Philadelphia-negative Myeloproliferative Neoplasms Research Articles

Combination of ligand‑based and structure‑based virtual screening for the discovery of novel Janus kinase 2 inhibitors against philadelphia-negative myeloproliferative neoplasms.

The activating V617F mutation in Janus kinase 2 (JAK2) has been shown to be the major cause for classic Philadelphia-negative myeloproliferative neoplasms (MPNs). Thus, the development of pharmacologic JAK2 inhibitors is an essential move in combating MPNs. In this study, screening methods examining both ligands and their structures were developed to discover novel JAK2 inhibitors from the ChemDiv database with virtual screening identifying 886 candidate inhibitors. Next, these compounds were further filtered using ADMET, drug likeliness, and PAINS filtering, which reduced the compound number even further. This consolidated list of candidate compounds (n = 49) was then evaluated biologically at molecular level and the highest performing inhibitor with a novel scaffold was selected for further examination. This candidate inhibitor, CD4, was then subjected to molecular dynamics studies, with complex stability, root-mean-square deviation, radius of gyration, binding free energy, and binding properties all examined. The result suggested that CD4 interacts with JAK2 and that the CD4-JAK2 complex is stable. This study was able to identify a candidate inhibitor that warrants further examination and optimization and may potentially serve as a future MPN treatment.

Chromosome 9p trisomy increases stem cells clonogenic potential and fosters T-cell exhaustion in JAK2-mutant myeloproliferative neoplasms

JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells.

Transformation into acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 from JAK2-mutated essential thrombocythemia: a case report

BackgroundBlast transformation is a rare but well-recognized event in Philadelphia-negative myeloproliferative neoplasms associated with a poor prognosis. Secondary acute myeloid leukemias evolving from myeloproliferative neoplasms are characterized by a unique set of cytogenetic and molecular features distinct from de novo disease. t(8;21) (q22;q22.1); RUNX1::RUNX1T1, one of the most frequent cytogenetic abnormalities in de novo acute myeloid leukemia, is rarely observed in post-myeloproliferative neoplasm acute myeloid leukemia. Here we report a case of secondary acute myeloid leukemia with t(8;21) evolving from JAK2-mutated essential thrombocythemia.Case presentationThe patient was a 74-year-old Japanese woman who was referred because of thrombocytosis (platelets 1046 × 109/L). Bone marrow was hypercellular with increase of megakaryocytes. Chromosomal analysis presented normal karyotype and genetic test revealed JAK2 V617F mutation. She was diagnosed with essential thrombocythemia. Thrombocytosis had been well controlled by oral administration of hydroxyurea; 2 years after the initial diagnosis with ET, she presented with leukocytosis (white blood cells 14.0 × 109/L with 82% of blasts), anemia (hemoglobin 91 g/L), and thrombocytopenia (platelets 24 × 109/L). Bone marrow was hypercellular and filled with 80% of myeloperoxidase-positive blasts bearing Auer rods. Chromosomal analysis revealed t(8;21) (q22;q22.1) and flow cytometry presented positivity of CD 13, 19, 34, and 56. Molecular analysis showed the coexistence of RUNX1::RUNX1T1 chimeric transcript and heterozygous JAK2 V617F mutation in leukemic blasts. She was diagnosed with secondary acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 evolving from essential thrombocythemia. She was treated with combination chemotherapy with venetoclax and azacytidine. After the first cycle of the therapy, blasts disappeared from peripheral blood and decreased to 1.4% in bone marrow. After the chemotherapy, RUNX1::RUNX1T1 chimeric transcript disappeared, whereas mutation of JAK2 V617F was still present in peripheral leukocytes.ConclusionsTo our best knowledge, the present case is the first one with JAK2 mutation preceding the acquisition of t(8;21). Our result suggests that t(8;21); RUNX1::RUNX1T1 can be generated as a late event in the progression of JAK2-mutated myeloproliferative neoplasms. The case presented typical morphological and immunophenotypic features associated with t(8;21) acute myeloid leukemia.

