Positive Myeloproliferative Neoplasms Research Articles

Myeloid Sarcoma of Conjunctiva in a patient with JAK2-positive myeloid neoplasm: two distinct myeloid neoplasms sequentially arising from a common precursor

Abstract Introduction/Objective Myeloid sarcoma (aka granulocytic sarcoma or chloroma), a rare type of myeloid neoplasm is characterized by the presence of myelogenous blastic cells outside of the bone marrow either before, during or after the development of leukemia. Presentation in the orbit is extremely rare, unlike other body parts. We report a case of myeloid sarcoma of conjunctiva in a patient with JAK2-positive myeloid neoplasm. Methods/Case Report An 84-year-old man presented with bilateral conjunctival masses which on biopsy showed diffuse involvement by blasts, immunohistochemically expressing positivity for myeloperoxidase, CD33, CD68, CD34 (partial), CD117 (partial) but were non-reactive to CD15, CD3 and PAX5. Next generation sequencing (NGS) revealed mutations in NRAS, ASXl1 and TET2 genes, consistent with the diagnosis of myeloid sarcoma (MS) of the conjunctiva. While patient’s past medical history included status post radiation and chemotherapy for prostate cancer and colonic adenocarcinoma respectively. it was also significant for a JAK 2 positive myeloproliferative neoplasm (MPN), six-years prior to current presentation. At the time he was treated with hydroxyurea for 5 years but developed neutrophilic leukocytosis and transfusion-dependent anemia. Subsequently a treatment regimen including ruxolitinib and pacritinib was initiated but without clinical and hematological improvements. NGS of the initial JAK2-positive MPN revealed ASXL1, JAK2 and TET2 mutations. Of note are the similarities and variations in the molecular signatures of patient’s initial MPN and current conjunctival MS. While the two neoplasms carried an identical TET2 mutation with highest variant allelic frequency, they are different in other mutations. Results (if a Case Study enter NA) NA Conclusion Our case illustrates an unusual manifestation of myeloid sarcoma localized to the conjunctiva. The identification of clonal relatedness on NGS with distinct mutations, adds complexity to our understanding of tumor origin. It highlights a series of events which may have contributed to this phenomenon, including consequence of therapy, clonal evolution with molecular discordance or origin as a totally independent entity. This report underscores the importance of genetic profiling in guiding prognosis and treatment strategies in such settings.

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F–positive myeloproliferative neoplasms

AbstractPegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F–positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α–treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F–positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F–positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Analysis of risk factors for thromboembolism in patients with JAK2V617F gene mutation positive myeloproliferative neoplasms

Objective: To analyze the risk factors of thrombosis in patients with JAK2V617F mutation positive myeloproliferative neoplasms (MPN). Methods: A total of 223 MPN patients with JAK2V617F mutation in the Second Hospital of Tianjin Medical University from September 2017 to May 2023 were retrospectively enrolled, including 111 males and 112 females, aged [M(Q1,Q3)] 57(21,66) years. According to the presence or absence of thromboembolism during follow-up, the patients were divided into thrombosis group (n=102) and non-thrombosis group (n=121). The clinical characteristics, laboratory characteristics, cytogenetics and other disease progression and survival of the two groups of patients were analyzed. As of March 31, 2023, the follow-up period [M (Q1, Q3)] was 6 (3, 10) years. The influencing factors of thrombosis in JAK2V617F positive MPN patients were analyzed by using the Cox risk model. Results: Among 223 JAK2V617F positive MPN patients, 144 were polycythemia vera (PV), 51 were essential thrombocythemia (ET) and 28 were primary myelofibrosis (PMF). The mutation rates of ASXL1 and BCORL1 genes in the thrombosis group were 19.6% (20/102) and 6.9% (7/102), respectively, which were higher than those in the non-thrombosis group [9.1% (11/121) and 0.8% (1/121)] (both P<0.05). The proportion of monocytes, C-reactive protein (CRP), interleukin-1β (IL)-1β, IL-8 and tumor necrosis factor-β (TNF-β) increased in the thrombosis group were higher than those in the non-thrombosis group (all P<0.05). Multivariate analysis showed that age≥60 years (HR=2.132, 95%CI: 1.376-3.303, P=0.001), history of thrombosis (HR=3.636, 95%CI: 2.121-6.202, P<0.001), ASXL1 mutation positive (HR=2.245, 95%CI: 1.093-3.231, P=0.022) and elevated TNF-β (HR=2.009, 95%CI: 1.113-3.624, P=0.021) were risk factors for thrombosis in JAK2V617F positive MPN patients. Conclusions: In addition to age, history of thrombosis and positive ASXL1 mutation, elevated TNF-β is also an influencing factor of thrombosis in JAK2V617F positive MPN patients. Intervention of inflammation may have a certain effect on the prevention and treatment of thrombosis.

