Chronic Neutrophilic Leukemia Research Articles

Chronic neutrophilic leukemia and atypical chronic myeloid leukemia: 2024 update on diagnosis, genetics, risk stratification, and management.

Chronic neutrophilic leukemia (CNL) is a rare BCR::ABL1-negative myeloproliferative neoplasm (MPN) defined by persistent mature neutrophilic leukocytosis and bone marrow granulocyte hyperplasia. Atypical chronic myeloid leukemia (aCML) (myelodysplastic "[MDS]/MPN with neutrophilia" per World Health Organization [WHO]) is a MDS/MPN overlap disorder featuring dysplastic neutrophilia and circulating myeloid precursors. Both manifest with frequent hepatosplenomegaly and less commonly, bleeding, with high rates of leukemic transformation and death. The 2022 revised WHO classification conserved CNL diagnostic criteria of leukocytosis ≥25 × 109/L, neutrophils ≥80% with <10% circulating precursors, absence of dysplasia, and presence of an activating CSF3R mutation. ICC criteria are harmonized with those of other myeloid entities, with a key distinction being lower leukocytosis threshold (≥13 × 109/L) for cases CSF3R-mutated. Criteria for aCML include leukocytosis ≥13 × 109/L, dysgranulopoiesis, circulating myeloid precursors ≥10%, and at least one cytopenia for MDS-thresholds (ICC). In both classifications ASXL1 and SETBP1 (ICC), or SETBP1 ± ETNK1 (WHO) mutations can be used to support the diagnosis. Both diseases show hypercellular bone marrow due to a granulocytic proliferation, aCML distinguished by dysplasia in granulocytes ± other lineages. Absence of monocytosis, rare/no basophilia, or eosinophilia, <20% blasts, and exclusion of other MPN, MDS/MPN, and tyrosine kinase fusions, are mandated. Cytogenetic abnormalities are identified in ~1/3 of CNL and ~15-40% of aCML patients. The molecular signature of CNL is a driver mutation in colony-stimulating factor 3 receptor-classically T618I, documented in >80% of cases. Atypical CML harbors a complex genomic backdrop with high rates of recurrent somatic mutations in ASXL1, SETBP1, TET2, SRSF2, EZH2, and less frequently in ETNK1. Leukemic transformation rates are ~10-25% and 30-40% for CNL and aCML, respectively. Overall survival is poor: 15-31 months in CNL and 12-20 months in aCML. The Mayo Clinic CNL risk model for survival stratifies patients according to platelets <160 × 109/L (2 points), leukocytes >60 × 109/L (1 point), and ASXL1 mutation (1 point); distinguishing low- (0-1 points) versus high-risk (2-4 points) categories. The Mayo Clinic aCML risk model attributes 1 point each for: age >67 years, hemoglobin <10 g/dL, and TET2 mutation, delineating low- (0-1 risk factor) and high-risk (≥2 risk factors) subgroups. Management is risk-driven and symptom-directed, with no current standard of care. Most commonly used agents include hydroxyurea, interferon, Janus kinase inhibitors, and hypomethylating agents, though none are disease-modifying. Hematopoietic stem cell transplant is the only potentially curative modality and should be considered in eligible patients. Recent genetic profiling has disclosed CBL, CEBPA, EZH2, NRAS, TET2, and U2AF1 to represent high-risk mutations in both entities. Actionable mutations (NRAS/KRAS, ETNK1) have also been identified, supporting novel agents targeting involved pathways. Preclinical and clinical studies evaluating new drugs (e.g., fedratinib, phase 2) and combinations are detailed.

Analysis of CSF3R mutations in atypical chronic myeloid leukemia and other myeloid malignancies

We report a series of patients with CSF3R-mutant (CSF3Rmut) atypical chronic myeloid leukemia (aCML), chronic neutrophilic leukemia (CNL) or other hematologic malignancies. We included 25 patients: 5 aCML and 4 CNL CSF3Rmut patients; 1 aCML, 2 CNL, and 2 myelodysplastic/myeloproliferative neoplasm, not otherwise specified patients without CSF3R mutation; and 11 CSF3Rmut patients with other diseases [8 acute myeloid leukemia (AML), 1 chronic myelomonocytic leukemia (CMML), 1 myelodysplastic syndrome (MDS), and 1 acute lymphoblastic leukemia (ALL)]. Patients with aCML or CNL were tested by Sanger sequencing and pyrosequencing to identify CSF3R T618I. Twenty-two patients underwent gene panel analysis. CSF3R mutations, mostly T618I (8/9), were found at high frequencies in both aCML and CNL patients [5/6 aCML and 4/6 CNL]. Two aCML patients in early adulthood with CSF3R T618I and biallelic or homozygous CEBPA mutations without other mutations presented with increased blasts and exhibited remission for >6 years after transplantation. The other 7 CSF3Rmut aCML or CNL patients were elderly adults who all had ASXL1 mutations and frequently presented with SEBP1 and SRSF2 mutations. Five AML patients had CSF3R exon 14 or 15 point mutations, and 6 other patients (3 AML, 1 CMML, 1 MDS, and 1 ALL) had truncating mutations, demonstrating differences in leukocyte counts and mutation status. In conclusion, CSF3R mutations were found at a higher frequency in aCML patients than in previous studies, which might reflect ethnic differences. Additional studies are needed to confirm these findings and the relationship between CSF3R and CEBPA mutations.

