MPL Mutations Research Articles

Myeloproliferative Neoplasms: Challenging Dogma.

Myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis, and primary myelofibrosis are a unique group of clonal hematopoietic stem cell neoplasms that share somatic, gain-in-function driver mutations in JAK2, CALR, and MPL. As a consequence, these disorders exhibit similar phenotypic features, the most common of which are the ceaseless production of normal erythrocytes, myeloid cells, platelets alone or in combination, extramedullary hematopoiesis, myelofibrosis, and a potential for leukemic transformation. In the case of polycythemia vera and essential thrombocytosis, however, prolonged survival is possible. With an incidence value in the range of 0.5-2.0/100,000, myeloproliferative neoplasms are rare disorders, but they are not new disorders, and after a century of scrutiny, their clinical features and natural histories are well-defined, though their individual management continues to be controversial. With respect to polycythemia vera, there has been a long-standing dispute between those who believe that the suppression of red blood cell production by chemotherapy is superior to phlebotomy to prevent thrombosis, and those who do not. With respect to essential thrombocytosis, there is a similar dispute about the role of platelets in veinous thrombosis, and the role of chemotherapy in preventing thrombosis by suppressing platelet production. Linked to these disputes is another: whether therapy with hydroxyurea promotes acute leukemia in disorders with a substantial possibility of longevity. The 21st century revealed new insights into myeloproliferative neoplasms with the discovery of their three somatic, gain-of-function driver mutations. Almost immediately, this triggered changes in the diagnostic criteria for myeloproliferative neoplasms and their therapy. Most of these changes, however, conflicted with prior well-validated, phenotypically driven diagnostic criteria and the management of these disorders. The aim of this review is to examine these conflicts and demonstrate how genomic discoveries in myeloproliferative neoplasms can be used to effectively complement the known phenotypic features of these disorders for their diagnosis and management.

The First Case of Cerebral Infarction Associated with Essential Thrombocythemia and MPL Mutation in South Korea

The First Case of Cerebral Infarction Associated with Essential Thrombocythemia and MPL Mutation in South Korea

Molecular Profile of BCR-ABL1 Negative Myeloproliferative Neoplasm in a Moroccan Population.

Myeloproliferative neoplasms (MPN) are associated with clonal hematopoiesis, genomic instability, hemostasis dysregulation, and immune response. Classic BCR-ABL1 negative myeloproliferative neoplasms (BCR-ABL1 negative MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are frequently associated with somatic abnormalities in JAK2, CALR, and MPL. Mutant clones induce an inflammatory immune response leading to immuno-thrombosis. The aim of this research was to investigate JAK2/STAT5 mutations in 49 Moroccan patients and to study their correlation with demographic and hematological parameters. Genomic DNA was isolated from peripheral blood samples, and JAK2V617F, JAK2 exon 12, CALR exon 9, and MPL exon 10 mutations were tested by qPCR, AS-PCR, and Sanger sequencing. Statistical analysis was performed using SPSS version 22. Our results showed that 33 (67.35%) of our cohort was mutated for one of the investigated genes. JAK2V617F was detected in 25 cases, as well as two previously described CALR mutations: c.1092_1143del52 and c.1154_1155insTTGTC. Mutations in JAK2 exon 12 (c.1622G>A/C and c.1641+6T>C) were observed and in PV and ET. MPL substitution c.1544G>T was detected in two cases. Mutations in JAK2, CALR, and MPL were mutually exclusive and benign single nucleotide variations/polymorphisms (SNV/SNP) have been identified in CALR and JAK2. The JAK2-PV subgroup was associated with higher red mass cell, hemoglobin, and hematocrit levels. Higher platelet counts correlated with the JAK2-ET subgroup, while higher leukocyte and neutrophil counts were associated with the JAK2-PMF subgroup. Complete blood count revealed hyperleukocytosis accompanied by hyperplateletosis and a lower level of hemoglobin in CALR-ET mutated patients, compared to JAK2-ET mutated or triple negative-ET cases. In conclusion, our study provides valuable insights into the prevalence and characteristics of JAK2, CALR, and MPL mutations in BCR-ABL1 negative MPNs in the Moroccan population, highlighting the importance of genetic characterization to optimize the clinical management of these diseases.