High-resolution melting assay for rapid, simultaneous detection of JAK2, MPL and CALR variants

AimsIdentification of recurrent genetic alterations in JAK2, MPL and CALR remains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing,...

Mechanobiology and Primary Cilium in the Pathophysiology of Bone Marrow Myeloproliferative Diseases.

Philadelphia-Negative Myeloproliferative neoplasms (MPNs) are a diverse group of blood cancers leading to excessive production of mature blood cells. These chronic diseases, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), can significantly impact patient quality of life and are still incurable in the vast majority of the cases. This review examines the mechanobiology within a bone marrow niche, emphasizing the role of mechanical cues and the primary cilium in the pathophysiology of MPNs. It discusses the influence of extracellular matrix components, cell-cell and cell-matrix interactions, and mechanosensitive structures on hematopoietic stem cell (HSC) behavior and disease progression. Additionally, the potential implications of the primary cilium as a chemo- and mechanosensory organelle in bone marrow cells are explored, highlighting its involvement in signaling pathways crucial for hematopoietic regulation. This review proposes future research directions to better understand the dysregulated bone marrow niche in MPNs and to identify novel therapeutic targets.

Myeloproliferative neoplasms: young patients, current data and future considerations.

The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders predominantly occurring in elderly, whereas in children and young adults are quite infrequent. Therefore, less is known about clinical presentation, genetic abnormalities, prognosis and best management strategies for this groups of patients. Currently, more cases of younger MPN patients are diagnosed. Nevertheless, diagnosis of MPNs, especially in childhood, may be difficult due to lower incidence of JAK2V617F and CALR mutations and differences in peripheral blood counts between adults and children. Challenges for younger MPN patients are longer life expectances, specific psychosocial need, fertility and pregnancy need and a long term therapy side effect (including second cancers). The most severe MPNs complication is transformation to secondary myelofibrosis (MF) or acute myeloid leukemia (AML). Optimal management of young MPNs remains a challenge as the classical risk scores fail in young MPNs. Moreover, the main objective of young MPNs therapy should be the disease outcome modification. Therefore, international collaborative work between pediatricians and "adult hematologists" is required to measure outcomes and generate protocol of management of young MPNs.

Evaluation of Philadelphia-negative myeloproliferative neoplasms in Erbil Iraq; analysis of JAK 2, CALR, and MPL mutations

Introduction: The prevalence of Janus kinase 2 V617F, Jak 2-Exon12, calreticulin (CALR) gene mutations and myeloproliferative leukemia mutations in myeloproliferative neoplasms (MPNs) without BCR-ABL1 gene fusion rearrangement in Iraq remains unknown due to inadequate data. Objectives: The aim of this study was to ascertain the prevalence of these mutations and assess the clinical characteristics of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) in Erbil, Iraq. Patients and Methods: In this cross-sectional study, 80 patients were evaluated for the presence of JAK 2 V617, CALR, and MPL mutations. Specific primers were utilized to amplify MPNs-associated mutations (JAK 2 V617F, Jak 2 -Exon12, CALR, and MPL) using DNA extracted from whole blood. Results: The presence of the JAK 2 V617F mutation was identified in 96% of patients diagnosed with PV and 35% of patients diagnosed with ET. Additionally, JAK 2 exon 12 was detected in 4% of PV patients. CALR mutation was identified in 65% of ET patients. No detection of MPL mutation was observed. A higher prevalence of the CALR mutation was observed in younger age groups, whereas the JAK 2 V617F mutation was predominantly found in older age groups. Conclusion: The incorporation of genetic tests for JAK 2 V617F, JAK 2 exon 12, MPL exon 10, and CALR exon 9 significantly improves the diagnostic effectiveness for BCR ABL1-gene fusion negative MPNs.

Cardiovascular Risk in Philadelphia-Negative Myeloproliferative Neoplasms: Mechanisms and Implications-A Narrative Review.