Enhanced MAPK signaling induced by CSF3R mutants confers dependence to DUSP1 for leukemic transformation

AbstractElevated MAPK and the JAK-STAT signaling play pivotal roles in the pathogenesis of chronic neutrophilic leukemia and atypical chronic myeloid leukemia. Although inhibitors targeting these pathways effectively suppress the diseases, they fall short in providing enduring remission, largely attributed to the cytostatic nature of these drugs. Even combinations of these drugs are ineffective in achieving sustained remission. Enhanced MAPK signaling besides promoting proliferation and survival triggers a proapoptotic response. Consequently, malignancies reliant on elevated MAPK signaling use MAPK feedback regulators to intricately modulate the signaling output, prioritizing proliferation and survival while dampening the apoptotic stimuli. Herein, we demonstrate that enhanced MAPK signaling in granulocyte colony-stimulating factor 3 receptor (CSF3R)–driven leukemia upregulates the expression of dual specificity phosphatase 1 (DUSP1) to suppress the apoptotic stimuli crucial for leukemogenesis. Consequently, genetic deletion of Dusp1 in mice conferred synthetic lethality to CSF3R-induced leukemia. Mechanistically, DUSP1 depletion in leukemic context causes activation of JNK1/2 that results in induced expression of BIM and P53 while suppressing the expression of BCL2 that selectively triggers apoptotic response in leukemic cells. Pharmacological inhibition of DUSP1 by BCI (a DUSP1 inhibitor) alone lacked antileukemic activity due to ERK1/2 rebound caused by off-target inhibition of DUSP6. Consequently, a combination of BCI with a MEK inhibitor successfully cured CSF3R-induced leukemia in a preclinical mouse model. Our findings underscore the pivotal role of DUSP1 in leukemic transformation driven by enhanced MAPK signaling and advocate for the development of a selective DUSP1 inhibitor for curative treatment outcomes.

Myeloproliferative Neoplasms: Diseases Mediated by Chronic Activation of Signal Transducer and Activator of Transcription (STAT) Proteins.

Myeloproliferative neoplasms (MPNs) are hematopoietic diseases characterized by the clonal expansion of single or multiple lineages of differentiated myeloid cells that accumulate in the blood and bone marrow. MPNs are grouped into distinct categories based on key clinical presentations and distinctive mutational hallmarks. These include chronic myeloid leukemia (CML), which is strongly associated with the signature BCR::ABL1 gene translocation, polycythemia vera (PV), essential thrombocythemia (ET), and primary (idiopathic) myelofibrosis (PMF), typically accompanied by molecular alterations in the JAK2, MPL, or CALR genes. There are also rarer forms such as chronic neutrophilic leukemia (CNL), which involves mutations in the CSF3R gene. However, rather than focusing on the differences between these alternate disease categories, this review aims to present a unifying molecular etiology in which these overlapping diseases are best understood as disruptions of normal hematopoietic signaling: specifically, the chronic activation of signaling pathways, particularly involving signal transducer and activator of transcription (STAT) transcription factors, most notably STAT5B, leading to the sustained stimulation of myelopoiesis, which underpins the various disease sequalae.

Commentary: Case report: Chronic neutrophilic leukemia associated with monoclonal gammopathies. A case series and review of genetic characteristics and practical management.

Commentary: Case report: Chronic neutrophilic leukemia associated with monoclonal gammopathies. A case series and review of genetic characteristics and practical management.

A Phase 2 Study of Fedratinib in Patients with MDS/MPN and Chronic Neutrophilic Leukemia

