Colony-stimulating Factor 3 Receptor Mutations Research Articles

Hereditary chronic neutrophilic leukemia in a four-generation family without transformation to acute leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood neutrophilia, marrow granulocyte hyperplasia, hepatosplenomegaly, and driver mutations in the colony-stimulating factor 3 receptor (CSF3R). Designation of activating CSF3R mutations as a defining genomic abnormality for CNL has led to increased recognition of the disease. However, the natural history of CNL remains poorly understood with most patients reported being of older age, lacking germline data, and having poor survival, in part due to transformation to acute leukemia. CSF3R driver mutations in most patients with CNL have been reported to be acquired, although rare cases of germline mutations have been described. Here, we report the largest pedigree to date with familial CNL, spanning four generations with affected family members ranging in age from 4 to 53 years, none of whom have transformed to acute leukemia. A heterozygous T618I CSF3R mutation was identified in peripheral blood and mesenchymal stromal cells from the proband and in all affected living family members, while the unaffected family members tested were homozygous wild type. We show that the T618I mutation also confers a survival advantage to neutrophils in an MCL1-dependent manner. Collectively, these data provide additional insights into the natural history of familial CNL arising from T618I CSF3R mutations and suggest that enhanced neutrophil survival also contributes to the high neutrophil count observed in patients with CNL.

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.

Truncated CSF3 receptors induce pro-inflammatory responses in severe congenital neutropenia.

Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.

The presence of Auer rods in neutrophils and monocytes besides blasts in a case of acute myeloid leukemia with CSF3R mutation.

An 81-year-old male with a history of diabetes, hypertension, hypercholesterolemia, previous DVT not on anticoagulation, and arthritis presented to the emergency department with a chief complaint of generalized weakness. Complete blood counts revealed white blood cells (WBC) 165.3 × 109/L, hemoglobin (Hb) 4.4 g/dl, and platelet (PLT) < 10 × 109/L, with 62% blasts. Review of peripheral blood showed blasts of variable sizes with a moderate amount of basophilic cytoplasm (Figure 1A–D; peripheral blood; Leishman stain; 100× objective), and pale-salmon-pink-colored cytoplasmic granules in a subset (Figure 1A, see arrows). Auer rods are easily identified in blasts (Figure 1A,B, see asterisk), occasionally seen in monocytes (Figure 1C, see asterisk) and neutrophils (Figure 1d, see asterisk). Bone marrow was examined to establish diagnosis, showing marked hypercellularity with 72% blasts on aspirate smears with morphologic features similar to those in peripheral blood (Figure 1E [bone marrow biopsy; hematoxylin and eosin, 40× objective] and F [bone marrow aspirate; Leishman stain; 50× objective]). Granulocytic dysplasia with pelgeroid morphology and abnormal nuclear chromatin clumping was seen in both peripheral blood and bone marrow (> 10% and < 50%). Concurrent flow cytometry showed increased myeloblasts with expression of CD34, CD13, CD 117, CD33, HLA-DR, CD19 (dim; minor subset), CD11b (dim; subset), CD14 (dim; minor subset), CD15 (dim; minor subset), myeloperoxidase (dim; subset), and terminal deoxynucleotidyl transferase (dim; minor subset). Conventional cytogenetics showed normal male karyotype. Concurrent FISH studies using acute myeloid leukemia (AML) and myelodysplastic syndrome panels of probes are negative for all loci studied, including 8q22 and 21q22 probes for RUNX1-RUNX1T1 rearrangement. Next generation sequencing showed mutation on the CSF3R gene (p.T640N, variant allele frequency 48.4%). An integrated diagnosis of AML with maturation was established (French–American–British Classification Subtype M2). Auer rods are a morphologic feature in leukemic blasts defining myeloid differentiation. The presence of Auer rods in neutrophils and monocytes are rarely reported, most commonly in association with acute promyelocytic leukemia (APL) with PML-RARA and AML with RUNX1-RUNX1T1, occasionally seen in other subtypes of AML and myelodysplastic syndrome (MDS) in the literature as well [1]. We presented a case of AML with Auer rods in non-blast cells as well as morphologic and immunophenotypic features resembling AML with RUNX1-RUNX1T1: blasts with basophilic cytoplasm, salmon-pink (pale) cytoplasmic granules and perinuclear Hof besides Auer rods as well as granulocytic dysplasia in the background. However, no recurrent cytogenetic and molecular genetic abnormalities as defined by World Health Organization (WHO) classification are detected, while a CSF3R mutation seen. Although mutations in the colony stimulating factor 3 receptor (CSF3R) occurs rarely in AML, comprising only about 1% of adults with AML, it is suggested that CSF3R can be a potential therapeutic target in AML [2]. In summary, we presented for the first time a case of AML with a few rare morphologic features including the presence of Auer rods in non-blast cells coinciding with a potential therapeutic target of CSF3R mutation. Whether the association between the morphologic features and the CSF3R mutation can be established warrants additional studies. In addition, a potential diagnostic pitfall to be taken into consideration in this case is that no reverse transcription-polymerase chain reaction (RT-PCR) was done to further exclude RUNX1-RUNX1T1 rearrangement, although the likelihood of having undetected rearrangement by karyotyping and FISH studies is extremely low. What is the new aspect of your work? Here, we for the first time presented a case of AML with morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells, coinciding with CSF3R mutation, which is a rare mutation in AML while representing a potential novel therapeutic target. What is the central finding of your work? A rare case of CSF3R-mutated AML with no recurrent cytogenetic abnormality, showing unusual morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells. What is (or could be) the specific clinical relevance of your work? The rare morphological features present in this AML could potentially serve as a trigger to identify cases of AML with CSF3R mutation for targeted therapy. None. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. Not applicable. Not applicable. Not applicable. The data that support the findings of this study are available from the corresponding author upon reasonable request.

