CALR Mutations Research Articles

Triple immunostaining demonstrates the possible existence of segregated-nucleus-containing atypical monocytes in human primary myelofibrosis bone marrow: a case report

BackgroundSegregated-nucleus-containing atypical monocytes have recently been identified in mice. Segregated-nucleus-containing atypical monocytes are thought to originate from the bone marrow and induce fibrosis in the drug-injured lung. The Lyc6c− murine monocyte subset is the counterpart to human CD14−CD16++ non-classical monocytes; however, the human counterpart to murine segregated-nucleus-containing atypical monocytes has not yet been identified. Primary myelofibrosis is a well-known disease of progressive marrow fibrosis, and atypical megakaryocytes are thought to be closely related to fibrosis in primary myelofibrosis bone marrow. However, recently, monocytes have been reported to play an important role in marrow fibrosis in primary myelofibrosis. We speculated that, if there is a human counterpart to murine segregated-nucleus-containing atypical monocytes, it would present the same markers as murine segregated-nucleus-containing atypical monocytes, such as CD14−CD16+ macrophage-1 antigen (CD11b/CD18 complex)+, MSR1+, and CEACAM1+, and it might exist in the bone marrow of patients with primary myelofibrosis.Case presentationA 74-year-old Japanese male visited our hospital for clinical follow-up after total prostatectomy for prostatic cancer. Anemia, thrombocytosis, and elevated lactate dehydrogenase were suddenly observed in a periodic examination. CALR mutation type 2 (p.K385fs*47) was observed. The histological features of the patient’s bone marrow were consistent with fibrotic primary myelofibrosis. We immunohistochemically studied the bone marrow in an attempt to identify a human counterpart to murine segregated-nucleus-containing atypical monocytes. We detected a few CD16+MSR1+CEACAM1+ cells, but not CD14+MSR1+CEACAM1+ cells, by triple immunostaining. The patient is in a good condition and does not require treatment for primary myelofibrosis.ConclusionThere is a possibility that human segregated-nucleus-containing atypical monocytes exist in the bone marrow of primary myelofibrosis patients and might be related to marrow fibrosis.

Myeloproliferative Neoplasm-like Mutations of Calreticulin Induce Phenotypes Associated with Calreticulin Dysfunction in C. elegans.

In previous research, we created a C. elegans model with homozygous mutations in calreticulin similar to those found in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), two myeloproliferative neoplasms (MPNs). This model, lacking JAK orthologs, enabled us to examine the transcriptomic effects caused by mutant calreticulin without the influence of JAK/STAT activation, the primary pathogenic mechanism associated with calreticulin mutations known to date. Most of the gene expression changes observed seemed to be due to a partial loss of protein function, with the alteration of the extracellular matrix being particularly notable. In this study, our aim was to determine whether this model exhibited any phenotype related to these transcriptomic alterations. The results demonstrate that these strains exhibit multiple phenotypes related to the alteration of the extracellular matrix, fat levels, and fertility, which could be a possible consequence of a partial loss of calreticulin function. These phenotypes resemble some of the clinical and molecular characteristics described in patients with MPNs, but they had never before been linked to a loss of protein function in humans. Thus, these results collectively suggest that CALR mutations could have significant effects on MPNs due to loss of protein function. Delving deeper into these effects to develop innovative therapies for these patients offers considerable potential and interest, given that targeted therapies for these patients have not yielded very promising results so far.

Differential modulation of mutant CALR and JAK2 V617F-driven oncogenesis by HLA genotype in myeloproliferative neoplasms.

It has been demonstrated previously that human leukocyte antigen class I (HLA-I) and class II (HLA-II) alleles may modulate JAK2 V617F and CALR mutation (CALRmut)-associated oncogenesis in myeloproliferative neoplasms (MPNs). However, the role of immunogenetic factors in MPNs remains underexplored. We aimed to investigate the potential involvement of HLA genes in CALRmut+ MPNs. High-resolution genotyping of HLA-I and -II loci was conducted in 42 CALRmut+ and 158 JAK2 V617F+ MPN patients and 1,083 healthy controls. A global analysis of the diversity of HLA-I genotypes revealed no significant differences between CALRmut+ patients and controls. However, one HLA-I allele (C*06:02) showed an inverse correlation with presence of CALR mutation. A meta-analysis across independent cohorts and healthy individuals from the 1000 Genomes Project confirmed an inverse correlation between the presentation capabilities of the HLA-I loci for JAK2 V617F and CALRmut-derived peptides in both patients and healthy individuals. scRNA-Seq analysis revealed low expression of TAP1 and CIITA genes in CALRmut+ hematopoietic stem and progenitor cells. In conclusion, the HLA-I genotype differentially restricts JAK2 V617F and CALRmut-driven oncogenesis potentially explaining the mutual exclusivity of the two mutations and differences in their presentation latency. These findings have practical implications for the development of neoantigen-based vaccines in MPNs.