Analysis of Molecular Testing for Suspected Myeloproliferative Neoplasm at a Hybrid Community-Academic Health System

Testing for somatic mutations in JAK2, MPL, and CALR genes is a crucial element in the diagnosis of myeloproliferative neoplasms (MPN). This may have inadvertently led to increased requests for testing to “rule out MPN,” including clinical situations with low pre-test probability. This paper examines JAK2, MPL, and CALR testing by next generation sequencing (NGS) with the goal of formulating practical guidelines to make test utilization more efficient and effective. NGS results from 1482 patients tested between 2015 and March 2022 were retrieved, along with corresponding bone marrow biopsies and CBC results performed within 90 days prior to NGS and 245 (16.5%) cases were positive for pathogenic variants in JAK2, MPL, or CALR genes.The findings showed an increase in the proportion of positive cases with patient age, and a statistically significant difference in RBC counts and platelet counts among patients with positive versus negative results. Utilizing these factors, simple algorithms were constructed to predict positive results with a maximum sensitivity of 91%, while potentially eliminating 28% of negative tests. However, these models still failed to identify ∼9% of patients with MPNs. Among these “missed” patients, many had either primary myelofibrosis or MDS/MPN. Considering a simple triage model to help guide MPN testing could represent a more cost effective approach, particularly if “missed” patients could be further reduced.

Possibilities for diagnosing essential thrombocythemia at the stage of primary health care

This clinical case presents a 39-year-old woman diagnosed with essential thrombocythemia, accompanied by a review of the epidemiology, clinical and diagnostic criteria, and patient management strategies. The case also includes a discussion of potential complications and prognosis associated with the condition. The importance of thorough outpatient monitoring is emphasized, which should include regular assessments of clinical symptoms, changes in blood count parameters, bone marrow trephine biopsy, and molecular genetic testing for MPL and CALR gene mutations. The report highlights gaps in the current medical examination process, suggesting that addressing these deficiencies could improve early detection of essential thrombocythemia and enable timely pathogenetic therapy with antiplatelet agents to prevent complications.

Effects of Down-Regulation of PAK1 on Differentiation and Apoptosis of MPN Cells with MPLW515L Gene Mutation and Survival of 6133/MPL Mice

To investigate the effects of down-regulation of p21 activated kinase 1 (PAK1) on the proliferation, differentiation, and apoptosis of myeloproliferative neoplasm (MPN) cells (6133/MPL) with thrombopoietin receptor MPL mutation at codon 515 (MPLW515L) and survival of 6133/MPL mice. Interference with the protein level of PAK1 in 6133/MPL cells was assessed using lentivirus-mediated shRNA transfection technology. CCK-8 assay was used to detect the effect of down-regulation of PAK1 on the proliferation viability of 6133/MPL cells, and colony-forming ability was measured by cell counting. Flow cytometry was used to detect the PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in 6133/MPL cells. The expression of cyclin D1, cyclin D3 and apoptosis-related protein Bax was detected by Western blot. The infiltration of tumor cells in spleen and bone marrow of 6133/MPL mice were detected by HE staining. Down-regulation of PAK1 inhibited the proliferation and reduced the ability of cell colony formation of 6133/MPL cells. After knocking down PAK1, the content of polyploid DNA in 6133/MPL cells increased from 31.8 to 57.5% and 48.0%, and the proportion of apoptosis increased approximately to 10.8%. Down-regulation of PAK1 led to a reduction of infiltration of tumor cells in liver and bone marrow of 6133/MPL mice, thereby prolonging survival time. Down-regulation of PAK1 can significantly inhibit the growth of 6133/MPL cells, promote the formation of polyploid DNA, induce 6133/MPL cell apoptosis, and prolong the survival time of 6133/MPL mice.