The prognostic factors in acute myeloid leukaemia with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm).

The prognostic factors to stratify acute myeloid leukaemia (AML) with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm) into different risk groups remains to be determined. In this retrospective study, we evaluated 171 consecutive patients with newly diagnosed AML with CEBPAdm by a Cox proportional hazards regression model. In univariate analyses, colony stimulating factor 3 receptor (CSF3R) and Wilms tumour 1 (WT1) mutations were associated with poor relapse-free survival (RFS). The induction regimens including homoharringtonine (omacetaxine mepesuccinate) or intermediate-dose cytarabine was associated with favourable RFS and overall survival (OS). The induction regimen including both homoharringtonine and intermediate-dose cytarabine was associated with the most favourable RFS (3-year RFS 84.7%) and OS (3-year OS 92.8%) compared to the conventional cytarabine and daunorubicin regimen (3-year RFS 27.7%, hazard ratio [HR] 0.126, 95% confidence interval [CI] 0.051-0.313, Wald p<0.001; and 3-year OS 56.4%, HR 0.179, 95% CI 0.055-0.586, Wald p=0.005). In multivariate analyses, the induction regimen including intermediate-dose cytarabine (HR 0.364, 95% CI 0.205-0.646, Wald p<0.001) and CSF3R mutations (HR 2.667, 95% CI 1.276-5.572, Wald p=0.009) were independently associated with RFS. Taken together, we found that induction regimen and CSF3R mutations were independent prognostic factors for AML with CEBPAdm.

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

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. 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. 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. 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.

Differential Implications of CSF3R Mutations in t(8;21) and CEBPA Double Mutated Acute Myeloid Leukemia