Impaired fibrinolysis in JAK2V617F-related myeloproliferative neoplasms

BackgroundMyeloproliferative neoplasms (MPNs) are characterized by a high rate of thrombotic complications that contribute to morbidity and mortality. MPN-related thrombogenesis is assumed to be multifactorial, involving both procoagulant and proinflammatory processes. Whether impaired fibrinolysis also participates in the prothrombotic phenotype of MPN has been poorly investigated. ObjectivesWe determined whether MPN, particularly JAK2V617F-positive MPN, is associated with fibrinolytic changes. MethodsTissue-type plasminogen activator (tPA)–mediated fibrinolysis was evaluated both in whole blood and plasma from mice with a hematopoietic-restricted Jak2V617F expression compared with wild-type (WT) mice (Jak2WT) using (1) halo clot lysis, (2) front lysis, and (3) plasmin generation assays. tPA clot lysis assay was performed in the plasma from 65 MPN patients (JAK2V617F mutation, n = 50; CALR mutations, n = 9) compared with 28 healthy controls. ResultsIn whole blood from Jak2V617F mice, we observed a decreased fibrinolysis characterized by a significantly lower halo clot lysis rate compared with Jak2WT (95 ± 22 vs 147 ± 39 AU/min; P < .05). Similar results were observed in plasma (halo clot lysis rate, 130 ± 27 vs 186 ± 29 AU/min; front lysis rate, 2.8 ± 1.6 vs 6.1 ± 1.2 μm.min−1; P < .05). Plasmin generation was significantly decreased both in plasma clots and standardized fibrin clots from Jak2V617F mice compared with Jak2WT mice. Among MPN patients, impaired tPA-related fibrinolysis with prolonged clot lysis time was observed in JAK2V617F and CALR patients. Plasminogen activator inhibitor-1 and α2-antiplasmin were significantly increased in plasma from JAK2V617F patients compared with controls. ConclusionOur results suggest that impaired tPA-mediated fibrinolysis represents an important prothrombotic mechanism in MPN patients that requires confirmation in larger studies.

MPN-176 Clinical Significance of CALR Mutation in Patients With Essential Thrombocythemia

MPN-176 Clinical Significance of CALR Mutation in Patients With Essential Thrombocythemia

Clinical Significance of CALR Mutation in Patients With Essential Thrombocythemia

Clinical Significance of CALR Mutation in Patients With Essential Thrombocythemia

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.

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.

Momelotinib - a promising advancement in the management of myelofibrosis in adults with anemia.

Myelofibrosis (MF) is a rare BCR-ABL negative myeloproliferative neoplasm characterized by clonal proliferation of stem cells, with mutations in JAK2, CALR, or MPL genes. MF presents in primary and secondary forms, with common symptoms including splenomegaly, anemia, and thrombocytopenia. Diagnostic criteria involve bone marrow examination and mutation studies. Current treatments are limited, with allogeneic stem cell transplant as the only curative option. Recent FDA approval of Momelotinib (MMB) offers new promise for MF patients with anemia. MMB, a JAK1/2 and ACVR1 inhibitor, effectively reduces spleen size, improves hemoglobin levels, and decreases transfusion dependency. The MOMENTUM trial compared MMB to danazol in JAK inhibitor-treated MF patients with anemia, showing MMB's superior symptom relief and transfusion independence rates. Additionally, the SIMPLIFY-1 and SIMPLIFY-2 trials evaluated MMB in JAK inhibitor-naïve and experienced patients, respectively, confirming MMB's non-inferiority to ruxolitinib in spleen volume reduction and highlighting its benefits in transfusion requirements. MMB's unique dual inhibition mechanism addresses anemia by suppressing hepcidin production, thus enhancing erythropoiesis. These trials collectively suggest MMB as an effective treatment for MF, improving quality of life and offering a survival advantage for patients with anemia. Despite challenges, such as trial design limitations and adverse events, MMB represents a significant advancement in MF management, providing a new therapeutic option for a previously underserved patient population.

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.

Aspirin therapy is associated with a lower risk of pregnancy loss in both JAK2- and CALR-mutated essential thrombocythemia-A Mayo Clinic study of 200 pregnancies.

Two-hundred pregnancies involving 100 women with essential thrombocythemia (ET) were accessed from Mayo Clinic databases (1990-2023). Median platelet count displayed a decline during pregnancy, nadiring at 48% of baseline, in the third trimester: 704-369 × 109/L. Live birth rate was 72%. Of 53 (27%) unintentional pregnancy losses, 48 (24%) occurred in the first trimester. Other fetal complications included preterm birth 3%, intrauterine growth retardation 3%, and stillbirth 1%. Maternal complications included major hemorrhage (7%), preeclampsia (6%), thrombosis (1%), and placental abruption (0.5%). Antepartum management included no specific therapy in 52 (26%), aspirin alone in 112 (56%), aspirin combined with cytoreductive drugs or systemic anticoagulants in 23 (12%), and other permutations in the remaining. Postpartum systemic anticoagulation was documented in 29 (15%) pregnancies. Unintentional first-trimester loss was predicted by prior fetal loss (43% vs. 18%; p < .01), diabetes mellitus (DM; 67% vs. 23%; p = .02), and absence of aspirin therapy (45% vs. 14%; p < .01); the salutary effect of aspirin therapy was independent of the other two risk factors and apparent in both high (presence of ≥1 risk factor; 33% vs. 61%; p = .07) and low (absence of both risk factors; 10% vs. 34%; p < .01) risk scenarios. The benefit of aspirin therapy, in preventing first-trimester loss, was significant in both JAK2-mutated (18% vs. 50%; p < .01) and CALR-mutated (8% vs. 43%; p < .01) cases. Aspirin use was also associated with a lower risk of venous thrombosis (0% vs. 3%; p = .03). By contrast, the use of systemic anticoagulation, antepartum or postpartum, did not influence fetal or maternal complication rates. CALR mutation and DM predicted maternal hemorrhage (13% vs. 4%; p = .05) and preeclampsia (33% vs. 5%; p = .03), respectively. The current study demonstrates the protective role of aspirin in preventing first-trimester loss in ET, independent of driver mutation status or other risk factors.

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 &gt;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 &lt;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 (&lt;1800 cm3 vs &gt;1800 cm3 by MRI/CT scan), and baseline platelet counts (100-200x109/L vs &gt;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 &gt;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 .

Immunophenotype of myeloid granulocytes in Chinese patients with BCR::ABL1-negative myeloproliferative neoplasms

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15–CD11b– was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

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.

CALR but Not JAK2 Mutations Are Associated with an Overexpression of Retinoid X Receptor Alpha in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a blood cancer caused by mutations in JAK2 and CALR. It is widely recognized that both mutations lead to the constitutive activation of JAK2/STAT signaling, although other JAK/STAT-independent pathogenic mechanisms triggered by these alterations have also been described in ET. In an attempt to study JAK2/STAT-independent mechanisms derived from CALR mutations, our research group created a C. elegans model with patient-like mutations in calreticulin that lacks JAK counterparts. The introduction of patient-like mutations in the calreticulin of C. elegans leads to an increase in the transcriptional expression of nhr-2, independently of JAK2/STAT activation. In the present study, we aim to verify if this mechanism is conserved in patients with ET harboring CALR mutations. To do so, we evaluated the expression of potential orthologs of nhr-2 in human cell lines of interest for the study, as well as in bone marrow (BM) or peripheral blood (PB) mononuclear cells from patients with CALR or JAK2 mutations. The results revealed that this mechanism is conserved in CALR-mutated ET patients, since CALR, but not JAK2 mutations, were associated with an overexpression of RXRA in patients with ET. The use of drugs targeting the activation or blockade of this target in the analyzed cell lines did not result in changes in cell viability. However, RXRA might be relevant in the disease, pointing to the need for future research testing retinoids and other drugs targeting RXRα for the treatment of ET patients.

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.