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.

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.

Chronic neutrophilic leukemia: 2022 update on diagnosis, genomic landscape, prognosis, and management.

DISEASE OVERVIEW: Chronic neutrophilic leukemia (CNL) is a rare, often aggressive myeloproliferative neoplasm (MPN) defined by persistent mature neutrophilic leukocytosis, bone marrow granulocyte hyperplasia, and frequent hepatosplenomegaly. The 2013 seminal discovery of oncogenic driver mutations in colony-stimulating factor 3 receptor (CSF3R) in the majority of patients with CNL not only established its molecular pathogenesis but provided a diagnostic biomarker and rationale for pharmacological targeting. DIAGNOSIS: In 2016, the World Health Organization (WHO) recognized activating CSF3R mutations as a central diagnostic feature of CNL. Other criteria include leukocytosis of ≥25 × 109 /L comprising >80% neutrophils with <10% circulating precursors and rare blasts, and absence of dysplasia or monocytosis, while not fulfilling criteria for other MPN. MANAGEMENT: There is currently no standard of care for management of CNL, due in large part to the rarity of disease and dearth of formal clinical trials. Most commonly used therapeutic agents include conventional oral chemotherapy (e.g., hydroxyurea), interferon, and Janus kinase (JAK) inhibitors, while hematopoietic stem cell transplant remains the only potentially curative modality. DISEASE UPDATES: Increasingly comprehensive genetic profiling in CNL, including new data on clonal evolution, has disclosed a complex genomic landscape with additional mutations and combinations thereof driving disease progression and drug resistance. Although accurate prognostic stratification and therapeutic decision-making remain challenging in CNL, emerging data on molecular biomarkers and the addition of newer agents, such as JAK inhibitors, to the therapeutic arsenal, are paving the way toward greater standardization and improvement of patient care.

Chronic neutrophilic leukemia complicated with monoclonal gammopathy of undetermined significance: A case report and literature review.

BACKGROUND: Study of the molecular biological characteristics of chronic neutrophilic leukemia complicated with plasma cell disorder (CNL-PCD) and lymphocytic proliferative disease (CNL-LPD). METHODS: The clinical data of a patient with chronic neutrophilic leukemia complicated with monoclonal gammopathy of undetermined significance (CNL-MGUS) in our hospital were reviewed, and the Chinese and/or English literature about CNL-PCD and CNL-LPD in PubMed and the Chinese database CNKI in the past 10 years was searched to analyze the molecular biological characteristics of this disease. RESULTS: A 73-year-old male had persistent leukocytosis for 18 months. The white blood cell count was 46.77 × 109/L and primarily composed of mature neutrophils; hemoglobin: 77 g/L; platelet count: 189 × 109/L. Serum immunofixation electrophoresis showed IgG-λ monoclonal M protein. A CT scan showed splenomegaly. Next-generation sequencing (NGS) showed that CSF3R T618I, ASXL1 and RUNX1 mutations were positive. It was diagnosed as CNL-MGUS. We summarized 10 cases of CNL-PCD and 1 case of CNL-LPD who underwent genetic mutation detection reported in the literature. The CSF3R mutational frequency (7/11, 63.6%) was lower than that of isolated CNL. The ASXL1 mutations were all positive (3/3), which may represent a poor prognostic factor. The SETBP1 mutation may promote the progression of CNL-PCD. We also found JAK2, RUNX1, NRAS, etc. in CNL-PCD. CONCLUSIONS: Chronic neutrophilic leukemia may be more inclined to coexist with plasma cell disorder. The CSF3R mutation in CNL-PCD is still the most common mutated gene compared with isolated CNL. Mutations in SETBP1 and ASXL1 may be poor prognostic factors for CNL-PCD.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Case and review: Cutaneous involvement by chronic neutrophilic leukemia vs Sweet syndrome- A diagnostic dilemma.