BackgroundFew data are available exploring mutations of the colony-stimulating factor 3 receptor (CSF3R) in acute myeloid leukemia (AML) in an all-round and systematic manner. The purpose of this study was to analyze the CSF3R mutations (CSF3Rmut) in AML with recurrent genetic abnormalities for potential synergistic pathomechanism. Patients and MethodsWe retrospectively screened 1102 adult de novo AML patients with available next-generation sequencing (NGS) information on 132 genes related to hematologic disorders. The χ2, Mann-Whitney U tests were used to analyze their associations with clinicopathologic characteristics, and a propensity score matching (PSM) followed by Kaplan-Meier method was applied to measure their prognostic effects. ResultsOverall, CSF3Rmut were detected in 40 (3.6%) of 1102 patients with adult de novo AML. CSF3Rmut were predominantly enriched in AML with the CEBPA double mutations (CEBPAdm) (16/122, 13.1%), t(8;21) (12/186, 6.5%) and mutated RUNX1 (3/50, 6.0%), respectively. The CSF3Rmut loci and types differed according to AML subtypes, with frameshift-indels and premature stop confined in the t(8;21) AML [10/12 (83.3%)], and missense recurrently aggregated in the CEBPAdm AML [16/16 (100%)]. Cases with CSF3Rmut had a lower WBC count versus those with CSF3R wild-type (CSF3Rwt) in the t(8;21) AML cohort, with a borderline significance [median 5.45 (range 0.94-20.30) × 109/L) vs. 8.80 (range 0.96-155.00) × 109/L, P = .046]. CSF3Rmut were non-significantly associated with higher WBC counts [median 33.6 (range 6.8-287.6) × 109/L vs. 18.1 (range 1.7-196.0) × 109/L, P = .156] and significantly with lower immunophenotypic CD15 positivity [0/8 (0%) vs. 44/80 (55%), P = .009] as compared to CSF3Rwt in the CEBPAdm AML cohort. After propensity score matching followed by Kaplan-Meier analysis, CSF3Rmut cases had comparable disease-free survival (DFS) and overall survival (OS) to those with CSF3Rwt (P = .607 and P = .842, respectively) in the t(8;21) AML cohort. By contrast, CSF3Rmut showed an inclination towards inferior DFS compared to CSF3Rwt in the CEBPAdm AML cohort [median DFS 19.8 (95%CI 3.1-36.5) months vs. not reached (NR), P = .086]. No significant difference was found for OS between CSF3Rmut and CSF3Rwt cases (P = .943). ConclusionWe concluded that CSF3Rmut were frequently enriched in patients with t(8;21) and CEBPAdm subtypes among AML, but showed divergent clinicopathologic features, mutation loci and types and differing prognostic aspects.

The clinical characteristics and prognosis of Chinese acute myeloid leukemia patients with CSF3R mutations.

The colony-stimulating factor 3 receptor (CSF3R) controls the proliferation of myeloid progenitors and differentiation into neutrophils. However, the clinical features and prognostic significance of CSF3R mutations in primary acute myeloid leukemia (AML) patients are still unclear. 158 newly diagnosed AML patients were retrospectively evaluated in our study. Amplicon-based next-generation sequencing (NGS) and multiplex-nested reverse-transcription polymerase chain reaction (RT-PCR) were used to investigate the 34 genes and 43 fusion genes associated with leukemia. In addition, clinical features, mutation incidence, and survival outcomes were compared between patients with CSF3R mutation and patients with wild-type CSF3R. In our study, CSF3R mutations were found in 7.6% (12/158) cases. The membrane-proximal amino acid substitution T618I (58.3%) was the most frequent mutation. CSF3R mutations were associated with higher WBC counts (P=.035). CEBPA mutation, TET2 mutation, and RUNX1-RUNX1T1 translocation were the most common co-mutations of CSF3R. The CSF3R gene was mutually exclusive with signal transduction genes (P=.029), while positively associated with TET2 mutations (P=.014). CSF3R mutations had no effect on CR1 (P=.935), R (P=.625) and OS (P=.1172). Patients with CSF3R mutations had a worse DFS (P=.0352) than those with wild-type CSF3R. Multivariate survival analysis showed that CSF3R mutation was an independent risk factor for DFS of primary AML patients (HR=2.048, 95%CI: 1.006-4.170, P=.048). AML patients with CSF3R mutations had unique clinical features and gene co-mutation spectrum. CSF3R mutation was an independent risk factor for DFS and could be a potential prognostic marker and therapeutic target for Chinese primary AML patients.

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A).The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date.Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members.Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C).In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. [Display omitted] DisclosuresVoorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Efficacy of Low-Dose rhGM-CSF Treatment in a Patient With Severe Congenital Neutropenia Due to CSF3R Deficiency: Case Report of a Novel Biallelic CSF3R Mutation and Literature Review.

