAK2V617F mutation. Simultaneously, the patient manifested multiple arteriovenous thromboses, necessitating treatment with edoxaban, aspirin, and hydroxyurea. Following thrombosis resolution, he was transferred to a transplantation center. This report delves into the thrombogenicity linked to the JAK2V617F mutation, while also examining documented instances of adrenal infarction in myeloid neoplasms. We should consider evaluating for JAK2V617F mutation even in cases of thrombosis at unusual sites, including adrenal infarction, even if there are no considerable abnormalities in blood counts.

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

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Impact of CALR and JAK2V617F Mutations on Clinical Course and Disease Outcomes in Essential Thrombocythemia: A Multicenter Retrospective Study in Turkish Patients.

Objective: In this study, we investigated the effects of calreticulin (CALR) and JAK2V617F mutational status on clinical course and disease outcomes in Turkish patients with essential thrombocythemia (ET). Materials and Methods: Seventeen centers from Türkiye participated in the study and CALR- and JAK2V617F-mutated ET patients were evaluated retrospectively. Results: A total of 302 patients were included, of whom 203 (67.2%) and 99 (32.8%) were JAK2V617F- and CALR-positive, respectively. CALR-mutated patients were significantly younger (51 years vs. 57.5 years, p=0.03), with higher median platelet counts (987x109/L vs. 709x109/L, p<0.001) and lower median hemoglobin levels (13.1 g/dL vs. 14.1 g/dL, p<0.001) compared to JAK2V617F-mutated patients. Thromboembolic events (TEEs) occurred in 54 patients (17.9%), 77.8% of which were arterial. Compared to CALR mutation, JAK2V617F was associated with a higher risk of thrombosis (8.1% vs. 22.7%, p=0.002). Rates of transformation to myelofibrosis (MF) and leukemia were 4% and 0.7%, respectively, and these rates were comparable between JAK2V617F- and CALR-mutated cases. The estimated overall survival (OS) and MF-free survival of the entire cohort were 265.1 months and 235.7 months, respectively. OS and MF-free survival durations were similar between JAK2V617F- and CALR-mutated patients. Thrombosis-free survival (TFS) was superior in CALR-mutated patients compared to JAK2V617F-positive patients (5-year TFS: 90% vs. 71%, respectively; p=0.001). Age at diagnosis was an independent factor affecting the incidence of TEEs. Conclusion: In our ET cohort, CALR mutations resulted in higher platelet counts and lower hemoglobin levels than JAK2V617F and were associated with younger age at diagnosis. JAK2V617F was strongly associated with thrombosis and worse TFS. Hydroxyurea was the most preferred cytoreductive agent for patients with high thrombosis risk.

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry.

BACKGROUND: Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. METHODS: Intracellular Ca2+ levels were measured over a time frame of 0-1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0-108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109-594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595-1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann-Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. RESULTS: Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. CONCLUSIONS: This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.

Exposure-efficacy and exposure-safety analyses of ropeginterferon alfa-2b treatment in patients with polycythaemia vera.

AIMS: To investigate the exposure-response (E-R) relationship, including exposure-efficacy and exposure-safety, of ropeginterferon alfa-2b treatment in patients with polycythaemia vera (PV). METHODS: Based on the results of the phase II trial A20-202 regarding ropeginterferon alfa-2b in patients with PV, E-R analyses were performed to evaluate the efficacy and safety of the given dosing regimen. The E-R analyses were based on logistic and linear regression and the relationship between exposure to ropeginterferon alfa-2b and key efficacy and safety variables. The key efficacy variables included complete haematologic response (CHR) and reduction of the driver mutation JAK2V617F. The safety variable was treatment-related adverse events (TRAEs). RESULTS: A clear relationship between the exposure to ropeginterferon alfa-2b and CHR was observed, with an increase in drug exposure resulting in an increased probability of achieving CHR. Similar CHR probabilities were observed in the third and fourth quantiles of the average concentration at Week 24. The results from the exposure-JAK2V617F model indicated that the JAK2V617F allele burden decreased with increasing exposure to ropeginterferon alfa-2b and baseline body surface area. Exposure-safety analysis revealed a risk of AEs associated with transaminase abnormalities, which were not associated with clinical significance. CONCLUSIONS: Our analyses have shown that patients with PV treated with ropeginterferon alfa-2b had an increased probability of achieving CHR and a molecular response with acceptable safety risks at the 250-350-500 µg titration dosing regimen. This study has provided the relevant data for the application of a biologics licence of ropeginterferon alfa-2b for PV treatment in China.

Clinical features of extrahepatic portal vein obstruction: Myeloproliferative neoplasms eliminate hypersplenic hematologic changes in extrahepatic portal vein obstruction.

Extrahepatic portal vein obstruction (EHPVO) is a rare disease. Most EHPVO patients are usually referred to a gastroenterologist for intestinal bleeding and hypersplenic thrombocytopenia; however, hypercoagulative diseases may be occult in these patients and require anticoagulation. The purpose of this study was to elucidate the clinical characteristics of EHPVO. We conducted a retrospective analysis of the hospital database, evaluating the medical records of 15 patients (7 males, 8 females, mean age of onset 42.0 years, range 5-74 years). Thirteen of 15 EHPVO patients (86.7%) had intestinal varices. These included 10 esophageal (66.7%), 12 gastric (80.0%), and 6 ectopic varices (40.0%). Nine (60.0%) of 15 had a history of intestinal bleeding. Regarding comorbidities, 5 of 15 (33.3%) suffered from vascular diseases, including acute myocardial infarction, cerebral infarction, pulmonary embolism, Budd-Chiari syndrome, and mesenteric vein thrombosis. The former 3 vascular commodities manifested at less than 32 years of age. Four patients (26.7%) with JAK2V617F mutation were diagnosed as myeloproliferative neoplasm (MPN). 72.3% of EHPVO patients without MPN experienced thrombocytopenic state. No EHPVO patients with MPN experienced thrombo-leukocytopenia. The elevation of white blood cell and platelet counts, and decrease of protein S were seen in EHPVO with MPN, compared with EHPVO without MPN. EHPVO is frequently associated with underlying hypercoagulative factors, causing a dilemma between thrombotic complications and portal hypertensive bleeding. Most EHPVO patients experience an evident thrombocytopenic state due to severe hypersplenism; however, hypersplenic hematologic changes are eliminated in EHPVO with MPN. MPN should be suspected in EHPVO patients negative for thrombo-leukocytopenia.

From primary myelofibrosis to chronic myeloid leukemia, BCR::ABL1+ B-Lymphoblastic leukemia, and back to primary myelofibrosis: An illustration of dynamic clonal evolution.

The simultaneous detection of BCR::ABL1 and JAK2 V617F was rarely reported and their clonal relationship and dynamic clonal shift were not characterized. Here, we described a unique case with the initial presentation as JAK2 V617F+ primary myelofibrosis, followed by the emergence of BCR::ABL1+ chronic myeloid leukemia. The patient then developed BCR::ABL1+ B-lymphoblastic leukemia. Treatment for B-lymphoblastic leukemia prompted a regression to the state of primary myelofibrosis. In light of these observations, we proposed a clonal evolution model for this case.