Chronic neutrophilic leukemia (CNL) is a rare leukemia with approximately 150 total cases reported. Cutaneous neutrophilic infiltrates, including Sweet syndrome (SS) and leukemia cutis (LC), have been reported in six patients with CNL. In the setting of CNL, these two conditions are difficult to differentiate due to clinical and histopathological similarities, but it is important to do so because LC is associated with a worse prognosis. In general, SS is distinguished by its tenderness, fever, and improvement with steroids (vs chemotherapy for LC). Biopsy of LC reveals immature leukocytes, whereas SS shows almost exclusively mature leukocytes, but morphology alone may not be sufficient in some cases. Here, we report a case of a 72-year-old male with CNL and a cutaneous eruption with clinical and pathological features which made the distinction between the two diseases difficult.

Diagnosis and management of neutrophilic myeloid neoplasms.

Chronic neutrophilia is commonly seen with persistent infections, inflammatory disorders, smoking, solid tumors, and specific medications. However, after reactive causes have been excluded, a workup for primary (clonal) neutrophilic disorders, such as myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative overlap syndromes, should be pursued. Except for chronic myeloid leukemia, which is defined by the presence of the Philadelphia (Ph) chromosome, and the classic Ph chromosome-negative MPNs (polycythemia vera, essential thrombocythemia, and primary myelofibrosis), clonal neutrophilic neoplasms historically have been challenging to diagnose and classify. The 2016 revised World Health Organization classification of these disorders has been based mainly on clinicopathologic features. However, recent discoveries of the molecular alterations underlying these disorders have served to supplement our knowledge of their morphologic and clinical features, opening new therapeutic avenues. In this review, we discuss the diagnostic approach, prognostic features, and treatments of neutrophilic myeloid neoplasms, with a focus on chronic neutrophilic leukemia, atypical chronic myeloid leukemia, and chronic myelomonocytic leukemia.

Coexisting of bone marrow fibrosis, dysplasia and an X chromosomal abnormality in chronic neutrophilic leukemia with CSF3R mutation: a case report and literature review.

BACKGROUND: Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) with less than 40 cases of patients being reported or clinically suspected meeting with 2008 World Health Organization ("WHO") diagnostic criteria. The current diagnosis of CNL remains to exclude other diseases. Recently, a new biomarker of CSF3R mutations that is almost invariably present in CNL has been identified. There is no effective treatment for CNL, therefore prognosis of the disease is poor, but it may be attributed to the presence of both SETBP1 and CSF3R gene mutations. The presence or absence of CSF3R mutation did not affect survival, whereas a trend for shortened survival was observed among patients with SETBP1-mutation. CASE PRESENTATION: Here we report a 65-year old woman patient who presented with leukocytosis without sign of fever and tumors. Bone marrow aspirates showed a markedly hypercellular feature with 76%-92% myeloid and the dysplastic changes were found in about 7% of neutrophils cells. The bone marrow biopsy demonstrated marrow fibrosis with Gomori staining positive (+++~++++). Cytogenetic analysis showed 46,X,del (X) (q22). No molecular markers of BCR/ABL1 rearrangement (P210, P230, P190 and variably), JAK2V617F, FIP1L1-PDGFRA, TEL-PDGFRB, ZNF198-FGFR1 and SETBP1 mutations were identified, however, the CSF3R gene membrane proximal mutation (c.1853C > T/p.T618I sites) was detected by PCR techniques. The patient was diagnosed with CNL and died in about 2 months after disease diagnosis. CONCLUSION: In clinical course, the CNL concurrently with severe bone marrow fibrosis and dysplastic features as well as X chromosomal abnormality may predict a worsening prognosis regardless of SETBP1 mutation status.

Chronic neutrophilic leukemia: 2018 update on diagnosis, molecular genetics and management.

DISEASE OVERVIEW AND DIAGNOSIS: Chronic neutrophilic leukemia (CNL) is a potentially aggressive myeloproliferative neoplasm, for which current WHO diagnostic criteria include leukocytosis of ≥ 25 x 109 /L of which ≥ 80% are neutrophils, with < 10% circulating neutrophil precursors with blasts rarely observed. In addition, there is no dysplasia, nor clinical or molecular criteria for other myeloproliferative neoplasms. UPDATE ON DIAGNOSIS: Previously the diagnosis of CNL was often as one of exclusion based on no identifiable cause for physiologic neutrophilia in patients fulfilling the aforementioned criteria. The 2016 WHO classification now recognizes somatic activating mutations of CSF3R (most commonly CSF3RT618I) as diagnostic, allowing for an accurate diagnosis for the majority of suspected cases through molecular testing. These mutations are primary driver mutations, accounting for the characteristic clinical phenotype and potential susceptibility to molecularly targeted therapy. RISK STRATIFICATION: Concurrent mutations, common to myeloid neoplasms and their precursor states, most frequently in SETBP1 and ASXL1, are frequent and appear to be of prognostic significance. Although data are evolving on the full genomic profile, the rarity of CNL has delayed complete understanding of its full molecular pathogenesis and individual patient prognosis.