Features of immune mediated diseases in JAK2 (V617F)-positive myeloproliferative neoplasms and the potential therapeutic role of JAK inhibitors

ObjectiveThe Janus Kinase (JAK) 2 (V617F) mutation is the most frequently detected in myeloproliferative neoplasms (MPN). JAK2(V617F) mutation displays a pro-inflammatory phenotype that may be associated to a higher risk of immune mediated diseases (IMIDs), thromboembolic complications or other cancers. We aimed to evaluate the prevalence and main features of both rheumatic and non-rheumatic IMIDs in a cohort of MPNs patients with JAK2 (V617F) mutation. MethodsStudy of all patients diagnosed with MPNs and JAK2 (V617F) mutation at a tertiary hospital in Northern Spain from 2004 to 2022. We focused on patients with rheumatic IMIDs to assess the time from IMIDs diagnosis to the detection of JAK2V617F mutation, the clinical course and severity of the disease, potential thrombotic complications, malignancies and therapeutic response. Results130 patients (73 men/57 women; mean age, 70.1 ± 14.5 years) were identified. Fifty-four (41.5 %) patients were diagnosed with at least one IMID. The prevalence of rheumatic IMIDs was 7.7 % (n = 10), including rheumatoid arthritis (n = 4), polymyalgia rheumatica (n = 3), Sjögren syndrome (n = 1), antiphospholipid syndrome (n = 1) and autoinflammatory syndrome with WDR1 mutation (n = 1). Thrombotic complications were observed in 4 of these 10 patients. The clinical course of the rheumatic IMID was mild in most cases and responded to conventional immunosuppressive therapy. One patient was successfully treated with Baricitinib, a JAK1/JAK2 inhibitor. ConclusionsA high prevalence of rheumatic IMIDs is observed in patients with MPNs and JAK2 (V617F) mutation. JAK inhibitors might be a targeted therapy option in these patients.

Proteomic Profiling and Cytokine Analysis Reveals Molecular Signatures and Inflammatory Factors Associated with Thrombosis in JAK2-V617F Positive Myeloproliferative Neoplasm (MPN) Cohorts

Background: Myeloproliferative neoplasms (MPNs), a hematological malignancy commonly associated with the JAK2-V617F mutation, exhibit a considerable burden of comorbidities resulting from inflammation-mediated processes. The JAK2-V617F malignant clones instigate systemic inflammation by upregulating various pro-inflammatory cytokines. This leads to dysregulated immune and inflammatory responses, thereby driving a process called thrombo-inflammation. Neutrophils play a crucial role as inflammatory mediators and effectors of pro-thrombotic activity in MPNs. We, therefore, hypothesize that perturbations in the neutrophil proteome may alter the molecular communication networks within neutrophils, thereby potentially contributing to the pathological thrombus formation in veins and arteries. Aim: The study was aimed to elucidate (i) the pathological significance of dysregulated protein expression profile of neutrophils carrying the JAK2-V617F mutation, and (ii) the impact of pro-inflammatory cytokines on neutrophils during thrombo-inflammation. Methods: In this study, which was conducted at the University Hospital Magdeburg, Germany, 11 patients carrying the JAK2-V617F mutation (6 with thrombosis; 5 without thrombosis) and 9 healthy volunteers were enrolled. Neutrophils were isolated from freshly collected whole blood using a negative selection kit. The isolated neutrophils were then lysed, and protein extraction was performed. High-resolution liquid chromatography - mass spectrometry (LC-MS/MS) was employed to investigate the protein inventory of neutrophils. Furthermore, platelet-poor plasma was isolated from the same cohort to analyse plasma cytokine/chemokine levels using a bead-based multiplex assay. Results: LC-MS/MS identified 2,006 proteins in neutrophils, which were then used for quantitative comparative analysis. Amongst these, 28 and 26 proteins exhibited significant up-regulation in the JAK2-V617F positive patients ‘with thrombosis’ and ‘without thrombosis’, respectively. Notably, ATP2A3 and ROCK1 were significantly upregulated in the JAK2-V617F thrombotic group, ranking among the top 10-upregulated proteins. Of note, ROCK1 activation has previously been implicated in cell proliferation, survival and cell cycle control. Functional annotation analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that upregulated proteins in the JAK2-V617F cohorts were significantly enriched for proteins involved in the calcium and TGF-ß signaling pathways. Furthermore, we assessed the levels of 60 cytokines/chemokines and soluble adhesion molecules in the same cohort, wherein significantly increased levels of ALCAM, ICAM-2 and VCAM-1 were observed in the JAK2-V617F thrombotic group. These molecules are known to play a critical role in vasculature-related inflammation and endothelial cell activation. Additionally, the JAK2-V617F thrombotic group exhibited significant upregulation of free active TGF-ß and pro-inflammatory cytokines such as IL-6 and IL-8 compared to both healthy subjects and JAK2-V617F non-thrombotic patients, indicating their contribution to the thrombo-inflammatory response. Moreover, the thrombotic cohort displayed elevated plasma levels of CXC chemokines and myeloperoxidase (MPO), a key enzyme linked to NETosis for neutrophil extracellular trap (NET) formation. Conclusions: The proteomic profile of neutrophils in MPN patients exhibit significant alterations, particularly between JAK2-V617F patients with and without thrombosis. The observed elevated expression of markers related to NETosis and calcium signaling pathways point towards the underlying mechanisms that could potentially contribute to the differential behavior of neutrophils, thereby facilitating the development of a thrombotic state in MPN patients.