Different Shades of Desmoid-Type Fibromatosis (DTF): Detection of Noval Mutations in the Clinicopathologic Analysis of 32 Cases.

Desmoid-type fibromatosis (DTF) is a locally aggressive myofibroblastic/fibroblastic neoplasm with a high risk of local recurrence. It has a variety of histologic features that might confuse diagnosis, especially when detected during core needle biopsy. The Wnt/β-catenin pathway is strongly linked to the pathogenesis of DT fibromatosis. This study examined 33 desmoid-type fibromatoses (DTFs) from 32 patients, analyzing its clinical characteristics, histologic patterns, occurrence rates, relationship with clinical outcomes, immunohistochemical and molecular findings. The DTFs exhibit a range of 1 to 7 histologic patterns per tumor, including conventional, hypercellular, myxoid, hyalinized/hypocellular, staghorn/hemangiopericytomatous blood vessels pattern, nodular fasciitis-like, and keloid-like morphology. No substantial association was found between the existence of different histologic patterns and the clinical outcome. All thirty-three (100%) samples of DTF had a variable percentage of cells that were nuclear positive for β-catenin. An NGS analysis detected novel non-CTNNB1 mutations in two DTFs, including BCL10, MPL, and RBM10 gene mutations. This study reveals a diverse morphology of DTFs that could result in misdiagnosis. Therefore, surgical pathologists must comprehend this thoroughly. Also, the importance of the newly identified non-CTNNB1 gene mutations is still unclear. More research and analyses are needed to completely grasp the clinical implications of these mutations.

Decoding Endothelial MPL and JAK2V617F Mutation: Insight Into Cardiovascular Dysfunction in Myeloproliferative Neoplasms.

Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential face a significantly elevated risk of cardiovascular diseases. Endothelial cells carrying the JAK2V617F mutation have been detected in many patients with MPN. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in patients with MPN. We investigated the impact of endothelial JAK2V617F mutation on cardiovascular disease development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. Our investigations revealed that JAK2V617F mutant endothelial cells promote cardiovascular diseases under stress, which is associated with endothelial-to-mesenchymal transition and endothelial dysfunction. Importantly, we discovered that inhibiting the endothelial TPO (thrombopoietin) receptor MPL (myeloproliferative leukemia virus oncogene) suppressed JAK2V617F-induced endothelial-to-mesenchymal transition and prevented cardiovascular dysfunction caused by mutant endothelial cells. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function. JAK2V617F mutant endothelial cells play a critical role in the development of cardiovascular diseases in JAK2V617F-positive MPNs, and endothelial MPL could be a promising therapeutic target for preventing or ameliorating cardiovascular complications in these patients.

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.

Structural basis of MPL activation by thrombopoietin

AbstractMyeloproliferative leukemia protein (MPL), also known as thrombopoietin (TPO) receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and differentiation toward the megakaryocyte lineage and is critical for normal platelet production. Mutations in MPL, TPO, or Janus kinase 2 (JAK2) have been implicated in multiple diseases from congenital thrombocytopenias to myeloproliferative neoplasms. The ligand for MPL, TPO, stimulates platelet production by inducing MPL dimerization and results in an active conformation that allows downstream JAK2/STAT5 signaling. Despite the biological importance of this pathway, the molecular signaling mechanism remained unclear. Here, we present a 3.39-Å cryo-electron microscopy structure of the ectodomain of mouse MPL bound to TPO. The structure revealed both low and high affinity sites between MPL and TPO that contain several pathologic mutations. To better understand TPO-driven MPL signaling, we expanded upon this structure by molecular dynamic (MD) simulations to model the full-length human MPL/TPO complex, and showed that MPL D4-D4 domain interactions are functionally relevant in activity assays. To build on our understanding of downstream activation, we added JAK2 to the MPL/TPO complex by MD simulations. This ternary complex illustrates JAK2 dimerization through the pseudokinase domain, illustrates residues important for MPL interactions, and reveals the constitutive activation mechanism of patient mutant V617F. The model also suggests the mechanism of JAK2 tyrosine kinase domain transphosphorylation. Overall, our studies illuminate TPO/MPL/JAK2 signaling mechanisms and provide additional insight into the nature of receptor signaling, which will further benefit human health.

NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 "triple-negative" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

Outcomes of blast-phase MPN and JAK2, MPL, and CALRmutated de novo AML: A propensity score-adjusted cohort study.

6526 Background: Progression to blast phase (BP) occurs in about 10% of patients with BCR-ABL-negative myeloproliferative neoplasms (MPN) and is associated with an extremely poor prognosis with no defined standard of care. The VIALE-A trial, which showed that venetoclax (VEN) improves overall survival (OS) in patients with acute myeloid leukemia (AML) when combined with azacitidine, excluded patients with MPN-BP. Rarely, patients develop de novo AML with a genetic profile like an MPN, harboring JAK2, MPL, or CALR mutations. However, there are a lack of data on the outcomes of these patients. Methods: We conducted a single-center retrospective cohort study to compare composite complete response (CCR), OS and event-free survival (EFS) of patients with MPN-BP or de novo AML with underlying JAK2, MPL, or CALR mutations (AML-D-MPN). CCR was defined as complete response (CR) or CR with incomplete count recovery. Propensity score (PS) modeling was used to adjust for baseline characteristics at AML diagnosis: age, sex, race, comorbidities, ECOG status, cytogenetics, and underlying mutations. Results: Of 750 patients treated from 2015 to 2023, 61 had MPN-BP and 12 had AML-D-MPN. The table describes baseline characteristics. The median time for MPN to BP transformation was 8 years (IQR: 2.8-13.6). With a median follow-up of 8.2 months (mo) (95CI: 1.6-59.6), the median OS for MPN-BP was 4.8 mo (95CI: 2.7-8) and for AML-D-MPN was 6.1 mo (95CI: 1.6-NC). In the MPN-BP, 40 received therapy, which included: intensive regimen [7+3 (n=13); CPX-351 (n=1)], hypomethylating agents (HMA) (n=11), HMA+VEN (n=11) and others (n=4). All AML-D-MPN received therapy, which included 7+3 (n=4), HMA (n=1), HMA+VEN (n=2), and others (n=5). In the whole treated population (n=52), the unadjusted CCR was 60% in intensive regimen, 20% in HMA, 20% in HMA+VEN and 0% in others (p=0.3). The PS-adjusted median (m) OS in mo (95%CI) was: 2.6 (1.4-NC) in intensive regimen, 6.2 (1.2-NC) in HMA, 15 (10-15) in HMA+VEN and 1.7 (0.46-NC) in others (p=0.1). The adjusted m-EFS in the same treatment groups was 1.5 (0.43-NC), 6.2 (1.2-NC), 15 (4.3-15) and 0.92 (0.46-NC) (p=0.16), respectively. In MPN-BP group, the adjusted m-EFS in mo (95%CI) was: 0.62 (0.43-NC) in the intensive regimen, 6.2 (1.2-NC) in HMA, 15 (4.2-15) in HMA+VEN and 0.92 (0.92-NC) in others (p=0.25). Conclusions: Patients with MPN-BP and AML-D-MPN have poor survival with similar responses to therapy. These patients have variable outcomes with different treatments; however, there is a trend toward improved survival with the use of HMA+VEN. [Table: see text]

Phase 3 study of the lysine-specific demethylase 1 (LSD1) inhibitor bomedemstat in patients with essential thrombocythemia (ET).