Myeloproliferative neoplasms (MPNs), encompassing disorders like polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by clonal hematopoiesis without the Philadelphia chromosome. The JAK2 V617F mutation is prevalent in PV, ET, and PMF, while mutations in MPL and CALR also play significant roles. These conditions predispose patients to thrombotic events, with PMF exhibiting the lowest survival among MPNs. Chronic inflammation, driven by cytokine release from aberrant leukocytes and platelets, amplifies cardiovascular risk through various mechanisms, including atherosclerosis and vascular remodeling. Additionally, MPN-related complications like pulmonary hypertension and cardiac fibrosis contribute to cardiovascular morbidity and mortality. This review consolidates recent research on MPNs' cardiovascular implications, emphasizing thrombotic risk, chronic inflammation, and vascular stiffness. Understanding these associations is crucial for developing targeted therapies and improving outcomes in MPN patients.

Trends in the thrombotic complications of myeloproliferative neoplasms: A nationwide analysis.

6581 Background: Thrombotic complications are a well known cause of morbidity and mortality in Philadelphia-negative Myeloproliferative Neoplasms[Polycythemia Vera, Essential thrombocythemia, Primary Myelofibrosis]. Current study evaluated thrombotic events incidence and outcome trends in Myeloproliferative Neoplasms[MPN] from 2012 to 2020. Methods: This is a longitudinal study of thrombotic events in MPN using the Nationwide Inpatient Sample. All patients with a diagnosis of MPN admitted with any thrombotic event including arterial or venous thrombosis were included in the study. Arterial thrombotic events included Myocardial infarction[MI], Ischemic stroke[IS], Transient Ischemic Attack[TIA] while venous thrombotic events included Deep Vein Thrombosis[DVT], Pulmonary Embolism[PE], Budd-Chiari Syndrome[BCS], Portal Vein Thrombosis[PVT]. Thrombosis related inpatient mortality rate, incidence, length of hospital stay, and total hospital charges were calculated. Results: Mortality rate from any thrombotic event overall[arterial plus venous] increased from 2.3% to 2.6% from 2012 to 2020. There was an increase in mortality rates from 2.8% to 3.8% among arterial thrombosis while there was a decrease in venous thrombosis related mortality from 1.7 to 0.4%. Specifically MI and IS related mortality increased from 3.7% to 4.3% and 3% to 4% respectively over the same period. The incidence of any thrombotic event declined from 2 to 1.3 cases/100,000 persons from 2012 to 2020. A similar decline in incidence of arterial, venous events, MI, IS were observed. The mean length of stay decreased from 5.8 days in 2012 to 5.2 days in 2020 among MPN patients with venous thrombosis which was statistically significant. Similar decline was noted in MI patients as well. Though the mean length of stay increased from 7.9 to 9.5 days among MPN patients with IS, this increase was not statistically significant. The mean hospitalization charges for thrombotic events increased from $53,421 in 2012 to $101,598 in 2020 after adjusting for inflation. Similar trends were noted in all types of thrombotic admissions which were all statistically significant. Upon age based stratification of MPN, mortality for those less than 60 years reduced from 2% to 0.3%, while it increased from 4.1 to 5.6% in those more than 60 years from 2012 to 2020. Conclusions: Inpatient mortality from thrombotic events in MPN has shown an increasing trend from 2012 to 2020 though there was a decline in overall incidence of thrombosis during the same period. Despite the decreasing trend in length of stay, the total economic burden of thrombotic complications in MPN is increasing in the US. Specifically the effects were more pronounced in those greater than 60 years. This points to the need for revisiting guidelines regarding thromboprophylaxis in MPN.