Chronic neutrophilic leukemia, an extremely rare cause of neutrophilia in childhood: Cure with hematopoietic stem cell transplantation.

CNL is a rare myeloproliferative disorder frequently seen in older adults. A significant proportion of patients show progression to AML. Here, we report the case of a patient with FA who was monitored for leukopenia but who developed leukocytosis during the follow-up and was diagnosed with CNL probably after an acquired CSF3R mutation. Because the patient had FA, which could accelerate the progression to AML, an HSCT was performed, which resulted in cure. This patient (aged 12 years) is one of the youngest patients reported to develop CNL as well as the first FA patient with a diagnosis of CNL.

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML.

BACKGROUND: The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS: To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS: We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS: Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

Chronic neutrophilic leukemia 2016: Update on diagnosis, molecular genetics, prognosis, and management.

Chronic neutrophilic leukemia (CNL) is a potentially aggressive myeloproliferative neoplasm, for which current WHO diagnostic criteria include leukocytosis of ≥25 × 10(9) /L (of which >80% are neutrophils) and with <10 and <1% circulating immature granulocytes and blasts, respectively without dysplasia, clinical, or molecular criteria for other myeloproliferative disorders, nor an identifiable cause for physiologic neutrophilia in the absence of markers of myeloid clonality. Such a pathogenic clonal marker has now been identified as a somatic activating mutation of CSF3R, most commonly CSF3R T618I, thus demanding revision of the current WHO diagnostic classification to include the molecular criterion of mutated CSF3R. The clinical presentation, disease course and prognosis of CSF-R mutated CNL have been recently outlined. Co-operative mutations in SETBP1 and ASXL1 appear to be of prognostic significance and correlate with disease progression. Advances in the understanding of the molecular pathogenesis of CNL, have not yet fully translated into satisfactory therapeutic strategies, but the foundations for these are strengthening. Am. J. Hematol. 91:342-349, 2016. © 2015 Wiley Periodicals, Inc.

Chronic neutrophilic leukemia: novel mutations and their impact on clinical practice.

PURPOSE OF REVIEW: Chronic neutrophilic leukemia (CNL) is a rare BCR-ABL1-negative myeloid malignancy that is characterized by mature granulocytosis without dysgranulopoiesis. Differential diagnosis of CNL includes reactive or secondary granulocytosis and other myeloid neoplasms, such as atypical chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML). Herein, we focus on recently described mutations in CNL and their impact on diagnosis, prognosis and treatment. RECENT FINDINGS: In 2013, membrane-proximal CSF3R mutations, most frequently CSF3RT618I, were described in CNL and aCML. Subsequent studies confirmed the presence of such mutations in nearly all patients with CNL but not in aCML. Furthermore, the majority of the patients with CSF3R-mutated CNL also expressed other mutations, such as SETBP1 and ASXL1, which might be prognostically detrimental. Laboratory studies revealed that CSF3RT618I induced JAK inhibitor-sensitive activation of JAK-STAT and CNL-like disease in mice. Case reports have indicated palliative but not disease-modifying activity of JAK inhibitor therapy in CSF3R-mutated CNL. SUMMARY: CNL is now a morphologically and molecularly defined myeloid malignancy, and no longer a diagnosis of exclusion. The identification of CNL-specific molecular markers provides a much needed pathogenetic insight and also offers the opportunity to revise current diagnostic criteria and identify prognostic biomarkers and potential drug targets.

Chronic neutrophilic leukaemia and plasma cell-related neutrophilic leukaemoid reactions.