TPS6596 Background: ET is a myeloproliferative neoplasm (MPN) driven by mutations in JAK2, CALR, and MPL that is characterized by thrombocytosis and megakaryocyte hyperplasia. Currently available treatments can prevent thrombotic complications but do not substantially alter the natural history of ET. LSD1 is an enzyme that regulates hematopoietic stem and progenitor cell proliferation and is overexpressed in MPNs. In a phase 2 study, the LSD1 inhibitor bomedemstat was generally well tolerated and improved symptoms, durably reduced platelet and white blood cell counts, and reduced mutation burden in patients with ET. Here, we describe the methods for a randomized, open-label, phase 3 study (NCT06079879) that has been designed to evaluate bomedemstat versus best available therapy in patients with ET who had an inadequate response to or were intolerant of hydroxyurea. Methods: Key eligibility criteria include age ≥18 years, a diagnosis of ET per World Health Organization 2016 diagnostic criteria, a bone marrow fibrosis score of grade 0 or 1, a platelet count of >450 x 109/L, and an absolute neutrophil count of ≥0.75 x 109/L. All patients must have a history of inadequate response to or intolerance of hydroxyurea per modified European LeukemiaNet (ELN) criteria. Patients will be randomly assigned 1:1 to bomedemstat 50 mg/day by mouth (starting dose) or investigator’s choice of best available therapy (anagrelide, busulfan, interferon alfa/pegylated interferon alpha, or ruxolitinib). Bomedemstat dose will be titrated to a target platelet count of ≥150 to ≤350 x 109/L. After 52 weeks, patients receiving best available therapy can cross over to bomedemstat at the investigator’s discretion and patients in the bomedemstat arm can continue on treatment (maximum of 156 weeks on study). Randomization will be stratified by hydroxyurea history (inadequate response vs intolerance) and MFSAF v4.0 baseline score (≥4 vs <4). Clinic visits will occur every 2 weeks until week 12 and monthly thereafter. Adverse events will be graded per NCI CTCAE v5.0 criteria and monitored for up to 30 days after last dose of study drug. The primary end point is durable clinicohematologic response rate per modified ELN criteria. Secondary end points are duration of clinicohematologic response, duration of hematologic remission, change from baseline in total symptom and fatigue score per MFSAF v4.0 criteria, change from baseline in total fatigue score per the PROMIS Fatigue SF-7a scale, incidence of thrombotic or major hemorrhagic events, transformation to post-ET myelofibrosis or myelodysplastic syndrome/acute myeloid leukemia, event-free survival, and safety. Exploratory end points include pharmacokinetics, the proportion of patients reporting stability or improvement versus decline on the MSAF v4.0 and PROMIS Fatigue SF-7a domains, and molecular biomarkers. Approximately 300 patients will be enrolled. Clinical trial information: NCT06079879 .

Phase 3 randomized double-blind study evaluating selinexor, an XPO1 inhibitor, plus ruxolitinib in JAKi-naïve myelofibrosis.