Renal Affection in Philadelphia Negative Myeloproliferative Neoplasms

Renal Affection in Philadelphia Negative Myeloproliferative Neoplasms

The characteristics of CALR mutations in myeloproliferative neoplasms: a clinical experience from a tertiary care center in Qatar and a literature review

ABSTRACT Background Myeloproliferative neoplasms (MPNs) are hematological disorders characterized by abnormal production of myeloid cells due to genetic mutations. Since 2013, researchers have identified somatic mutations in the Calreticulin (CALR) gene, primarily insertions or deletions, in two Philadelphia chromosome-negative MPNs; essential thrombocytosis (ET) and primary myelofibrosis (PMF), and occasionally in chronic myelomonocytic leukemia (CMML). This study aims to identify the various types of CALR mutations and their impact on CALR-positive MPN patients’ clinical manifestations and outcomes. Methods A single-center retrospective study was conducted. The data was collected from pre-existing records. The study was carried out on Philadelphia-negative MPN patients who were being followed up on at the NCCCR (National Center for Cancer Care and Research) to assess the clinical manifestation and outcome of disease treatment. All patients included, were followed in our center between January 1, 2008, and November 20, 2021. Results A total of 50 patients with CALR-positive MPN were reviewed with a median follow-up of three years (1–11). This cohort included 31 (62%) patients with ET, 10 (20%) patients with PMF, and 9 (18%) patients with prefibrotic myelofibrosis (pre-MF). The study involved 38 (76%) male and 12 (24%) female patients. There were 16 (32%) patients diagnosed before the age of 40, 24 (48%) patients diagnosed between the ages of 40 and 60; and 10 (20%) patients diagnosed after the age of 60. Molecular analysis showed 24 (48%) patients with CALR type 1, 21 (42%) patients with CALR type 2, and 5 (10%) patients with none Type 1, none Type 2 CALR mutations. Two patients have double mutations; 1(2%) with none Type 1, none Type 2 CALR and JAK2 mutations, and 1(2%) with CALR type 1 and MPL mutations. The thrombotic events were 3 (6%) venous thromboembolisms, 3 (6%) abdominal veins thromboses, 2 (4%) strokes, and 4 (8%) ischemic cardiac events. Only 4 (8%) patients progressed to Myelofibrosis and were carrying CALR 1 mutations, and 1 (2%) patient progressed to AML with CALR 2 mutation. Conclusion The data shows a significant rise in CALR-positive MPN diagnoses in younger people, emphasizing the need for a better assessment tool to improve disease management and reduce complications.

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

Carotid Plaque Burden and Neutrophil-Lymphocyte Ratio in Patients with Philadelphia Negative Myeloproliferative Neoplasms and Their Relation to JAK2 V617F Mutation

Carotid Plaque Burden and Neutrophil-Lymphocyte Ratio in Patients with Philadelphia Negative Myeloproliferative Neoplasms and Their Relation to JAK2 V617F Mutation

Retinal Vessel Analysis and Microvascular Abnormalities in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms.

Background: Philadelphia-negative chronic myeloproliferative neoplasms are a group of clonal hematopoietic disorders including polycythemia vera, essential thrombocythemia, and primary myelofi-brosis. These neoplasms are characterized by an increased risk of thrombotic complications. Several studies have highlighted that the study of vessels of the retina offers the opportunity to visualize, in vivo, the damage to microcirculation that is common in various systemic pathologies. Methods: in our study, forty patients underwent an ophthalmological examination, using non-invasive imaging tech-niques, for analyses of their retinal vascularization. The objective was to correlate the findings ob-tained from this study of the retina with different markers of thrombotic risk, to demonstrate the usefulness of studying retinal vessels as a possible new prognostic biomarker of thrombotic risk in patients affected by Philadelphia-negative chronic myeloproliferative neoplasms. Results: retinal imaging demonstrated changes in the microcirculation, with a reduced vascular density of the deep and superficial capillary plexuses with respect to a normal group, and a correlation between retinal changes and blood parameters. Conclusions: additional research will allow us to determine whether retinal changes in individuals with chronic myeloproliferative neoplasms could be predictive of the development of thrombotic events in these subjects.

Essential thrombocythaemia.