Introduction MDS/MPNs are clinically and molecularly complex diseases that exhibit proliferative symptoms and aggressive clinical courses. Evaluation of mutational patterns and gene expression profiles suggest these diseases should be viewed as a spectrum rather than distinct disease entities. Treatment options are limited and poorly defined as patients (pts) are often excluded from clinical trials. The JAK1/JAK2 inhibitor, ruxolitinib, has shown clinical benefit in pts with MDS/MPN and pts harboring CSF3R mutations. The experience of JAK2 inhibitors in myelofibrosis (MF) has shown that non-JAK2 kinase targets of JAK inhibitors may result in unique profiles of clinical benefit. Fedratinib is a JAK2 inhibitor approved for higher-risk MF. Compared to ruxolitinib, it has a broader kinase inhibition profile which may convey enhanced efficacy in high-risk, molecularly complex disease. Fedratinib potently inhibits FLT3 and BRD4 and potently suppresses c-Myc expression which may have biologic relevance in MDS/MPN. Study Design This is a phase 2, multi-institutional, investigator-initiated clinical trial (NCT05177211) assessing the efficacy of fedratinib in pts with atypical chronic myeloid leukemia (aCML), chronic neutrophilic leukemia (CNL), MDS/MPN-unclassifiable (MDS/MPN-U), and MDS/MPN-ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) per 2016 WHO classification. Inclusion criteria included splenomegaly (≥ 5 cm below left costal margin or ≥ 450 cc) and/or significant disease-related symptoms (MPN TSS ≥ 10). Pts with a platelet count &amp;lt; 35 x 10 9/L or peripheral/marrow blasts &amp;gt; 10% were excluded. There was no exclusion based on prior treatment. The primary endpoint of this ongoing study is overall response rate defined as complete or partial response or clinical benefit at 24 weeks per proposed MDS/MPN IWG response criteria. C-Myc (a potential biomarker for response) was stained in the bone marrow collected at baseline and week 24. C-Myc expression product was scored by multiplying % positive cells by intensity (0 = none, 1 = mild, 2 = moderate, 3 = marked). Fedratinib was given at a dose of 400 mg daily. Planned enrollment is 25 pts with an interim analysis completed after 9 pts are evaluable for efficacy. Results At time of data cut-off, 10 pts have been enrolled with a median follow-up of 5 months. Eight pts remain on treatment. Baseline demographics, genetic makeup, and treatment history are shown in table 1. Enrolled pts include 1 with aCML, 4 with CNL, 4 with MDS/MPN-RS-T, and 1 with MDS/MPN-U. Three or more mutations were present in 7 (70%) pts. Three of 5 (60%) evaluable pts responded at week 24. This included 3 (75%) symptom responses and 1 (20%) spleen response (1 pt with both). Six pts have completed 12 weeks of treatment with 1 spleen response and 2 symptoms responses (Figure 1). Among 6 pts with baseline splenomegaly who received 12 weeks of treatment, spleen volume decreased in 5 (83%) by an average of -23% (+5% to -71%). Among 5 pts with significant baseline symptom burden who received 12 weeks of treatment, 4 (80%) experienced an improvement in symptom burden by an average of -43% (range 0% to -76%). One pt who discontinued treatment prior to week 8 for reasons unrelated to disease or treatment was considered a spleen and symptom non-responder despite experiencing a 48% symptom improvement at week 4. At baseline, C-Myc expression was demonstrated by IHC staining in a median of 10% of cells (5-15%). Average baseline c-Myc expression product (% positive cells * staining intensity) was 26.5 (range 10-37.5). In pts with paired samples (n = 4), c-Myc expression product decreased in all cases by an average of 51% (25%-85%), p = 0.02. Ten pts were evaluable for safety. The most common AEs occurring in &amp;gt;2 pts were anemia, platelet count decrease, diarrhea, nausea, muscle cramp, and constipation. Grade ≥3 AEs were anemia (40%) and neutropenia (10%). There were no grade ≥ 3 non-hematologic AEs. Two pts discontinued study treatment: one due to disease progression after initial response and one due to pt decision unrelated to disease or treatment. Conclusion: Fedratinib demonstrates promising clinical efficacy in MDS/MPN and CNL pts with proliferative features. The safety profile is consistent with prior experience. Fedratinib's unique kinase inhibition profile may provide a mechanism for enhanced effectiveness in this pt population. Updated results will be presented at the meeting.

Cumulative Incidence of Myeloid Neoplasms in Patients with Nodal T-Follicular Helper Cell Lymphoma