TPS6594 Background: Myelofibrosis (MF) is a myeloproliferative neoplasm with common somatic gene driver mutations in JAK2, CALR, and MPL. Selinexor, an investigational oral XPO1 inhibitor, may inhibit MF-relevant JAK/STAT and non-JAK/STAT pathways. Preclinical studies have shown potential synergy with ruxolitinib treatment. In the phase 1 portion of XPORT-MF-034 evaluating selinexor plus ruxolitinib in JAKi-naïve patients with MF, the most common AEs in the 60 mg cohort were nausea (79%), anemia (64%), thrombocytopenia (64%), and fatigue (57%). Nausea was predominantly Grade 1 and transient in nature. Treatment-related AEs leading to treatment discontinuation were thrombocytopenia (n=1) and neuropathy (n=1). SVR35 and TSS50 was achieved by 79% and 58% of the 60 mg cohort intent-to-treat population at Week 24, respectively. Response rates were consistent across subgroups, including sex and regardless of ruxolitinib starting dose. These data provide strong support to further evaluate selinexor (60 mg) and ruxolitinib in patients with JAKi-naïve MF. Methods: The XPORT-MF-034 (NCT04562389) trial includes a global, Phase 3 randomized, double-blind, placebo-controlled study designed to evaluate selinexor and ruxolitinib. JAKi-naïve patients with MF will be randomized 2:1 to receive oral selinexor 60 mg or placebo once weekly (28-day cycle) and twice daily ruxolitinib. Randomization will be stratified by DIPSS risk category (int-1 vs int-2 or high-risk), spleen volume (<1800 cm3 vs >1800 cm3 by MRI/CT scan), and baseline platelet counts (100-200x109/L vs >200x109/L). Dual anti-emetics for nausea prophylaxis will be required for the first two cycles. Select eligibility criteria include ≥18 years of age, spleen volume ≥450 cm3 by MRI or CT, DIPSS intermediate-1, intermediate-2, or high-risk, active symptoms of MF (MFSAF v4.0), currently not eligible for stem cell transplantation, ECOG≤2, and platelet count ≥100 x 109/L. Select exclusion criteria include >10% blasts in peripheral blood or bone marrow; previous treatment with JAKi for MF, or previous treatment with selinexor or other XPO1 inhibitors. The co-primary study endpoints are SVR35 and TSS50 at Week 24 and will be tested hierarchically. The key secondary endpoint is anemia response at Week 24 per the IWG-MRT and ELN criteria. The XPORT-MF-034 Phase 3 trial is currently open for enrollment; a total of 306 JAKi-naïve MF patients will be enrolled and the study was initiated on June 28, 2023. Clinical trial information: NCT04562389 .

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.

Clonal evolution process from essential thrombocythemia to acute myeloid leukemia in the original patient from whom the CALR-mutated Marimo cell line was established.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

A comparative analysis of the clinical and genetic profiles of blast phaseBCR::ABL1-negative myeloproliferative neoplasm and acute myeloid leukemia, myelodysplasia-related.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (Ph (-) MPNs), have variable potential for progression to the blast phase (MPN-BP) of the disease. Except initiated by distinct driver mutations, MPN-BP frequently carry similar genetic abnormalities defining acute myeloid leukemia myelodysplasia-related (AML-MR). Because of dissimilar initial pathogenesis, MPN-BP and AML-MR are retained under different disease categories. To determine if separately classifying these entities is justified, we compare MPN-BP with AML-MR patients based on mutational landscape and clinical parameters. 104 MPN-BP patients and 145 AML-MR patients were identified with available clinical, cytogenetic, and genetic data. AML-MR patients presented with a higher blast count (median, 51% vs. 30%) while MPN-BP patients had higher WBC counts, platelet counts and bone marrow cellularity (all p<0.0001). Patients with MPN-BP showed similar genetic mutations with similar mutation pattern (functional domain, hotspot and locus involved by the mutations) but a different mutation rate from AML-MR, with more frequent JAK2, CALR, MPL, ASXL1, IDH2, SETBP1 and SRSF2 mutations and less frequent TP53 and DNMT3A mutations. The overall survival (OS) of MPN-BP (OS post-BP-progression) is comparable to that of AML-MR (median OS, 9.5 months vs. 13.1 months, p=0.20). In addition, the subgroups of MPN-BP show similar OS as AML-MR. When harboring certain mutation such as TP53, ASXL1, DNMT3A, TET2, RUNX1, IDH1, IDH2, EZH2, U2AF1, BCOR and SRSF2, MPN-BP and AML-MR patients carrying the same somatic mutation show no difference in OS. MPN-BP and AML-MR harbor similar somatic mutations and clinical outcomes, suggesting a unified clinical disease entity.

Clinical laboratory characteristics and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR.