Increase in Total Protein Level of Bruton's Tyrosine Kinase in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by atherosclerosis and cardiovascular risk. The mechanisms that connect MPNs with atherothrombosis remain elusive. It is known that atherosclerotic plaque causes Glycoprotein VI (GPVI)-dependent platelet activation and hence activation of bruton kinase (Btk) and that Btk inhibitors in vivo and ex vivo suppress GPVI-mediated paltelet activation. Aim of this study is to measure the levels of GPVI and Btk. In addition, we measured the CXCL12 as marker of atherothrombosis. We studied 25 JAK2V617F positive MPNs patients (WHO criteria) (mean age 58 years) including PV 12, ET 8, and PMF 5. We measured platelet count by automated analyzer, GPVI by flow cytometry, total Btk (T-Btk) protein by immunoblot and total CXCL12 (T-CXCL12) protein by ELISA. All patients had thrombocytosis (630±40x109/L) and allele burden ≥ 50%. We found high GPVI expression (mean fluorescence intensity (MFI) 20±5.8) and high T-Btk (mean value 1.0) as well as high T-CXCL12 (15±2 μg/ml). A positive correlation there was between GPVI and Btk. We further investigated Btk mRNA tran script levels using RT-PCR assay in triplicate from each samples and found mean values ≥ 1.0. On the basis of these results the use of GPVI- and Btk- inhibiting agents as atherothrombosis-focused antiplatelet drugs is conceivable. Therefore, clinical trials are eagerly awaited.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Meth- ods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

Molecular analysis of phenotypic heterogeneity in JAK2V617F-positive myeloproliferative neoplasms reveals a potential target for therapy.

JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/β-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/β-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/β-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/β-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/β-catenin signalling as a potential therapeutic target for MPN.

Diagnostic and Therapeutic Challenges of Chronic Portal Vein Thrombosis Secondary to Myeloproliferative Neoplasm in Sub-Saharan Region: A Series of Cases and Literature Review

Myeloproliferative neoplasms (MPN) are the commonest causes of non-cirrhotic abdominal vein thrombosis. MPN was observed in 30-40% with Budd Chiari Syndrome (BCS) and portal vein thrombosis (PVT), whereas this is the cause in only a minority of other types of venous thrombosis. Herein, we report 10 cases of chronic PVT secondary to Janus Kinase 2 gene (JAk 2) positive myeloproliferative neoplasm (MPN). The median age was 35 years with M:F 1:1. Portal hypertension was present at presentation in all of them. Nine patients have esophageal varices, of which 6 have bleeding at presentation. Classical MPN phenotype on complete blood count (CBC) was not present in 6 of them. Management of patients with anticoagulation was difficult with 4 patients experiencing bleeding during anticoagulation, and bleeding occurring despite the target international normalized ratio (INR) in 2 of them. Portal hypertension leading to hypersplenism and hemodilution may mask erythrocytosis and thrombocytosis, which are the main criteria used to diagnose MPN. The resulting absence of a typical MPN phenotype in routine laboratory studies alongside accessibility issues, and the need for a multidisciplinary treatment approach imparts a diagnostic as well as a therapeutic challenge for chronic PVT associated with Jak 2 positive MPN in sub-Saharan patients.

Effects of Ruxolitinib on Myeloproliferative Neoplasms via the Negative Regulators.