Introduction: Certain T-cell lymphomas (TCL), in particular nodal T-follicular helper (TFH) cell lymphomas, including angioimmunoblastic TCL (AITL), commonly develop on a background of mutations identified in clonal hematopoiesis (CH). Divergent clonal evolution can result in both nodal TFH cell lymphomas and myeloid neoplasms (MN). As peripheral TCLs are commonly treated with combination and dose intensive chemotherapy, there is a risk that these therapies could promote an evolution to MN. The frequency at which this occurs is unclear. Methods: We conducted a retrospective search for any patient with an International Classification of Disease (ICD) code referring to a TCL. Then, we identified patients with a concomitant ICD code referring to either myelodysplastic syndromes (MDS), acutemyeloid leukemia (AML), or a myeloproliferative neoplasm (MPN). Once the initial query was performed, we individually reviewed each patient and included only patients meeting the following criteria: (1) A histologically-confirmed diagnosis of either peripheral TCL, not otherwise specified (PTCL-NOS) or nodal TFH cell lymphoma by an MSK hematopathologist between 1/1/02 and 7/1/23; (2) Presentation to MSK at lymphoma diagnosis; (3) At least 6 months of follow-up (unless death from lymphoma); (4) if relevant, a histologically-confirmed diagnosis of a MN by an MSK hematopathologist. Histological diagnoses were recorded as documented in the pathology electronic record in accordance with the WHO criteria in place at the time of diagnosis. In particular, at our center, prior to 2016, cases of PTCL-NOS were not routinely assessed for TFH phenotype, as nodal TFH cell lymphoma was only added to the WHO classification in 2016. Cases documented herein as PTCL-NOS were not re-reviewed to determine TFH phenotype. Only patients who received systemic treatment were included (systemic steroids were not considered systemic treatment). CI of MN was evaluated using the reverse Kaplan-Meier method treating death as a competing risk. Results: We identified a total of 376 patients with either PTCL-NOS (N=178) or nodal TFH cell lymphoma (N=198; AITL N=184; nodal TFH cell lymphoma, NOS N=13; nodal TFH cell lymphoma, follicular-type=1). In total, 24 patients with a MN and either PTCL-NOS or nodal TFH cell lymphoma were identified. Nine had a MN that preceded the TCL diagnosis (AML: 2; MDS: 1; myelofibrosis: 2; CMML: 2; MDS/MPN: 1; polycythemia vera: 1)-these patients were excluded for CI calculation. Fifteen patients were diagnosed with a MN subsequent to lymphoma diagnosis and treatment (AML: 5; MDS: 8; CMML: 1; chronic neutrophilic leukemia: 1). Of the 198 patients with nodal TFH cell lymphoma, nine developed a MN. The CI of developing a MN among all patients at 2, 5, 10, and 15 years was 1.1%, 4.4%, 4.8%, and 5.8%. The CI of developing a MN among those with known nodal TFH cell lymphoma at the same timepoints were 1.6%, 4.5%, 5.3% and 7.1%. The median follow up among survivors was 4.1 years (range: 0.5-18). The median age at MN diagnosis was 72.6 years (range: 45-81), and the median time from lymphoma diagnosis to MN diagnosis was 2.6 years (range: 0.3-10.4). Four patients had either prior RT or chemotherapy for non-lymphoma conditions. Prior to MN diagnosis, ten patients (67%) received etoposide and seven patients (47%) received autologous stem cell transplant (ASCT) (six with BEAM, one with cyclophosphamide plus total body irradiation). The median number of therapies (including ASCT) prior to MN diagnosis was 2 (range: 1-5). At the time of MN diagnosis, 10 patients had relapsed TCL and were receiving lymphoma therapy. No patients had undergone alloSCT prior to the diagnosis of a MN. Next generation mutational profiling with MSK-IMPACT-Heme had previously been performed for eight of the 15 patients (on lymphoma tissue). All except one had mutations in TET2 and/or DNMT3A. Conclusions: Herein we report the CI of MN in a cohort of patients with TCL, with particular attention to nodal TFH cell lymphomas given known CH mutations and the genotoxic stress of combination chemotherapy. As more patients have prolonged survival after initial therapy for TCL, further analysis, including baseline genetics and prospective characterization for clonal expansion and acquired mutations during therapy, could identify those at highest risk for developing a MN. Further characterization of our cohort and comparison to patients who did not develop a MN is ongoing.

Research progress of additional pathogenic mutations in chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a rare type of myeloproliferative neoplasm (MPN). Due to its nonspecific clinical symptoms and lack of specific molecular markers, it was previously difficult to distinguish it from other diseases with increased neutrophils. However, the discovery of the CSF3R mutation in CNL 10years ago and the update of the diagnostic criteria by the World Health Organization (WHO) in 2016 brought CNL into a new era of molecular diagnosis. Next-generation sequencing (NGS) technology has led to the identification of numerous mutant genes in CNL. While CSF3R is commonly recognized as the driver mutation of CNL, other mutations have also been detected in CNL using NGS, including mutations in other signaling pathway genes (CBL, JAK2, NARS, PTPN11) and chromatin modification genes (ASXL1, SETBP1, EZH2), DNA methylation genes (DNMT3A, TET2), myeloid-related transcription factor genes (RUNX1, GATA2), and splicing and RNA metabolism genes (SRSF2, U2AF1). The coexistence of these mutated genes and CSF3R mutations, as well as the different evolutionary sequences of clones, deepens the complexity of CNL molecular biology. The purpose of this review is to summarize the genetic research findings of CNL in the last decade, focusing on the common mutated genes in CNL and their clinical significance, as well as the clonal evolution pattern and sequence of mutation acquisition in CNL, to provide a basis for the appropriate management of CNL patients.