Essential thrombocythemia (ET) is a chronic Philadelphia-negative myeloproliferative neoplasm that has its main involvement in the megakaryopoietic lineage, generating sustained thrombocytosis in peripheral blood and an increase in the number of mature megakaryocytes in the bone marrow. In addition to marked thrombocytosis, it is characterized by increased thrombotic or hemorrhagic risk and the presence of constitutional symptoms. Patients with ET have a low but known risk of disease progression to myelofibrosis and/or acute leukemia. The diagnosis is made based on the 2016 WHO criteria. At present, available treatments for patients with ET are mainly aimed at minimizing the risk of thrombosis and/or bleeding.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical “MF-like” morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

Polycythemia vera and management of the thrombotic risk: an update

Polycythemia vera (PV) is a chronic Philadelphia-negative myeloproliferative neoplasm caused by JAK2 mutation and characterized predominantly by the overproduction of red blood cells. The current treatment strategies of PV are based on periodic phlebotomies aimed at preventing thrombotic events associated with increased hematocrit levels. Additional therapies to mitigate the thrombotic burden, which represents the most important predictor of reduced survival in PV patients, include cytoreductive therapies, low-dose aspirin, and systemic anticoagulation. This concise review summarizes the current knowledge on the management of the thrombotic risk in PV patients.

Combined Myeloid and Lymphoid Malignancies in 13 Elderly Patients

Disclosures Myeloid malignancies such as myelodysplasia (MDS) often evolve from age related clonal hematopoiesis (CH) with progression of variant allele frequencies. The Philadelphia negative myeloproliferative neoplasms (MPN) polythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are associated with mutations in JAK2, CALR, or MPL. Multiple myeloma (MM) and monoclonal gammopathies (MGUS) are felt to be age related, and the malignant cell is of lymphoid origin. Burkitt's Lymphoma is associated with EBV exposure and dysregulation of c-myc. However the etiology of most lymphoproliferative disorders (LPD) is not defined, but immune dysregulation may be causative. The combination of myeloid and lymphoid malignancies are not recognized to be a common occurrence; however within a three year period 13 patients with combined myeloid and lymphoid malignancies were detected in a single community practice. DATA Five patients had combined myeloid and lymphoid malignancies diagnosed concurrently. Three had a lymphoid disorder and then developed a myeloid disorder. Five had a myeloid disorder followed by a lymphoid disorder. Three patients had multiple myeloma (MM) and two had MGUS. Initially all patients responded to standard therapies, One patient at 87 years died from end stage renal disease likely related to her myeloma. Another patient with CLL and PV died at 92 years from an epithelial malignancy. Concurrent diagnoses Age at diagnosis (years) CLL ET 76MGUS MDS 77MM MDS 78MM MDS (RSBA) 85 5) DLBCL MF. 60 LPD/MM followed by a Age at initial diagnosis (years) and time to second diagnosis myeloid disorder. 1) MZL MF 58 15 months 2) MM ET 83 7 months 3) CLL PV 86 4 years Myeloid disorder followed by a LPD/MGUS MDS DLBCL 76 3 monthsMDS MGUS 78 30 monthsTP CLL 15 60 yearsET MyF 77 65 monthsET CLL 60 15 years (CLL chronic lymphocytic leukemia, DLBCL diffuse large B cell lymphoma, MF myelofibrosis, MyF mycosis fungoides, MZL mantle zone lymphoma, RSBA ringed sideroblastic anemia, TP inherited thrombocytopenia) JAK2 mutations were found in three of the four ET patients and in the one PV patient. One MF patient and one ET patient were negative for the JAK2 and CALR mutations. The mutation status of the other MF patient was not known. At diagnosis the patient with inherited thrombocytopenia was suspected to have a myeloid disorder. Combined myeloid and lymphoid malignancies is not well defined and is a seemingly uncommon event. Yet an Italian study demonstrated that MPN patients with JAK2 mutations have a 2.8-3.4 fold increased risk of developing a LPD compared with an age matched population (1). The linkage is unclear. Immune senescence may be important for the development of LPD observed in the elderly. Also long term latency of many years between the JAK2V617F mutation acquisition and MPN presentation has been documented (2). The occurrence of both myeloid disorders and LPD is not well recognized. Future studies may provide insights into the mechanisms underlying these disorders. Increasing genetic complexity and immune senescence associated with aging likely plays a major role. The limitations of this study are its small size, single community investigator, retrospective clinical nature, and the lack of gene sequencing and other molecular studies in most if not all of the patients. This observation was noted over three years in a single community practice which strongly implies that combined occurrence may be more common than recognized and suggests that further investigation is warranted. Boxer none, Denes none