We evaluated the JAK2V617F mutation and p-JAK2, SOCS-1, SHP-1 expression in JAK2V617F positive myeloproliferative neoplasms (MPNs) patients and the role of JAK/STAT pathway in human erythroleukemia (HEL) cells, which had JAK2V617F mutation. Protein expression of p-JAK2, SOCS-1, SHP-1 in bone marrow biopsies (BMBs) were detected by immunohistochemical staining methods. Cell apoptosis and cell cycle were detected by flow cytometry and Caspase 3/7 assay kits. 1. The p-JAK2, SOCS-1, and SHP-1 expressions were significantly different between JAK2V617F positive MPN and control patients (p < 0.01); 2. After being treated for 3 months, the p-JAK2, SOCS-1, and SHP-1 expressions were significantly different compared with newly diagnosed patients (p < 0.01). 3. HEL cell viabilities were significantly different after being treated with different concentrations of ruxolitinib. Ruxolitinib had a significant effect on the cell apoptosis, viability, and the protein activity of caspase-3 and -7 of HEL cells. 3. The mRNA and protein expressions of JAK2 and the protein expression of p-JAK2 were gradually decreased (p ＜ 0.01, p ＜ 0.05), while the mRNA and protein expressions of SOCS1 and SHP1 were gradually increased (all p ＜ 0.01).

Global Hemostatic Assays for Risk Stratification of Thrombosis in Myeloproliferative Neoplasms (MPN): A Systematic Review and Meta-Analysis

Background: Arterial and venous thromboembolism are major complications of myeloproliferative neoplasms (MPN), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The pathogenesis of thrombosis in MPN is not fully understood. Global hemostatic assays, including thrombin generation assay (TGA), rotational thromboelastometry (ROTEM), and thromboelastography (TEG), have been evaluated as predictive markers of hypercoagulability in MPNs. However, validation of these and other laboratory surrogates is lacking. The objective of this systematic review is to summarize the literature and to examine the utility of global hemostatic assays as predictive markers for hypercoagulability and occurrence of thrombosis in MPNs. Methods: A systematic literature search was performed using MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews from inception to April 14, 2022, restricted to the English language. Criteria for eligible studies were studies that reported the results of TGA, ROTEM, and TEG in ET, PV, or PMF adult patients. Studies were required to have ≥10 subjects in each group. Two reviewers (A.T. and T.C.) independently searched the literature and extracted data from eligible studies. Disagreements were resolved by consensus or a third reviewer (C.K) when necessary. The methodological quality of included studies was appraised using the Newcastle - Ottawa Quality Assessment Scale (NOS) assessment tool. A meta-analysis was planned given sufficient data from included studies. The study protocol is available on PROSPERO (CRD42022325893). Results: A total of 815 records were retrieved from the literature search. After screening by title and abstract, 745 records were excluded. The remaining 70 references underwent full text review, 30 of which met eligibility criteria and were included in the analysis. These 30 studies (15 full-texts and 15 abstracts) collectively enrolled 926 controls, 700 ET, 556 PV, and 192 PMF patients. The number of studies that reported TGA, ROTEM, and TEG were 26, 3, and 3, respectively. The number of participants in each study ranged from 25 to 212 including the controls. Two studies originated from Oceania, 1 from North America, and 27 from Europe. JAK2 mutation was reported only in 15 studies and only two studies reported quantitative variant allele frequencies. There were 5 studies that did not have healthy controls. All of these studies were cross sectional analyses; no longitudinal prospective age/sex matched controlled studies have been published. Of the 26 TGA studies, comparison of TGA parameters were made between MPN vs. controls (15 studies), MPN with thrombosis vs. without thrombosis (3 studies), JAK2 positive vs. negative MPNs (11 studies), and MPN with vs. without treatment (6 studies). There is heterogeneity in the types of samples (platelet poor plasma or platelet rich plasma), trigger reagents (amounts of tissue factor, phospholipid, and thrombomodulin), and the automated platforms that were used. Significant differences in the endogenous thrombin potential (9 studies), peak height (10 studies), lag time (6 studies), and velocity index (3 studies) were found between MPN and controls. Thirteen studies reported rates of arterial and venous thrombotic outcomes in MPN patients, which ranged from 19 to 45%. Only 1 study found an association of TGA parameters and thrombosis. Two ROTEM studies showed ET having higher maximum clot firmness and shorter clot formation time compared to controls. Two TEG studies showed increased alpha angle between MPN and controls and between PV and controls. Risk of bias assessment with NOS showed summary scores ranging 1-3 for abstracts and 4-9 for full text studies. We were not able to perform meta-analysis due to insufficient data and the high heterogeneity in study objectives and laboratory methods. Conclusions: Studies were limited by small sample sizes and inconsistent results and do not comprehensively reflect the effects of treatment, duration of disease, or clonal status. The role of these global hemostatic assays in predicting thrombotic events remains inconclusive. Future studies are necessary to establish the usefulness and validity of these surrogate markers as predictors of thrombotic complications in the MPNs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Knockout of HMGA1 Promotes ATRA-Induced Differentiation in MLL-Rearranged Leukemia