Antineoplastic effects of pharmacological inhibitors of aurora kinases in CSF3RT618I-driven cells

Myeloproliferative neoplasms (MPN) are consolidated as a relevant group of diseases derived from the malfunction of the hematopoiesis process and have as a particular attribute the increased proliferation of myeloid lineage. Among these, chronic neutrophilic leukemia (CNL) is distinguished, caused by the T618I mutation of the CSF3R gene, a trait that generates ligand-independent receptor activation and downstream JAK2/STAT signaling. Previous studies reported that mutations in BCR::ABL1 and JAK2V617F increased the expression of the aurora kinase A (AURKA) and B (AURKB) in Ba/F3 cells and their pharmacological inhibition displays antineoplastic effects in human BCR::ABL1 and JAK2V617F positive cells. Delimiting the current scenario, aspects related to the AURKA and AURKB as a potential target in CSF3RT618I-driven models is little known. In the present study, the cellular and molecular effects of pharmacological inhibitors of aurora kinases, such as aurora A inhibitor I, AZD1152-HQPA, and reversine, were evaluated in Ba/F3 expressing the CSF3RT618I mutation. AZD1152-HQPA and reversine demonstrated antineoplastic potential, causing a decrease in cell viability, clonogenicity, and proliferative capacity. At molecular levels, all inhibitors reduced histone H3 phosphorylation, aurora A inhibitor I and reversine reduced STAT5 phosphorylation, and AZD1152-HQPA and reversine induced PARP1 cleavage and γH2AX expression. Reversine more efficiently modulated genes associated with cell cycle and apoptosis compared to other drugs. In summary, our findings shed new insights into the use of AURKB inhibitors in the context of CNL.

Chronic neutrophilic leukemia: A case report

Objectives. We present a case report of a patient with malaise, abdominal pain, and vomiting, later underwent bone marrow biopsy and found to have CNL with no genetic mutation. Case presentation. A 40 years old male came into hospital with malaise, abdominal pain, nausea, and vomiting. Furthermore there were bilateral lymphadenopathy in the neck, hepatomegaly and moderate splenomegaly in abdominal examination. Anemia, leukocytosis and increase of Lactat Dehydrogenase (LDH) were found. Bone marrow aspiration and biopsy was performed and showed predominant neutrophil and the bone marrow biopsy showed hypercellular bone marrow with increased myeloid-neutrophil, increased myeloid erythroid ratio. Outcome. After further investigation this patient was diagnosed with chronic neutrophilic leukemia based on bone marrow aspiration and biopsy result.

Chronic neutrophilic leukemia preceded by myelodysplastic syndromes.

Chronic neutrophilic leukemia (CNL) is primarily diagnosed by excluding myelodysplastic syndromes (MDS). We report the case of a patient who developed secondary CNL 3years after hypoplastic MDS. We used droplet digital polymerase chain reaction mutation detection assay to analyze genomic alterations during the progression from MDS to CNL. At the time of MDS diagnosis, U2AF1 Q157P and SETBP1 D868N were dominant and additional mutation of ASXL1 1934_insG was observed. CSF3R T618I and SETBP1 D868N were increasing at the time of CNL diagnosis. We revealed the accumulation of multiple gene mutations during CNL development from MDS. This suggests that CNL was clonally developed from the founding clone of MDS and CSF3R mutation contributes to the development of CNL in the present case. These findings provide insights into the pathology of CNL.

CSF3R-mutant chronic myelomonocytic leukemia is a distinct clinically subset with abysmal prognosis: a case report and systematic review of the literature

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) chacaterized by persistent peripheral blood monocytosis, hypercellular bone marrow and dysplasia at least in one myeloid lineage. CMML shares much of its molecular landscape with other myeloid neoplasms, while differs from others such as chronic neutrophilic leukemia (CNL), given the high frequency of CSF3R mutations in the latter. In this article, we report a case of CSF3R-mutated CMML and dissect this rare entity by reviewing the medical literature, with the intent to understand how this rare mutation shapes CMML's clinical and morphological phenotype. CSF3R-mutated CMML emerges as a rare entity meeting the ICC/WHO diagnostic criteria for CMML and simultaneously showing clinical-pathological and molecular traits of CNL and atypical chronic myeloid leukemia, rising an important and difficult diagnostic and therapeutical issue.