Many cases reported as 'chronic neutrophilic leukaemia' have had an associated plasma cell neoplasm. Recent evidence suggests that the great majority of such cases represent a neutrophilic leukaemoid reaction to the underlying multiple myeloma or monoclonal gammopathy of undetermined significance. We have analysed all accessible reported cases to clarify the likely diagnosis and to ascertain whether toxic granulation, Döhle bodies and an increased neutrophil alkaline phosphatase score were useful in making a distinction between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction. We established that all these changes occur in both conditions. Toxic granulation and Döhle bodies are more consistently present in leukaemoid reactions but also occur quite frequently in chronic neutrophilic leukaemia. The neutrophil alkaline phosphatase score is increased in both conditions and is of no value in making a distinction.

The new genetics of chronic neutrophilic leukemia and atypical CML: implications for diagnosis and treatment.

Although activation of tyrosine kinase pathways is a shared theme among myeloproliferative neoplasms, the pathogenetic basis of chronic neutrophilic leukemia (CNL) has remained elusive. Recently, we identified high-frequency oncogenic mutations in the granulocyte-colony stimulating factor receptor (CSF3R) in CNL and in some patients with atypical chronic myeloid leukemia. Inhibition of Janus kinase 2 or SRC kinase signaling downstream of mutated CSF3R is feasible and should be explored therapeutically. Herein, we discuss the potential impact of these findings for the classification and treatment of these disorders.

ASXL1 mutations are frequent and prognostically detrimental in CSF3R-mutated chronic neutrophilic leukemia.

Colony stimulating factor 3 receptor gene (CSF3R) mutations have recently been associated with chronic neutrophilic leukemia (CNL). Fourteen patients with CSF3R-mutated CNL (median age 67 years; 57% males) were screened for additional mutations; 8 (57%) and 5 (38%) harbored an ASXL1 and/or SETBP1 mutation (two patients expressed both), respectively. Two patients developed blastic transformation, both SETBP1-mutated and ASXL1-unmutated, whereas two other cases evolved into chronic myelomonocytic leukemia (CMML), both ASXL1-mutated and SETBP1-unmutated. Median survival was 23.2 months (10 deaths documented). On multivariable analysis mutated ASXL1 (P = 0.009; HR 19.6, 95% CI 2.1-184.1) and thrombocytopenia (P = 0.005; HR 28.8, 95% CI 2.8-298.2) were independently predictive of shortened survival. This study provides information on the natural history of CSF3R-mutated CNL and identifies mutant ASXL1 and thrombocytopenia as risk factors for survival. The study also suggests pathogenetic roles for SETBP1 and ASXL1 mutations in disease evolution into blast phase disease and CMML, respectively.

The molecular genetics of chronic neutrophilic leukaemia: defining a new era in diagnosis and therapy.

PURPOSE OF REVIEW: In the current WHO classification of myeloid disorders, chronic neutrophilic leukaemia (CNL) is recognized as a myeloproliferative neoplasm characterized by sustained neutrophilic leukocytosis, hepatosplenomegaly and bone marrow granulocytic hyperplasia without evidence of dysplasia, BCR-ABL1 or rearrangements of PDGFRA, PDGFRB or FGFR1. This diagnosis is contingent upon exclusion of underlying causes of reactive neutrophilia particularly if evidence of myeloid clonality is lacking. The lack of a specific molecular marker has left the diagnosis to be largely one of exclusion. Recently, the molecular landscape shifted with the discovery of specific oncogenic mutations in the colony-stimulating factor 3 receptor gene (CSF3R) in CNL patients. We review the implications for diagnosis, pathogenesis and potential for new therapeutic options. RECENT FINDINGS: In 2013, oncogenic mutations in CSF3R were identified in a majority of patients with CNL and demonstrated that their downstream signalling was sensitive to known kinase inhibitors. This discovery was then validated with the demonstration of 100% CSF3R mutational frequency (predominately CSF3RT618I) in strictly WHO-defined CNL. Simultaneously, novel somatic mutations in SETBP1 were found to be enriched in CNL with possible prognostic significance. SUMMARY: CNL appears to be driven by specific somatic activating CSF3R mutations. These bestow susceptibility to known kinase inhibitors, opening the door to novel specific therapeutic options for CNL. The diagnosis of CNL will no longer be one only of exclusion, and revision of the current WHO diagnostic criteria is expected to include the molecular criterion of CSF3R mutation positivity.