Real World, Single-Centre Experience of Pregnancy in Patients with Philadelphia Negative Myeloproliferative Neoplasms

Pregnancy in myeloproliferative neoplasms (MPN) is commonly associated with maternal and foetal complications due to increased risks of thromboembolic and haemorrhagic events and placental dysfunction. The incidence of UK pregnancies in MPN is estimated as 3·2/100 000 maternities per year (Alimam, S., et al 2016, Br J Haematol 2016; 175:31-36; Robinson et al, Br J Haematol 2020;189:625-34). Here we present pregnancy outcomes of MPN patients attending University College London Hospital (UCLH), a large UK tertiary referral centre, between 2015 and 2023. Twenty-three patients were identified with a median age at MPN diagnosis of 33 years (range (r), 13 - 46). The majority of patients (21/23, 91.3%) had a diagnosis of ET or triple negative (TN) thrombocytosis and 8.7% (2/23) a diagnosis of polycythaemia vera (PV). In 61.4% of pregnancies, diagnosis was known at conception. Regarding mutational status, JAK2V617F was present in 10/23 (43.5%) patients, CALR in 5/23 (21.7%) and MPL in 1/23 (4.3%). The remaining 7/23 (30.4%) patients were TN. Median VAF for JAK2V617F and CALR were 15.8% (r, 21.1 - 33%) and 25.9% (r, 5.7 - 46%), respectively. The cohort collectively experienced 57 pregnancies, 38 of which were managed at UCLH. Focusing on pregnancies managed at UCLH, median age at pregnancy was 34.5 years (r, 22 - 47). Conception was spontaneous in 31/38 (81.6%) pregnancies. Six (15.8%) patients required assisted conception and had a higher median age of 38.5 years (r, 35 - 47). Where gestation at delivery was known, 92.6% (25/31) of live births delivered at term (>37 weeks). Caesarean rate was significant at 41.9% (13/31), of which 61.5% were emergency sections. The live birth rate of the 57 overall pregnancies was 76.9% and 88.6% in the 38 managed at UCLH. At UCLH, we adopt an individualised approach to managing pregnant women with MPNs, working alongside obstetricians and obstetric haematology colleagues. Consequently, as shown in Table 1, there were bespoke regimens for individual patients. Low dose aspirin was prescribed throughout pregnancy in the majority (78.9%, 30/38) of pregnancies. In 18.4% (7/38), cytoreduction was established prior to pregnancy. In 42.9% (3/7), pegylated interferon (PegIFN) was commenced to optimise counts prior to pregnancy and in 42.9% (3/7) as long term count control. One patient was cytoreduced with hydroxycarbamide prior to pregnancy and subsequently switched to PegIFN. Low molecular weight heparin (LMWH) was administered at some point during pregnancy in 60.9% (14/23) of patients and 57.9% (22/38) of pregnancies. 65.8% (25/38) underwent monitoring with uterine artery (UA) dopplers. Median gestation for UA assessment was 20 weeks (r, 19 - 28). UA dopplers were reported as abnormal in 16% (4/25). In two patients, this was due to a raised combined pulsatility index, UA notching in one and was unspecified in one patient. Nine (39.1%) patients experienced foetal loss, with 12 miscarriages recorded across the total 57 pregnancies and 4 miscarriages in the 38 pregnancies managed at UCLH. Median maternal age was 31.5 (r, 24 - 42) and median gestation was 8 weeks (r, 5 - 24). Six (66.7%) patients experiencing foetal loss had a diagnosis of ET, 2 (22.2%) PV and 1 (11.1%) TN thrombocytosis. Three miscarriages occurred in one patient, who demonstrated a concurrent heterozygous prothrombin G20210A mutation in addition to ET. Eight (34.8%, 8/23) patients collectively experienced 11 haemorrhagic events. The majority (72.7%) were post-partum haemorrhage (PPH), 37.5% of which were classified as major or severe. Three (27.3%) bleeding events occurred whilst on aspirin monotherapy, 3 (27.3%) on combined LMWH and aspirin, 1 (9.1%) on LWMH alone and 1 (9.1%) on LMWH and PIFN. With regards to thrombosis, one patient with undiagnosed ET had a cerebral venous sinus thrombosis at 11-weeks gestation. A total of 2/23 (8.7%) patients' pregnancies resulted in hypertensive complications and pre-eclampsia, both resulting in delivery with caesarean section at 34 and 35 weeks. Although the live birth rate is encouraging, our data highlights that patients with MPN remain at increased risk of miscarriage, early delivery and haemorrhagic events. There remain inconsistencies in the management of pregnancy in MPN and, although we adopt an individualised patient-centred approach, there is an unmet need for further study and development of evidence based treatment algorithms.