Abstract A38: Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia

Abstract Chronic Neutrophilic Leukemia (CNL) is a rare BCR-ABL negative myeloproliferative neoplasm that affects approximately 1 in a million individuals. The typical age of onset and disease presentation is in elderly patients with a median age of 63 and a range of 9-84. The oncogenic driver of CSF3R, the G-CSF receptor protein, in CNL has been classified over the last decade and has improved therapies for patients with targeted Tyrosine Kinase Inhibitors, such as Ruxolitinib (a potent JAK1/2 inhibitor). Currently, the only curative option is an allogeneic hemopoietic stem cell transplant with typical median overall survival of 2 years. Because of the low occurrence rate of CNL, there have only been 3 germline cases of CSF3R classified in literature. With these patients, they exhibit a lifelong skewing of the myeloid lineage causing gross neutrophilia. Our patient presented at 13 months with leukocytosis and neutrophilia, resulting in genetic testing revealing the diagnosis of the youngest documented case of CNL. A germline CSF3R T618I constitutive activating mutation was documented in our patient and in her father. Her father had documented lifelong leukocytosis of indeterminate origin and it was not until 33 that genetic testing was done, and he was diagnosed with CNL. He underwent an allogeneic stem cell transplant after being admitted with gross hepatosplenomegaly with a WBC of 322, the highest recorded in a germline CNL patient in literature. Our patient presented to clinic with leukocytosis ranging from 45-98 and hepatosplenomegaly. After a bone marrow aspirate and biopsy was taken to determine disease stage and severity, we immediately began Ruxolitinib therapy. We began 20 mg BID dosing based on platelets and monitored response to therapy while conducting targeted RNA-seq for myelodysplastic and myeloid leukemia genes on the bone marrow, which did not detect any secondary mutations. Ruxolitinib dosing was tapered down in a stepwise fashion, until the patient arrived at the current dose of 7.5 mg BID Ruxolitinib, resulting in stable leukocyte counts around 25. As part of monitoring, we are currently conducting 6-month BM procedures and NGS for AML/MDS genes and Ruxolitinib resistance mutations alongside monthly CBCs. Our patient has been on Ruxolitinib for 9 months and has not developed any secondary contributing mutations and remains sensitive to therapy. We continue to monitor our patient’s response to Ruxolitinib to determine if germline predisposition alters the transformative nature of the disease. Within T618I germline CNL cases, we are investigating the difference in phenotype and disease burden that we observe in our patient’s family compared to literature. Based on current cases, it seems that germline CNL does not transform during childhood and adolescence into AML without the influence of a secondary mutation. Retrospective studies into previous cases of CNL to determine if there is a higher frequency of germline origin could pave the way for potential early-stage therapies and detection of CNL prior to transformation. Citation Format: Kevin Zhang, Matthew Rees, Mahsa Khanlari, Laura Rooms, Raul Ribeiro, Marcin Wlodarski. Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A38.

Integrated transcriptome, proteome and single-cell sequencing uncover the prognostic and immunological features of colony-stimulating factor 3 receptor in pan-cancer.

Colony-stimulating factor 3 receptor (CSF3R) has been demonstrated to be associated with various hematological tumors, especially chronic neutrophilic leukemia; however, the detailed roles of CSF3R in other cancers remain to be explored. In the present study, we systematically analyzed the expression profiles of CSF3R in pan-cancer by comprehensive bioinformatics databases, such as Tumor Immune Estimation Resource, version 2 (TIMER2.0), Gene Expression Profiling Interactive Analysis, version 2 (GEPIA2.0), etc. GEPIA2.0 was also used to analyze the relationship between CSF3R expression and patients' survival prognosis. We found that the high expression of CSF3R was associated with a poor prognosis in the brain tumor patients, such as brain lower grade glioma and glioblastoma multiforme. In addition, we further investigated the genetic mutation and DNA methylation level of CSF3R in multiple cancers. Immune infiltration analysis showed that CSF3R expression was positively correlated with a variety of tumor-infiltrating immune cells in most cancers. Single cell sequencing indicated that CSF3R levels were correlated with several cancer-associated pathways, such as DNA damage, cell invasion, and stemness. Taken together, the role of CSF3R in multiple cancers might reveal its potential as a novel prognostic biomarker and therapeutic target for cancer patients.

Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.

Successful Allogeneic Stem Cell Transplantation with Ruxolitinib Maintenance Therapy for CSF3R T618I Mutant Chronic Neutrophilic Leukemia

Successful Allogeneic Stem Cell Transplantation with Ruxolitinib Maintenance Therapy for CSF3R T618I Mutant Chronic Neutrophilic Leukemia

Myeloproliferative neoplasms: changes since the WHO 2016 classification

A group of international experts, including haematopathologists, oncologists, and geneticists met on September 2021 in Chicago, IL, USA to update the 2016/17 World Health Organization classification system for haematopoietic tumours. After careful deliberation, the group introduced the new International Consensus Classification (ICC) for Myeloid Neoplasms and Acute Leukaemias. The classification was published in June 2022 (Arber DA, Orazi A, Hasserjian RP, et al. Blood 2022; 140: 1200–1228). This presentation focuses on the ICC-2022 category of chronic myeloid leukaemia (CML), BCR::ABL1 positive and the BCR::ABL1 negative myeloproliferative neoplasms (MPNs): essential thrombocythemia, polycythemia vera, primary myelofibrosis, and MPN, unclassifiable. In CML, ICC recognises three disease phases: chronic (CP), accelerated (AP), and blast (BP) phase. Their definition has been streamlined. For the MPN group, ICC chose to preserve the primary role of bone marrow morphology in disease classification and diagnostics, while also acknowledging the complementary role of genetic markers for establishing clonality, facilitating MPN subtype designation, and disease prognostication. Chronic neutrophilic leukaemia (CNL) can now be diagnosed with a WBC of > 13x 109/L. The diagnosis of chronic eosinophilic leukaemia, NOS, now requires a bone marrow showing dysplastic changes. Reference 1. Thiele J, Kvasnicka HM, Orazi A, et al. The international consensus classification of myeloid neoplasms and acute leukemias: myeloproliferative neoplasms. Am J Hematol 2022; doi: 10.1002/ajh.26751. Epub ahead of print. PMID: 36200127.