Introduction: HMGA1 is a small non-histone chromatin protein characterized by 3 AT-hooks, which modulates transcription by altering the chromatin architecture. It tends to be highly expressed in poorly differentiated cells including hematopoietic stem cells. In various kinds of solid tumors, HMGA1 has been reported to play as an oncogene, and its upregulation was reported to be associated with poorer clinical outcomes. However, although recent study demonstrated that HMGA1 was associated with progression of JAK2V617F positive myeloproliferative neoplasms to myelofibrosis or leukemia, the roles of HMGA1 in myeloid malignancies were still unclear. Therefore, we analyzed its function in myeloid malignancies using HMGA1 knockout (KO) mice. We could not detect clear differences in peripheral blood counts or bone marrow cells, and generated various kinds of models of acute myeloid leukemia (AML). Knockout of HMGA1 could not delay the onset of these leukemia, but we could demonstrate that leukemic cells with MLL-ENL fusion gene which lacked in HMGA1 gene were more susceptible to all-trans retinoic acid (ATRA)-induced expression of CD11b, which suggested that HMGA1 could be associated with the differentiation process induced by ATRA. Method: We generated Vav-iCre HMGA1flox/flox mice, and we first analyzed the fraction of peripheral blood cells and bone marrow cells of these mice using flow cytometry. Next, to evaluate roles of HMGA1 in AML, we made various murine models of AML. We collected c-kit positive fractions of bone marrow cells from the KO mice or Vav-iCre(-) HMGA1flox/flox mice and retrovirally introduced MLL-ENL, MOZ-TIF2, BCL2-MYC fusions. Primary transplantation was performed to sublethally irradiated (5Gy) mice with 4.0×105 infected cells for each fusion gene. Secondary transplantation was also performed for MLL-ENL and BCL2-MYC with 1.0×104 cells for each. Blood samples were collected weekly, and disease-free survivals were compared by Kaplan-Meier method with Vav-iCre(-) HMGA1flox/flox mice. Also, leukemic cells infected with MLL-ENL were collected, and these cells were cultured for four days with different concentrations (1μM, 5μM or 10μM) of ATRA. Flow cytometry analysis was performed to evaluate the differentiation induced by ATRA with antibodies to murine CD11b, c-kit and Gr-1. Result: There was no significant difference in birth rate between Vav-iCre(+) and Vav-iCre(-) mice, which suggested that HMGA1 was not essential for fetal hematopoiesis. Peripheral blood cell counts were measured at week 8 and week 20, and fractions of peripheral blood and bone marrow cells were assessed at week 8 using flow cytometry, but no significant differences were observed. As for AML models, there were no significant differences in survival periods after primary or secondary transplantation. Leukemic cells retrovirally induced with MLL-ENL were collected from primary recipients, and these cells were treated with ATRA. Although growth rates were not show significantly different, expression levels of CD11b after exposure to ATRA were significantly higher in Vav-Cre (+) mice (5μM: 70% vs 44%, respectively; p=0.035. 10μM: 80% vs 46%, respectively; p=0.0006) as shown in the following figure. Discussion: We established HMGA1 KO mice and assessed the function of HMGA1 both in normal and leukemic hemopoiesis. Although HMGA1 might have little effect on normal hematogenesis, it seemed that HMGA1 had inhibitory effect on ATRA-induced differentiation process of MLL-ENL leukemic cells. Though more conditions including AML types and differentiation methods should be tested and the molecular mechanisms should be scrutinized, there is a possibility that inhibition of HMGA1 is effective in combination with differentiation treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ropegiterferon Alfa-2b-Njft for Treatment Myeloproliferative Neoplasms: A Single Institution Experience

Background: While interferon alfa has been used for decades as a cytoreductive agent in myeloproliferative neoplasms (MPNs), tolerability and need for frequent parenteral administration have limited its widespread adoption. Ropeginterferon Alfa-2b-njft (ropeg) is monopegylated extended half-life interferon alpha product that received FDA approval for treatment of polycythemia vera (PV) in November 2021. A phase III clinical trial has demonstrated efficacy and tolerability of this novel agent as compared to hydroxyurea (HU) in PV patients with indication for cytoreductive therapy. Real-world experience (RWE) assessment of a newly available pharmaceutical can yield important insights regarding barriers to access, clinical feasibility, and usage in populations not yet evaluated in clinical trial. In this single institution cohort, we report the first RWE using ropeg for patients with MPNs, including essential thrombocythemia (ET), in the United States. Methods: Patients presenting to an MPN clinic at an academic medical center were evaluated for initiation of ropeg based individualized assessment, with patients engaged in shared decision-making regarding risks and benefits. Patients were screened for autoimmune, neuropsychiatric, or endocrine disorder on basis of history and limited laboratory investigation as appropriate, including thyroid stimulating hormone, triglycerides, and anti-nuclear antibody testing. The treating physician and pharmacist provided pre-treatment counseling including review of potential toxicities, administration teaching, and financial considerations. Patients on HU were started on a dose 50mcg every 2 weeks via subcutaneous injection with subsequent HU tapering; patients not on HU were started 100mcg every 2 weeks. Ropeg dosing was increased to target hematologic response. Complete blood counts and comprehensive metabolic panels were ordered every 2 weeks until hematologic response achieved, with less frequent monitoring thereafter. Results: Between December 2021 to June 2022, 13 patients with JAK2 V617F positive MPNs have been started on ropeg. Four patients were unable to start ropeg due to payor denial of coverage and ineligibility for manufacturer patient assistance programs. Patients were predominantly female (10/13, 77%) with average age of 60.9 years (range 36-73 years). Seven patients had PV (transformed from ET in 2 cases), 5 patients had ET, and 1 patient had post-ET myelofibrosis. Mean pre-treatment hematocrit, platelet count, and WBC were 42.5% (range 34.3% - 51.5%), 630 *109/L (range 127-2065*109/L), and 7.0*109/L (range 2.5 - 13*109/L) respectively. At treatment start, 7 patients were on HU (either alone or in combination with therapeutic phlebotomy (TP)), 1 was on TP alone, 1 was on ruxolitinib, 1 was on peginterferon alfa-2a, and 3 were on no cytoreductive treatment. Reasons for switching to ropeg included poor tolerance or efficacy of prior therapy (6/13, 46%), patient interest in potential disease modifying properties of interferon (6/13, 46%), and, in the case of patient on peginterferon alfa-2a, ease of administration (1/13, 7.6%). Patients received medication through insurance approval with copay assistance (11/13, 85%) or through the manufacturer patient assistance program (2/13, 15%). Average time to receipt of medication from prescription submission was 25 days. At mean follow-up time of 12.9 weeks, all patients remain on treatment. One patient required treatment interruption due to severe fatigue, two patients deferred scheduled doses due to cold-like symptoms. While efficacy data is immature, one patient with PV and 2 patients with ET obtained a hematologic response (hematocrit <45% without phlebotomy in 3 months and platelet count <450), with mean response time 6.9 weeks (range 5.7 -7.7). Two patients experienced rebound in hematocrit after HU stop and are requiring TP as bridging therapy. Conclusions: Medication access, patient counseling, and symptom and laboratory monitoring must be addressed in translating clinical trial findings to clinical practice with ongoing evaluation of drug efficacy and safety. In a single institution analysis, 13 patients with MPNs, including 5 patients with ET, were started on ropeg with good initial tolerance at mean follow-up of 12.9 weeks. Lengthy insurance approval process and payor coverage denials limit medication access.

Micheliolide Exerts Therapeutic Effect in Myeloproliferative Neoplasms through Binding to STAT3 and STAT5 Proteins and Inhibiting Their Activities

The JAK1/2 inhibitor ruxolitinib is a cornerstone of management for some subsets of BCR-ABL negative myeloproliferative neoplasms (MPN). While many patients benefit with reduced splenomegaly and symptoms, some patients respond suboptimally to ruxolitinib, and even patients with a good initial response often develop resistance to ruxolitinib after several years of therapy. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, in the JAK2V617F positive MPN entities. We firstly evaluated the effect of MCL treatment on cell growth and survival in human MPN cell lines expressing the JAK2V617F mutation. MCL alone reduced cell viability and induced apoptosis of UKE1 and SET2 cells in a dose-dependent manner, and obviously improved the effect of ruxolitinib as monotherapy. Importantly, treatment with MCL also induced significant apoptosis of UKE1 cells which were selected for resistance against ruxolitinib. We further explored the therapeutic potential of MCL in vivo using a Jak2V617F knock-in (Jak2VF) mouse model, and found that DMAMCL, an orally available derivative of MCL, significantly increased ruxolotinib efficacy in Jak2VF mice, manifested as amplified hematological and spleen responses. What's more, DMAMCL, whereas not ruxolitinib, contributed to reduce the in vivo Jak2V617F mutant allele burden reflected by the percentage of mutant hematopoietic cells in peripheral blood and bone marrow. To determine the molecular mechanisms by which MCL exerts efficacy in MPN, we firstly performed western blot (WB) anlyses to evaluate the effect of MCL on hyper-activated JAK-STAT signaling, which is the cornerstone of MPN pathogenesis. We observed a dose-dependent inhibition of STAT3 and STAT5 phosphorylation caused by MCL treatment in UKE1 and SET2 cells (Figure 1A), and as expected, combination MCL with ruxolitinib provided an enhanced inhibitory effect. By next performing the affinity pull-down experiment, we confirmed that MCL suppressed the activation of STAT3 and STAT5 mainly through directly binding to the proteins (Figure 1B). We further conducted RNA-sequencing analysis in UKE1 cells treated with MCL and/or ruxolitinib to gain insights into mechanisms involved in MCL effect. Gene set enrichment analysis (GSEA) showed that genes related to TNF-α-NF-κB signaling and inflammatory response pathway were significantly downregulated upon treatment with MCL, that were verified by WB analyses and real-time quantitative PCR. Additionally, GSEA also revealed significant upregulation of genes related to reactive oxygen species (ROS) and unfolded protein response (UPR) pathway, respectively. Both flow cytometry and fluorescent microscopy imaging confirmed the ROS accumulation by MCL treatment. We also determined the activation of PERK branch of UPR pathway caused by MCL, as assessed by increased phosphorylation of eIF2α, and upregulation of pro-apoptotic genes, including ATF4, CHOP and GADD34. Finally, by addition of antioxidant agent, the activation of UPR pathway and increase of apoptosis in UKE1 cells induced by MCL were significantly rescued, indicating that MCL exerts cytotoxic effects mainly through triggering the oxidative stress in MPN cells. In summary, our results demonstrate that MCL is an effective agent with multiple biological activities in MPN, especially inhibition of STAT3/5 activity via directly binding to the proteins, and may be a promising drug combined with ruxolitinib to benefit patients who have suboptimal response or acquire resistance to ruxolitinib. Figure 1. MCL inhibits STAT3 and STAT5 phosphorylation through binding to the proteins directly. (A) Western blot analyses are shown for phosphorylated-STAT3 (p-STAT3), STAT3, phosphorylated-STAT5 (p-STAT5) and STAT5 levels in total cell extracts from UKE1 and SET2 cells treated with MCL (5, 10, 20μM) for 3 hours. (B) The biotin-conjugated MCL (biotin-MCL) and its inactive analogue S-MCL (biotin-S-MCL) were prepared as the positive- and negative- probe respectively. UKE1 and SET2 cell lysates were incubated with 20μM biotin-MCL or biotin-S-MCL for 1 hour at room temperature, then the mixtures were separately pulled down with streptavidin-coated agarose beads, and STAT3/STAT5 were detected with WB analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Study of the Janus kinase 2 (JAK2) gene haplotype 46/1 association with driver mutations of chronic Ph-negative myeloproliferative neoplasms

Introduction. Haplotype JAK2 46/1 is associated with more frequent development of Ph-negative myeloproliferative neoplasms (MPN) and with an increased detection rate of the JAK2 V617F mutation. At the same time, the molecular mechanisms of such associations remain unclear. Previously, there were no studies of regional, age and gender aspects of the predictive value of carriage of the 46/1 JAK2 haplotype, which could assess this relationship in some observations.Aim — to analyze the degree of association between 46/1 haplotype and the V617F mutation of the JAK2 gene depending on the sex, age, and place of residence of patients examined for suspected MPN.Methods. The study included 949 DNA samples from patients with suspected MPN. Samples of 150 volunteers and blood donors were included in the control group. Haplotype 46/1 (rs10974944), V617F mutation in the JAK2 gene, mutations in the CALR gene (type 1: c.1092_1143del; L367fs*46, COSV57116546; type 2: c.1154_1155insTTGTC; K385fs*47, COSV57116551) and in the MPL gene (W515K, W515L) were determined for all samples using real-time polymerase chain reaction (PCR-RT).Results. The 46/1 JAK2 haplotype were shown to be associated with a clinically significant level (&gt; 2 %) of the allelic burden of the JAK2 V617F mutation. The odds ratio of the risk of developing a V617F positive MPN when carrying this haplotype variant did not depend on the main place of residence of the patients and was found to be most pronounced in men under 50 years of age. The odds ratio of the risk did not depend on the age of the examined women.Conclusion. The association of 46/1 haplotype with the presence of other drivers of MPN mutations in the CALR or MPL genes was also statistically significant, which confirms the hypothesis of “favorable soil” rather than “hypermutability” of the JAK2 gene.

Two myeloproliferative diseases in one patient - co-occurence of clones

Classical myeloproliferative diseases can be divided into Philadelphia chromosome-positive chronic myeloid leukemia and Philadelphia chromosome-negative myeloproliferative neoplasm. The driver mutations of the latter occur in the Janus kinase 2 or calreticulin genes. The coincidence of Philadelphia chromosome-negative and -positive myeloproliferative neoplasms in the same patient is exceptionally rare in the literature. During the long-term follow-up of our 120 patients with chronic myeloid leukemia, we investigated the clinical data of patients in whom Philadelphia chromosome-negative myeloproliferative disease was also confirmed. Philadelphia chromosome was detected by classical cytogenetic methods and/or fluorescence in situ hybridization. The amount of BCR-ABLI fusion RNA was monitored by quantitative real-time polymerase chain reaction. Mutations in the Janus kinase 2 and calreticulin genes were detected by quantitative allele-specific polymerase chain reaction and fragment analysis. The dynamics of disease development were inferred from the change in the amount of mutant clones over time and from the clinical data. We identified four cases carrying both Philadelphia chromosome and Janus kinase 2/calreticulin gene mutation. In some cases, competition between the clones, in other cases their co-occurrence in a common clone was observed. Isolated thrombocytosis at the time of diagnosis or persisting thrombocytosis during targeted therapy with good molecular response may call attention to the possibility of the co-occurrence of the two diseases. Co-occurrence of Philadelphia chromosome-positive and -negative myeloproliferative neoplasms is more frequent than the literature suggests. If the disease has an unusual appearance, the association of the two myeloproliferative dieseases may be suspected.

Clinical Analysis of Gene Mutation and Vascular Events in Patients with BCR/ABL Negative Myeloproliferative Neoplasms

To explore the relationship between clinical features, peripheral blood cell count, coagulation function, gene mutation and hemorrhagic events and thrombotic events in essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis(PMF) patients. Clinical data of 78 patients with ET, PV, and PMF who were admitted to the Second Affiliated Hospital of Chongqing Medical University between September 2019 and August 2020 were retrospectively analyzed. Information about sex, age, gene mutation, peripheral blood cell count, coagulation function, and hemorrhagic and thrombotic events was included, and the influence of these data on the occurrence of hemorrhagic and thrombotic events was estimated. Among the 78 patients with myeloproliferative neoplasms, there were 47 cases of ET, 15 cases of PV, and 16 cases of PMF.A total of 10 patients (12.82%) experienced hemorrhagic events and 27 (34.62%) experienced thrombotic events. Male,patients aged ≥ 60 years, and patients with a JAK2V617F mutation were more likely to experience thrombotic events (P<0.05). Patients with thrombotic events had higher platelet (PLT) counts and fibrinogen (FIB) levels than patients without hemorrhagic-thrombotic events (P<0.05).White blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) level, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and international normalized ratio (INR) showed no statistical difference between patients with thrombotic events and patients without hemorrhagic-thrombotic events (P>0.05). There was also no significant difference in the above-mentioned indexes between patients with hemorrhagic events and patients without hemorrhagic-thrombotic events (P>0.05). Among JAK2V617F positive myeloproliferative neoplasm patients, male patients were more likely to have thrombotic events (P<0.05), and patients with thrombotic events had higher platelet counts than those without hemorrhagic-thrombotic events (P<0.05). There was no significant difference in age, white blood cell count, red blood cell count, hemoglobin level, PT, APTT, FIB, TT or INR between patients with thrombotic events and patients without hemorrhagic-thrombotic events (P>0.05). Sex, age, JAK2V617F mutation and platelet count have a certain value for predicting thrombosis in patients with myeloproliferative neoplasms.

Study of Tumor Necrosis Factor-Alpha-Induced Protein 3 Gene Single-Nucleotide Variants in JAK2 V617F-Positive Myeloproliferative Disorders: A Case–Control Study

Background and Objectives: Myeloproliferative neoplasms (MPNs) are Philadelphia negative disorders involving polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF). Although JAK2 mutation is almost involved, other several mutations are linked to MPNs risk and prognosis. TNFAIP3 genetic mutations are related to several cancers and autoimmune diseases. Our study aimed to demonstrate the effects of rs2230926_T/G &amp; rs5029939_C/G SNVs of TNFAIP3 gene on the risk and prognosis of JAK2 V617F positive MPNs.&#x0D; Methods: Matched 2 groups in age, sex and race were enrolled in our research; 80 MPNs cases group and 130 normal healthy controls group with follow up of MPNs cases for 3 years. Taqman assay probes involved in real time polymerase chain reaction (PCR) were utilized for variants analysis.&#x0D; Results: The rs2230926 &amp; rs5029939 SNVs were in modest linkage disequilibrium (LD) in MPNs cases. The observed frequencies of G allele and its genotypes of both variants were more prevalent in MPNs patients than normal controls. The bleeding symptoms and the presence of splenomegaly were more existent in the heterozygous genotype and the combined G involved genotypes respectively. The overall survival (OS) was lower in G containing genotypes of both variants but the progression free survival (PFS) was affected only in rs5029939 SNV.&#x0D; Conclusion: Our study revealed the association of G containing genotypes of both rs2230926 &amp; rs5029939 SNVs to the increased MPNs incidence as well to poor clinical course and prognosis of JAK2 V617F positive MPNs disorders in Egyptian ethnic.