This study reports the clinical manifestations, genetics, and efficacy of treatment with the efficacy of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-GSF) of a 2-year-old female patient with severe congenital neutropenia (SCN) type 7 (SCN7) caused by novel biallelic mutations in the colony-stimulating factor 3 receptor (CSF3R) gene. Genetic diagnosis of the patient was performed by whole-exome and Sanger sequencing. Expression of the CSF3R gene in the peripheral neutrophils of the patient was detected by real-time PCR and Western blotting. The patient presented with recurrent suppurative tonsillitis and decreased absolute neutrophil count <0.5 × 109/L. Novel heterozygous mutations were found to be inherited from each parent (maternal c.690delC [p.met231Cysfs*32] and paternal c.64+5G>A). The patient's neutrophils had lower CSF3R mRNA and protein levels than those of the parents. Low-dose rhGM-CSF (3 μg/kg/day once a week) prevented recurrent infection in the patient. These results demonstrate that the clinical manifestations of SCN7 with biallelic CSF3R mutations and downregulated CSF3R can be effectively treated with rhGM-CSF.

Colony-stimulating factor 3 receptor (CSF3R) M696T mutation does not impact on clinical outcomes of a Ph+ acute lymphoblastic leukemia patient

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders. Although CSF3R point mutations (eg, T618I) are emerging as key players in chronic neutrophilic leukemia/atypical chronic myelogenous leukemia , the significance of rarer CSF3R mutations is unknown. Here, we report a 32-year-old female who was diagnosed as Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) with the CSF3R M696T mutation and was undergone unrelated donor hematopoietic stem cell transplantation. The patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. Meanwhile, we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. We confirmed the presence of a CSF3R M696T germline mutation in this patient which was inherited from her mother. The in vitro experiment results showed that the CSF3R M696T mutation contributes marginally to the tumor transformation of Ba/F3 cells, indicating that CSF3R M696T mutation was neutral in tumor transformation ability. We concluded that TKI is effective in patients with the CSF3R M696T mutation in Ph+ ALL and donors with CSF3R M696T mutation might still be selected as the candidate for transplantation.

Oncogenic SETBP1 Mutations Combine with Activating Mutations in CSF3R to Produce a Highly Proliferative, Lethal Leukemia through Aberrant Myc Signaling

SETBP1 (SET Binding Protein 1) mutations are associated with exceptionally poor prognosis in myeloid neoplasms. Despite this, SETBP1's role in oncogenesis remains incompletely understood. In this study, we find that SETBP1 leads to a marked upregulation of the Myc oncogene and associated pro-stem and progenitor programs through epigenetic dysregulation. We further identify an epigenetic modulatory drug that normalizes SETBP1-driven Myc overexpression and synergizes with disease-relevant therapy. SETBP1 has documented roles in both the regulation of tumor suppressor pathways and modulation of transcription. To understand how SETBP1 modulates leukemia biology and leverage this mechanistic insight to develop novel therapeutic strategies, we turned to a genetically well-defined model system, chronic neutrophilic leukemia (CNL). SETBP1 is mutated in approximately half of all cases of CNL, a myeloproliferative neoplasm characterized by the presence of signaling-activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R). By expressing SETBP1 and CSF3R mutations in mouse hematopoietic progenitors, we have generated models of CNL that can be leveraged for mechanistic studies and drug development. In a hematopoietic colony forming unit (CFU) assay, murine hematopoietic progenitors co-expressing mutant SETBP1 and mutant CSF3R have a high proliferation phenotype compared to those with mutant CSF3R alone. When cells co-expressing mutant SETBP1 and CSF3R are transplanted into lethally irradiated mice, they develop a rapidly lethal disease relative to the control mice. This is associated with an expansion of the granulocyte lineage, quantified by complete blood count, flow cytometry and histology. We find that SETBP1 is essential for the induction of a pro-proliferative transcriptional program. One of the most prominent SETBP1-associated signatures is that of Myc target genes. Myc itself is one of the top differentially expressed genes driven by SETBP1. Congruent with its increased expression, we also find higher Myc transcriptional activity in cells overexpressing SETBP1. To better understand the epigenetic regulation of Myc and progenitor pathways by SETBP1, we employed a low input profiling methods called CUT&amp;Tag to assess the activation of regulatory elements. We began by profiling two epigenetic marks associated with active enhancers-H3K4me1 and H3K27Ac. We also assessed activation of the Myc promoter by measuring H3K27Ac and H3K4me3. Together this data helps us to understand the epigenetic underpinnings for dysregulation of Myc-driven programs by SETBP1. Therapeutic strategies that normalize aberrant Myc activity may be effective against SETBP1-driven disease. We found that SETBP1 and CSF3R transformed hematopoietic progenitors are highly sensitive to inhibitors of the epigenetic regulator lysine specific demethylase 1 (LSD1). LSD1 inhibition restores Myc expression to physiological levels and represses Myc promotor activity in vitro. Furthermore, LSD1 inhibitors are highly synergistic with JAK inhibitors, which block signaling downstream of CSF3R. Together these data establish the importance of Myc activation in SETBP1-driven malignancies and identify a therapeutic approach to normalize aberrant Myc activity. In future, we will use the insight gained in this genetically well defined disease to inform studies on the mechanistic basis and therapeutic vulnerabilities of other SETBP1-mutant myeloid malignancies. Disclosures Maxson: Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

The Colony-Stimulating Factor 3 Receptor M696T Mutated in a Patient with Ph+ Acute Lymphoblastic Leukemia

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders, such as severe congenital neutropenia (SCN), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and atypical chronic myelogenous leukemia (aCML). Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. We report a case of philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) with the M696T mutation in CSF3R gene and assess the pathogenicity of the CSF3R M696T mutation in Ph+ ALL. Experimental Design: Here we report on a 32-year-old female who presented with asthenia. The initial hematological workup revealed white blood cell (WBC) count of 97 x 109/L (normal range 4-10 x 109/L). There was 84% prolymphocyte in the bone marrow. The immunophenotype of the blasts as judged from flow cytometry was in accordance with a B-ALL. The fusion gene for BCR-ABL P210 was positive. Hot mutation closely related to diseases was: CSF3R (nucleotide change c.2087 T&amp;gt;C, amino acid change p.M696T, mutation frequency 50.4%). Cytogenetic analysis showed 46, XX, t (9;22) (q34;q11). The patient was diagnosed as Ph+ ALL with the CSF3R M696T mutation and achieved Long-term survival after unrelated donor hematopoietic stem cell transplantation. Meanwhile we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. The phosphorylation of STAT3 was analyzed by G-CSF dependence assays and immunoblot analysis to evaluate the CSF3R M696T mutation contribution to the tumor transformation ability of Ba/F3 cells. Results: This patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. We confirmed the presence of a CSF3R M696T germline mutation in this patient, and the mutation was inherited from her mother. The experiments in vitro result showed the CSF3R M696T mutation harbors marginal contribution to the tumor transformation ability of Ba/F3 cells. CSF3R M696T mutation was neutral in tumor transformation ability. Conclusions: We believe that TKI is still effective in patients with the CSF3R M696T mutation in Ph+ ALL. Donor with CSF3R M696T mutation might still be selected. Disclosures No relevant conflicts of interest to declare.

Chronic neutrophilic leukemia: 2020 update on diagnosis, molecular genetics, prognosis, and management.

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 seminal discovery of oncogenic driver mutations in colony-stimulating factor 3 receptor (CSF3R) in the majority of patients with CNL in 2013 anchored a new scientific framework, deepening our understanding of its molecular pathogenesis, providing a diagnostic biomarker, and rationalizing the use of pharmacological targeting. In 2016, the World Health Organization (WHO) included the presence of 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. Increasingly comprehensive genetic profiling of CNL has disclosed a complex genomic landscape and additional prognostically relevant mutational combinations. Though prognostic determination and therapeutic decision-making remain challenging, emerging data on prognostic markers and the use of newer therapeutic agents, such as JAK inhibitors, are helping to define state-of-the-art management in CNL.

T618I CSF3R mutations in chronic neutrophilic leukemia induce oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor form

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL.

Analysis of gene mutation characteristics in patients with chronic neutrophilic leukaemia

ABSTRACTObjective: This study aims to investigate the gene mutation characteristics of chronic neutrophilic leukaemia (CNL).Method: This study retrospectively analyses the molecular biological characteristics, laboratory characteristics and clinical data of four patients with CNL that were admitted in the second Hospital of Shanxi Medical University from May 2014 to October 2016. On the basis of the molecular biological data of 22 patients with CNL and 4 patients with CNL, we further analysed the characteristics of CNL molecular mutation.Results: Two out of the four patients with CNL were carriers of colony-stimulating factor 3 receptor (CSF3R) mutation, among which two were carriers of CSF3R T618I mutation combined with ASXL1 mutation and SETBP1 mutation, and two were only carriers of JAK2 V617F mutation. According to the molecular biological data of 22 patients with CNL, 20 patients were positive for CSF3R mutation. Two patients were positive for JAK2 V617F mutation. A total of 10 patients were positive for SETBP1 mutation which was correlated with the CSF3R T618I gene mutation (P = 0.03). A total of 13 patients were positive for ASXL1 mutation. No patients carried mutations in ASXL2 and MPL genes.Conclusion and Discussion: CSF3R mutation is the main tumorigenic mutation in CNL, in which CSF3R T618I mutation is the main mutation, and an extremely small number of CNL patients may be caused by JAK2 V617F mutation. SETBP1 and ASXL1 are the most common concomitant mutations in CNL with CSF3R mutation, and SETBP1 and CSF3R T618Imutations may have a certain correlation.

Checkpoint Inhibition in CSF3R Mutated Chronic Neutrophilic Leukemia

Mutations in the colony-stimulating factor 3 receptor (CSF3R) have been identified in 90% of chronic neutrophilic leukemia (CNL) and lower frequency in atypical chronic myeloid leukemia (aCML). It was shown recently, that signaling of the different types of CSF3R mutations varies, but a central role of the JAK/STAT pathway activation by oncogenic CSF3R membrane proximal point mutations has been anticipated. However, the exact downstream signaling pathways mediated by CSF3R mutations are still not completely understood. Recent studies in JAK mutated myeloproliferative diseases uncovered the vital importance of JAK/STAT signaling in PD-L1 mediated immune escape, thereby suggesting a possible role and mechanistic link for PD-1/PD-L1 immune checkpoint regulation in CNL and aCML.To investigate the dependence of the oncogenic CSF3R signaling cascade on an activated JAK/STAT axis, we performed Ruxolitinib treatment in MTT assays with BaF3 cells retrovirally infected with pMIG-R1 vectors harboring either CSF3R point or truncation mutations. Our data indicate, that both, CSF3R point and truncation mutations induced cell growth could be inhibited by Ruxolitinib treatment. To narrow down the involvement of the different JAK family kinases, we included CSF3R mutation transduced JAK1 deficient U4C, JAK2 deficient y2A and TYK2 deficient U1A cell lines in combination with different JAK inhibitors. Interestingly, here we were able to show that CSF3R point mutations require functional JAK1/TYK2-STAT3 signaling for oncogenic potential, whereas truncation mutations depend on all JAK kinases through STAT3/5 for activation.To further study potential therapeutic strategies, we examined checkpoint inhibition in CSF3R mutated disease. In this regard we demonstrate a significant induction of PD-L1 expression in murine cell lines upon expression of CSF3R point mutations T615A or T618I. To investigate the impact of PD-L1 upregulation in vivo, we used a retroviral murine bone marrow (BM) transplantation model where we infected BM of Balb/c mice with a vector carrying CSF3R T618I point mutation and transplanted it into lethally irradiated recipients. Transplanted mice developed a CSF3R driven leukemia and died by leukocytosis and organ infiltration, whereas mice transplanted with BM harboring CSF3RT618I and additional ADA mutation failed to induce leukemia. To test checkpoint inhibition as a potential therapeutic strategy in vivo, CSF3RT618I transplanted mice were treated regularly with anti-PD-L1-antibody or isotype control. Interestingly, checkpoint inhibition led to reduced EGFP burden and prolonged survival of responder mice suggesting a treatment response of immunooncologic agents in these rare diseases.In immunophenotypic analyses of primary patient material, we were able to show increased PD-L1 expression in primary diseased myeloid cells isolated from peripheral blood of CNL and aCML patients, thereby defining this structure as a possible new therapeutic target.To summarize, our data expand the knowledge of oncogenic CSF3R signaling pathways and implicate an immunotherapeutic strategy by checkpoint inhibition in CSF3R driven CNL and atypical CML. DisclosuresLubbert:Teva: Other: Study drug; Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding.

CSF3R Mutations are frequently associated with abnormalities of RUNX1, CBFB, CEBPA, and NPM1 genes in acute myeloid leukemia

Mutations in the colony-stimulating factor 3 receptor (CSF3R) gene occur frequently in chronic neutrophilic leukemia and are rare in de novo acute leukemia. The objective of this study was to assess the incidence of CSF3R mutations in acute leukemia and their association with other genetic abnormalities. Amplicon-targeted, next-generation sequencing of 58 genes was performed retrospectively on 1152 patients (acute myeloid leukemia [AML], n = 587; acute lymphoid leukemia [ALL], n = 565). Reverse transcriptase-polymerase chain reaction analysis was used to detect 35 leukemia-specific gene fusions. CSF3R mutations (26 patients) were detected in 3.6% (13 of 364 patients), 4.6% (8 of 175 patients), and 8.3% (4 of 48 patients) of those with de novo, relapsed, and secondary AML, respectively, and in 0.2% (1 of 565 patients) of those with ALL. In total, 9 distinct CSF3R mutations were detected. Membrane-proximal missense mutations and cytoplasmic truncations were identified as mutually exclusive. The proportion of patients who had French-American-British subtypes M2 and M4 in the CSF3R-mutated group was significantly greater than that in the CSF3R wild-type group for both the de novo AML cohort (P = .001) and the relapsed AML cohort (P = .024). All de novo and relapsed AMLs with CSF3R mutations were associated with genetic alterations in transcription factors, including RUNX1-RUNX1T1, CBFB-MYH11, double-mutated CCAAT/enhancer binding protein α (CEBPAdm), and NPM1 mutations; and core-binding factor gene abnormalities and CEBPAdm accounted for 90.5% (19 of 21 patients). CSF3R mutations are uncommon in AML; however, when they occur, they are often associated with core-binding factor gene abnormalities and CEBPAdm. An in-depth understanding of the interaction between these genetic alterations could facilitate a clearer understanding of the role of CSF3R mutations in AML development and may be used for disease classification, prognosis, and the development of targeted therapy.

CSF3R Mutations Are Predominantly Subclonal Events in Intermediate Risk Karyotype AML and Prevalently Occur with CEBPA Mutations

Background: The colony stimulating factor 3 receptor(CSF3R) plays an important role in granulocyte differentiation and acquired mutations have been described in different myeloid malignancies. Besides chronic neutrophilic leukemia (CNL) and severe congenital neutropenia (SCN), mutations in CSF3R (CSF3Rmut) have also been reported in acute myeloid leukemia (AML) at low frequency and recently have been associated with CEBPA mutations (Lavallèe et al., Blood 2016; Maxson et al., Blood 2016) in adult and pediatric AML. First studies suggest sensitivity of CSF3Rmut CNL against JAK kinase inhibitors suggesting the possibility for targeted therapy for a subset of CSF3Rmut AML patients.Aims: The evaluation of CSF3Rmut in AML with intermediate-risk karyotype for frequency and association with other mutations with special focus on CEBPA mutation status.Methods: We analyzed 274 cases with intermediate risk de novo AML for CSF3Rmut by next generation sequencing (Illumina, San Diego, CA) of exon 14 and 17 (sensitivity: 1%), which contain the mutational hotspot regions of CSF3R. As CSF3R mutations were associated with CEBPA mutations in the above-mentioned studies, we analyzed a selected cohort of 179 CEBPA mutated cases in comparison to 95 cases with CEBPA wild type.202 cases (74%) had normal karyotype (CN-AML) and 72 (26%) had intermediate-risk aberrant cytogenetics according to MRC criteria. Female/male ratio was 127/147 and age ranged from 16- 88 y (median: 64 y). Mutation status of the following genes were available in all cases: ASXL1, CEBPA, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, KMT2A-PTD, NPM1, NRAS, RUNX1 and WT1. All mutations with a variant allele frequency (VAF) < 10% were defined as subclonal. 95 cases had wild type CEBPA (CEBPAwt), 92 cases were CEBPA single mutated (CEBPAsm), 81 cases were CEBPA double mutated (CEBPAdm), 6 cases showed CEBPA mutations withloss of heterozygosity (CEBPA LOH).Results: Overall, in 7/274 patients (3%) CSF3Rmut were detected. This is slightly higher than expected in an unselected AML cohort (Sano et al., Br J Haematol 2015). Cytomorphology revealed AML M0 (n=1), AML M1 (n=2), AML M2 (n=3) and AML M4 (n=1). With regard to immunophenotype CSF3Rmut cases did not differ from CSF3Rwt cases which may be due to limited case numbers. All CSF3Rmut cases had CN-AML. The activating transmembrane mutation p.Thr618Ile, which has been identified as common mutation in CNL was the most frequent mutation (4/7). In addition two mutations leading to a truncated receptor (p.Ser715* and p.Gln749*) and a previously not described frame-shift mutation leading to a defecting splice variant (p.Ser783Glnfs*6) were identified. The median CSF3R VAF was 7% (range 2-45%). In 5/7 cases, CSF3Rmut was subclonal (VAF <10%, see figure). CSF3Rmut cases showed a median of 2 concomitant mutations (range 1-4). The most frequent concomitantly mutated gene was CEBPA (5/7 cases; 2 cases CEBPAsm, 1 case CEBPAdm and 2 cases with CEBPA LOH), followed by NRAS (3/7), ASXL1 (2/7) and RUNX1 (2/7). IDH1, TET2 and WT1 were mutated in one case each (see figure). Mutations in NPM1, FLT3, KMT2A or GATA2 were never detected. Interestingly, in CEBPAdm and CEBPA LOH cases, CSF3R was the only concomitantly mutated gene, whereas CEBPAsm cases harbored mutations in at least 1 gene in addition to CSF3R. In 2/7 CSF3Rmut cases we analyzed changes in the patterns of genetic lesions between diagnosis and relapse. Case 1 harbored CEBPAdm and a subclonal CSF3Rmut (VAF: 7%) at diagnosis. At relapse the CEBPAdm persisted, whereas CSF3Rmut was lost. Case 2 harbored two germline CEBPAmut and one somatic CEBPAmut at diagnosis. In addition, mutations in CSF3R and WT1 were identified. At relapse both the somatic CEBPAmut and WT1mut persisted, whereas CSF3Rmut was lost. These results indicate again that the CSF3Rmut was present only in a subclone of CEBPAmut AML in both cases.Due to the limited number of CSF3Rmut cases, we did not perform survival analysis in this study.Summary: 1) CSF3R mutations are rare and predominantly subclonal events in AML and therefore may not be a suitable target for directed therapy.2) CSF3Rmut prevalently occur with CEBPA mutations but are not limited to CEBPAdm. [Display omitted] DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.

Colony Stimulating Factor 3 Mutations and Myeloid Malignancies.

Granulocyte colony stimulating factor (G-CSF) plays a major role in the proliferation, differentiation, and activation of neutrophil cell line hematopoietic cells. G-CSF exert the function depending on its binding to colony-stimulating factor 3 receptor (CSF3R), a homo-dimer receptor located on the surface of effector cells. Some recent studies have demonstrated that CSF3R mutations play a significant role in many diseases. Some of the hematopoietic diseases, especially myeloid malignancies (e.g. chronic neutrophilic leukemia) are related to the presence of various CSF3R mutations, which leads to abnormal G-CSF signal pathways. Also, the downstream kinases can be the treatment targets for these diseases. This review summarizes CSF3R mutations, mechanisms of mutations, and their contributions to the myeloid malignancies, with an attempt to further reveal the pathogenesis of myeloid malignancies, inform the diagnosis and clinical treatment of the myeloid malignancies, and provide clues for the research and development of new molecular target drugs.