To evaluate the incidence, clinical laboratory characteristics, and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR. We collected a total of 359 Ph-negative MPN patients with classical mutations in driver genes JAK2, MPL, or CALR, and divided them into two groups based on whether they had additional atypical variants of driver genes JAK2, MPL, or CALR: 304 patients without atypical variants of driver genes and 55 patients with atypical variants of driver genes. We analyzed the relevant characteristics of these patients. This study included 359 patients with Ph-negative MPNs with JAK2, MPL, or CALR classical mutations and found that 55 (15%) patients had atypical variants of JAK2, MPL, or CALR. Among them, 28 cases (51%) were male, and 27 (49%) were female, with a median age of 64 years (range, 21-83). The age of ET patients with atypical variants was higher than that of ET patients without atypical variants [70 (28-80) vs. 61 (19-82), p = 0.03]. The incidence of classical MPL mutations in ET patients with atypical variants was higher than in ET patients without atypical variants [13.3% (2/15) vs. 0% (0/95), p = 0.02]. The number of gene mutations in patients with atypical variants of driver genes PV, ET, and Overt-PMF is more than in patients without atypical variants of PV, ET, and Overt-PMF [PV: 3 (2-6) vs. 2 (1-7), p < 0.001; ET: 4 (2-8) vs. 2 (1-7), p < 0.05; Overt-PMF: 5 (2-9) vs. 3 (1-8), p < 0.001]. The incidence of SH2B3 and ASXL1 mutations were higher in MPN patients with atypical variants than in those without atypical variants (SH2B3: 16% vs. 6%, p < 0.01; ASXL1: 24% vs. 13%, p < 0.05). These data indicate that classical mutations of JAK2, MPL, and CALR may not be completely mutually exclusive with atypical variants of JAK2, MPL, and CALR. In this study, 30 different atypical variants of JAK2, MPL, and CALR were identified, JAK2 G127D being the most common (42%, 23/55). Interestingly, JAK2 G127D only co-occurred with JAK2V617F mutation. The incidence of atypical variants of JAK2 in Ph-negative MPNs was much higher than that of the atypical variants of MPL and CALR. The significance of these atypical variants will be further studied in the future.

Broad Next-Generation Integrated Sequencing of Myelofibrosis Identifies Disease-Specific and Age-Related Genomic Alterations.

Myeloproliferative neoplasms (MPN) are characterized by the overproduction of differentiated myeloid cells. Mutations in JAK2, CALR, and MPL are considered drivers of Bcr-Abl-ve MPN, including essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic primary myelofibrosis (prePMF), and overt myelofibrosis (MF). However, how these driver mutations lead to phenotypically distinct and/or overlapping diseases is unclear. To compare the genetic landscape of MF to ET/PV/PrePMF, we sequenced 1,711 genes for mutations along with whole transcriptome RNA sequencing of 137 patients with MPN. In addition to driver mutations, 234 and 74 genes were found to be mutated in overt MF (N = 106) and ET/PV/PrePMF (N = 31), respectively. Overt MF had more mutations compared with ET/PV/prePMF (5 vs. 4 per subject, P = 0.006). Genes frequently mutated in MF included high-risk genes (ASXL1, SRSF2, EZH2, IDH1/2, and U2AF1) and Ras pathway genes. Mutations in NRAS, KRAS, SRSF2, EZH2, IDH2, and NF1 were exclusive to MF. Advancing age, higher DIPSS, and poor overall survival (OS) correlated with increased variants in MF. Ras mutations were associated with higher leukocytes and platelets and poor OS. The comparison of gene expression showed upregulation of proliferation and inflammatory pathways in MF. Notably, ADGRL4, DNASE1L3, PLEKHGB4, HSPG2, MAMDC2, and DPYSL3 were differentially expressed in hematopoietic stem and differentiated cells. Our results illustrate that evolution of MF from ET/PV/PrePMF likely advances with age, accumulation of mutations, and activation of proliferative pathways. The genes and pathways identified by integrated genomics approach provide insight into disease transformation and progression and potential targets for therapeutic intervention.