Chronic neutrophilic leukemia/chronic eosinophilic leukemia

Chronic neutrophilic leukemia (CNL) is a clonal disorder that is characterized by increasing mature neutrophils. Colony stimulating factor 3 receptor (CSF3R) T618I mutation was frequently identified in patients with CNL and is defined as a molecular marker of the disease. Ruxolitinib, a JAK2 inhibitor, provided a promising therapeutic effect in a phase II study. In particular, ruxolitinib was more efficient for patients with CSF3R mutation. Allogeneic stem cell transplantation (Allo-SCT) may be a curative treatment for CNL. On the other hand, further studies are needed to define the optimal method of transplantation, source of donor, conditioning therapy, and timing of transplantation. Chronic eosinophilic leukemia (CEL) is a clonal disorder that is characterized by increasing eosinophils. In the World Health Organization Classification 5th edition, diagnostic criteria for CEL are renewed. Because the new criteria will be more specific for CEL than criteria in the older edition, "not otherwise specified (NOS) " is removed from the name of the disease. Anti-CD52 antibody, alemtuzumab, or anti-IL-5 antibody, mepolizumab, are promising drugs to control symptoms that are associated with hypereosinophilic syndrome. Allo-SCT is anticipated as a curative treatment for CEL, but the evidence of Allo-SCT for CEL is still limited. Further study is required to define the treatment strategy.

Metformin Inhibits STAT3 and MAPK Signaling through AMPK Activation in CSF3RT618I Cells

Introduction: In the light of the discovery of the CSF3R mutation in chronic neutrophilic leukemia (CNL) patients, the T618I mutation demonstrated to be a molecular marker and a possible target for new therapies due to the JAK/STAT signaling pathway activation [N Engl J Med (2013) 368(19):1781-90]. Since no curative treatment options are available for patients diagnosed with CNL, it is of interest the study new therapeutical approaches for these patients. In this regard, the treatment of JAK2V617F murine models with metformin, an FDA-approved drug for type 2 diabetes, reduced splenomegaly, extramedullary hematopoiesis, and colony formation of HSPC [Cell Death Dis. 2018;9(3):311]. Hence, we characterized the preclinical cellular and molecular effects of metformin in CSF3RT618I models. Methods: The in vitro experiments were performed in Ba/F3 cells transduced retrovirally with CSF3RT618I overexpression (OE) model. Cells were treated with metformin (1-20 mM) to access cell viability and survival. Protein expression was evaluated by western blot. For in vivo evaluation, eight-week-old female NSG mice were transplanted with 2x106 Ba/F3 CSF3RT618I cells, resuspended in Matrigel: PBS (1:1 ratio). After the tumor formation (2 days), mice were treated for 10 days by intraperitoneal injection of vehicle (PBS) or metformin (125 mg/kg, n=10/group). Tumor volume was measured using the formula (V = W2 × L × 0.52), where W and L are the smallest and largest diameters, respectively. Ex vivo primary cells from a patient diagnosed with CNL harboring the CSF3RT618I mutation were used to assess the effects of metformin treatment. The primary bone marrow mononuclear cells were evaluated regarding cell survival and clonogenic capacity upon metformin treatment. All experiments were approved by the Animal and Human Ethics Committee of the Institution. Statistical analyses were performed by Mann-Whitney or ANOVA test and Bonferroni post-test. A p-value < 0.05 was considered. Results: After 24 and 48 hours, Ba/F3 CSF3RT618I treated with 1, 5, 10 and 20 mM of metformin presented a reduction in cell viability in a dose- and time-dependent manner (p < 0.01). The IC50 for metformin treatment for 24, 48, and 72 hours was 17.44, 1.3, and 3.2 mM, respectively. Increased frequency of apoptotic cells was observed at 5, 10, and 20 mM of metformin at times of 24, 48, and 72 hours (p < 0.01). Molecularly, CSF3RT618I cells displayed reduced STAT3 and ERK phosphorylation and increased AKT/mTOR activation, upon metformin 5 mM treatment for 24 hours. AMPK activation was observed in cells treated with 1-20 mM of metformin compared to vehicle. In vivo, metformin treatment decreased the tumor burden of CSF3RT618I transplanted mice after 9 days of treatment (vehicle versus metformin mean ± SEM: 731.9±90.14 vs. 481.6±38.14; p = 0.035). At the protein level, we observed inhibition in Cyclin D1 and the presence of caspase 3 cleavage in the isolated tumor from the metformin-treated mice. Ex vivo metformin treatment of the primary cells isolated from the CNL patient harboring CSF3RT618I resulted in increased apoptosis, with 51.94% (p < 0.05), 52.40% (p < 0.05), and 63.1% (p < 0.001), of apoptosis induction upon treatment with 5, 10 and 20 mM. Metformin ex vivo treatment strongly reduced hematopoietic progenitor cells colony formation on primary CNL CSF3RT618I peripheral blood (83.5% at 10 mM and 100% at 20 mM) and bone marrow mononuclear cells (100% at 10 mM and 98% at 20 mM). Conclusions: We provide evidence that metformin exerts anticancer activity in CSF3RT816I cells, by downregulation of STAT3 and MAPK signaling through AMPK activation. These results support the idea that metformin can be repurposed in the CNL clinics, as a therapeutical option.