Lactate Transporters Modulate Stromal Cell Remodeling in Myeloproliferative Neoplasms (MPN)

Introduction. Philadelphia-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The BM morphology includes cellularity, erythropoiesis, and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early-stage myelofibrosis that present with thrombocytosis and clinically mimic ET. Since alteration of hematopoiesis is constantly associated with profound modifications of the bone marrow (BM), we hypothesize that CD34+ cells from PMF can actively produce lactate and thus rewiring metabolism of immune cells and mesenchymal stromal cells (MSCs). In turn, this could favor immune evasion and remodeling of BM niche. Methods.Cell proliferation was assessed in vitro on primary MSCs, HS-5 cell line, and PBMC cells. Protein expression was performed by Western blot and immunofluorescence assay. The NCBI GEO database was used to select transcriptome datasets and to analyze gene expression in PMF patients. Cytokine detections were performed by Multiplex immunobead assay technology. An adult TPO-mimetic-induced PMF Zebrafish model was used for in vivo evaluation. An immunohistology assay was performed in MPN BM biopsies. Results.CD34+ cells from PMF patients significantly upregulated the lactate monocarboxylate transporters (MCT-1 and -4) compared to healthy CD34+ cells. Consistently, lactate concentration was higher in PMF sera compared to healthy controls. Since PMF patients showed an increased percentage of circulating immunosuppressive cells such as G- and M-MDSC and Treg, we incubated healthy peripheral blood mononucleated cells (PBMNCs) with PMF sera in the presence or absence of MCT1inhibitor (AZD3965) to evaluate the role of lactate in the expansion of these immunological subsets. We found an expansion of both Treg and M-MDSCs, which was reverted after blocking of circulating lactate by AZD3965. In order to study the effect of lactate in BM remodeling, we treated in vitro healthy MSCs with lactate (20 mM) for 24h. Interestingly, lactate induced a modification of extracellular matrix organization, as demonstrated by increased reticulin and collagen deposition and metalloproteases (MMP9, MMP2). Moreover, lactate increased calcium deposit and soluble osteogenic molecular signals (i.e. osteoprotegerin, osteonectin). These results also were obtained after incubation with PMF sera and were reverted after AZD3965 treatment. Since it has been demonstrated the role of cancer-associated fibroblast (CAF) in cancer development through ECM remodeling, we next evaluated the expression of CAF markers in MSCs after lactate exposure. α-SMA, FAP1, and TGFβ were found upregulated and this effect was reverted by AZD3965. Finally, we further confirmed these results in a Zebrafish animal model of PMF. Interestingly, in Zebrafish model we observed a reduction of MCT4 expression that was confirmed in the PBMNCs of the PMF patients. Conclusions. Inhibition of lactate metabolism may represent a strategy to inhibit cancer cells and contribute to restoring the anti-cancer immune response. Therefore, lactate metabolism may represent a promising target to counteract inflammation, osteosclerosis, and fibrosis in PMF patients.