Epigenetic Priming with 5-Azacytidine Prior to Allogeneic Stem Cell Transplantation for Myeloid Malignancies with In Vivo T Cell Depletion: Results of a Phase II Trial

Background: Relapse is the main cause of death in patients who undergo allogeneic hematopoietic stem cell transplantation (HCT) for myeloid malignancies. Recent improvements in survival have been mainly due to reduction in non-relapse mortality (NRM). Acquired abnormalities of DNA methylation are frequent in myeloid malignancies and are particularly observed among patients with adverse risk features. Epigenetic priming with decitabine to induce DNA hypomethylation was investigated in a phase I trial for acute myeloid leukemia (AML) and has proven safe and feasible (Scandura et al Blood 2011). Applying the principle of chemotherapy-sensitization through hypomethylation in myeloid malignancy, we conducted a phase II trial (NCT02497404) of the epigenetic modulator -5-azacytidine given prior to a reduced intensity conditioning (RIC) regimen. Here we report the results of this trial. Methods: We designed an open label phase II study to determine the feasibility and efficacy of epigenetic priming with 5-Azacytidine prior to RIC for HCT with in-vivo T-cell depletion. Inclusion criteria were AML, myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) with an intermediate or poor risk profile by ELN and IPSS-R, respectively, and adequate organ function. Stem cell grafts were GCSF- mobilized peripheral blood stem cells from either a matched related donor (MRD) or matched unrelated donor (MUD), matched for HLA at loci A, B, C, DR, and DQ. The epigenetic priming regimen was 5-Azacytidine 75 mg/m2/daily X 5, given between days -11 to -7 (Dose reduction to 50 mg/m2/daily after 14 patients due to toxicity). The conditioning regimen consisted of Fludarabine 40 mg/m2/daily, days -6 to -3, and Melphalan 140 mg/m2, on day -3. Graft versus host disease (GVHD) - prophylaxis consisted of Alemtuzumab (total dose 60 mg for MUD and 30 mg for MRD), and Tacrolimus for 3-6 months post-transplant. The Primary objectives were overall survival (OS) and progression free survival (PFS) at 1 year. Secondary objectives were incidence of GVHD and NRM. Results: 39 adult patients with AML, MDS, and chronic neutrophilic leukemia (CNL) were enrolled prospectively between 2/2015 and 6/2018. The median age was 60 years (range, 26-74). The majority of patients had AML (33, 85%), or MDS (5, 13%). Nineteen AML patients (58%) had a poor risk karyotype. Nine cases (23%) had mutated TP53. The donor was MUD in 22 (44%), MRD in 17 (56%) of patients. Other patient characteristics are listed in Table 1. The median follow up for survivors is 6.7 years (5.8 - not reached). Thirty-eight patients engrafted neutrophils at a median of 13 days (9-16) and platelets at a median of 16 days (12-102). One patient was not evaluable due to early death on day 9. OS at 1 year was 64% (95% CI 51-81%) and 36% (95% CI 24-55%) at 4 years, (Figure 1). PFS at 1 year was 54% (95% CI 40-72%) and 33% (95% CI 21-52%) at 4 years. At 1 year, relapse was the cause of death in 21% (95% CI 18-22%) and NRM was 15% (95% CI 13-17%). At 4 years, relapse was the cause of death in 41% (95% CI 38-43%), while NRM reached 23% (95% CI 20-25%). The most common cause of NRM was infection in 5 patients (46%), followed by thrombotic microangiopathy and GVHD. The incidence of acute GVHD by day 100 was 29% (95% CI 13-43%) with grade III-IV acute GVHD found in 8% (95% CI 6-9%). The incidence of chronic GVHD at 1 year was estimated at 22% (95% CI 7.4-35%). No new cases of chronic GVHD were seen thereafter. The most common grade 3/4 toxicities were infections in 8 patients (21%) and renal failure (23%). We found 6/14 patients to have grade 3/4 renal toxicity with 2 requiring hemodialysis. After reducing the 5-azacytidine dose to 50 mg/m2/daily, we observed only 3/25 cases of grade 3/4 renal dysfunction. Conclusion: Preconditioning treatment with 5-azacytidine as epigenetic priming is tolerable with a toxicity profile overall similar to conditioning with fludarabine/melphalan. But the addition of azacytidine at 75 mg/m2/daily (total 5 doses) causes significant renal toxicity, which abates with dose reduction. The PFS in this very high-risk population with historically high relapse rates is promising. A larger number of patients is needed to further evaluate the efficacy of this approach. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal