Chronic Idiopathic Neutropenia Research Articles

Natural history of chronic idiopathic neutropenia of the adult

Chronic idiopathic neutropenia (CIN) is a rare benign condition caused by an immune attack against neutrophils, either primary or in the context of other autoimmune conditions, lymphoproliferative syndromes, and inborn errors of immunity. In this single-center prospective study, 131 adult CIN patients were enrolled (median age 55 years, range: 20–93). At baseline, 56% had anti-neutrophil autoantibodies and 31% had autoimmune comorbidities. Over a median 3-year follow-up, the rate of grade ≥ 2 infections was 42%, with 10% grade ≥ 3, irrespective of neutrophil counts, demographics, and anti-neutrophil antibodies positivity, and G-CSF was used in 6 patients only. No malignant evolution nor deaths were observed. Bone marrow evaluation showed a large granular lymphocyte (LGL) infiltrate in 52%, mostly polyclonal, and hypocellularity in 31% of cases. Immune-histochemistry highlighted deposits of IgG, IgM, and complement fractions C3 and C4d in most cases. Interestingly, 19% of tested patients displayed somatic mutations of myeloid genes with an association with age. In conclusion, adult CIN appears to be a benign condition without life-threatening infections, yet deserving an extensive hematologic evaluation including bone marrow assessment to inform the differential diagnosis.

Characterization of myeloid-derived suppressor cells in the peripheral blood and bone marrow of patients with chronic idiopathic neutropenia.

Characterization of myeloid-derived suppressor cells in the peripheral blood and bone marrow of patients with chronic idiopathic neutropenia.

Causes of acquired isolated neutropenia in adulthood: a single center study

Aims: Neutropenia, associated with several hereditary and acquired causes, is detected in a significant portion of patients presenting to the hematology outpatient clinic today. We conducted this study since we failed to find any Turkish publication on acquired isolated neutropenia in adults. Methods: This retrospective study examined data belonging to patients aged 18 years and older who applied to the adult hematology outpatient clinic whose absolute neutrophil count (ANC) was below the normal reference values (? 1.5 x 109/L). Results: 103 adult patients, 93 women and 10 men, were included in the study. Our study detected the cause of acquired isolated neutropenia as chronic idiopathic neutropenia (CINA) in 39.8% (n=41) of patients, autoimmune diseases in 18.4% (n=19), drugs in 14.6% (n=15), folate deficiency in 13.6% (n=14), infection in 9.7% (n=10), and hematological malignancy in 2.9% (n=3) and cyclic neutropenia in 1% (n=1). 92 patients (89.3%) were found to display mild neutropenia while 9 (8.7%) displayed moderate neutropenia and 2 (2%) displayed severe neutropenia. Interestingly, folate deficiency was detected in 6 of 9 (66.7%) moderately neutropenic patients. Conclusion: In our study, CINA was determined as the most common cause of acquired isolated neutropenia in adults, while autoimmune neutropenia was the second most common. In addition, the detection of folate deficiency in 6 of 9 patients (66.7%) with moderate neutropenia and the fact that these patients were in the advanced age group [mean 68 (range 62-81)] was a major finding of the study.

Heterogeneous Genetic Landscape of Chronic Idiopathic Neutropenia Revealed By Whole Exome Sequencing

Background: Adultpatients with persistent, unexplained neutropenia who do not fulfill the diagnostic criteria of any underlying disease are characterized as chronic idiopathic neutropenia (CIN) cases.It is an exclusion diagnosis that can be established after a thorough clinical/laboratory investigation for any underlying causes, including negative anti-neutrophil antibody testing, inconclusive bone marrow (BM) aspiration/biopsy and normal cytogenetics. CIN patients regardless of their absolute neutrophil counts (ANC) usually display a benign and uncomplicated clinical course. Although the pathogenesis of CIN remains largely unknown, we hypothesize that a constitutional/congenital background might exist, at least in some cases, that have potentially escaped diagnosis during childhood. Aim of the study: To investigate the genetic landscape of neutropenia in CIN by conducting whole exome sequencing (WES) analysis in a number of adult CIN patients. Methods: We have performed WES in 16 adult (median age 59 years, range 30-72 years) patients (11 females and 5 males) with CIN according to previously published criteria (Fioredda F, et al Hemasphere. 2023). The patients had median ANC 1.2 x 10 9/L (range 0.4-1.6 x 10 9/L) for prolonged period and unknown neutropenia onset in most cases. Genomic DNA was extracted from peripheral blood samples, coding fragment libraries were prepared, sequencing performed on Illumina NovaSeq 6000 System, and data subjected to an in-house pipeline for bioinformatic analysis for variant calling and annotations. Singleton analyses were performed focusing on genes associated with congenital defects of phagocytes, BM failure (BMF) and immune deficiency and dysregulation. Mean depth of coverage was 70x and the mean number of variants/sample was 22553. Results: WES analysis identified 10 pathogenic/likely pathogenic (P/LP) variants in 10 patients. The variants consisted of 4 frameshift mutations, 3 nonsense mutations, 1 missense mutation, 1 splice-site mutation and 1 splice-region mutation. Pathogenic variants in congenital neutropenia (CN)-related genes included only a heterozygous mutation in G6PC3:c.511C>T,p.Gln171Ter (previously detected in one of our CN patients; Nikolouzakis TK et al, Ann Hematol. 2022). Pathogenic variants in genes associated with BMF were detected in two genes, namely FANCM:c.1492C>T, p.Gln498Ter and CTC1:c.3058C>T,p.Gln1020Ter, respectively. The majority of the pathogenic variants were found in genes associated with immune deficiency and dysregulation. These involved: (i) a heterozygous mutation in DCLRE1C:c.1784dup,p.Ile596fs (associated with immunodeficiencies affecting cellular and humoral immunity); (ii) a LP mutation in ORAI1:c.132_133del,p. Arg45fs and a pathogenic variant in SPINK5:c.1302+4A>T (related to combined immunodeficiencies with associated or syndromic features); (iii) a pathogenic variant in MEFV:c.2080A>G, p.Met694Val and a LP variant in PSMG2:c.703-2A>T (both genes associated with autoinflammatory disorders); (iv) a LP variant in IRF7:c.1089dup,p.Pro364Alafs (associated with defects in intrinsic and innate immunity); and (v) a LP variant in PEPD:c.738_739insGT,p.Ser247fs (associated with immune dysregulation). Regarding variants of undetermined significance (VUS) an average of 4.5 VUS variants per patient were detected in genes related to CN ( HAX1, VPS13B, LYST), BMF ( GATA2, SAMD9, CXCR4) or immunodeficiency ( TCF3). However, none of these patients display clinical symptoms typical of these conditions. Specifically, the majority of the patients presented with mild/moderate neutropenia and rarely with severe neutropenia, they have relatively normal BM biopsies and do not display major symptoms or signs related to severe immunodeficiency or immune dysregulation syndromes. Conclusions: This study is the first attempt of WES analysis in adult CIN patients and shows that there is a heterogeneous genetic landscape underlying the disease. More studies are required so that the detected P/LP variants and VUS can be associated with the clinical phenotypes to more accurately interpret their potential contribution to the neutropenia phenotype.

Severe Chronic Idiopathic Neutropenia Managed with Chronic Filgrastim: A Case Report

Myelodysplastic syndrome (MDS) represents a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis and abnormal blood cell production. Chronic neutropenia is one such MDS and is classified into mild (Absolute Neutrophil Count (ANC): 1,000 to 1,500/µL), moderate (ANC: 500 to 1,000/µL), or severe (ANC: below 500/µL), with the latter posing the highest risk of infection-related complications. In an attempt to alleviate neutropenia, patients with MDS are often treated with colony stimulating factors (CSFs), such as filgrastim to promote growth and maturation of neutrophils. The target ANC is 1,500 to 2,000/µL for adequate protection against most infections. However, CSF treatments have the potential to stimulate the transformation of MDS into acute myeloid leukemia (AML) so CSF is often used with caution. Presented below is a case of severe chronic idiopathic neutropenia responsive to chronic filgrastim that has yet to show evidence of transformation. A 79 year-old female with a past medical history of distant tobacco use, hypertension, acute cerebrovascular accident, and Hashimoto's thyroiditis presents to the clinic for routine follow-up of her now stable chronic idiopathic neutropenia being managed with weekly filgrastim injections for three weeks with one week off in between cycles. She had originally presented to medical attention in 2004 complaining of chronic mouth sores and ulcers with leukopenia and a severely decreased ANC of <500/µL. Continued monitoring with more than 3 complete blood counts (CBC) with a differential consistently noted a decreased ANC in the setting of persisting oral mucosal erosion. Leukocytes were not noted to be dysplastic, quantitative IgG, IgA, IgM levels were noted to be within normal limits, anti-nuclear antibodies/ anti-neutrophil antibodies were noted to be negative argued against an etiology of large granular lymphocytic leukemia, immunodeficiency, and/or autoimmune disorders, respectively. A trial of glucocorticoids did not significantly increase her ANC which led to her ultimately receiving a bone marrow biopsy (BMB) in 2008. A mildly hypercellular marrow with trilineage hematopoiesis was found, with maturation of all lines, negative flow cytometry, and myelodysplasia. Comforted by the lack of MDS pathologically, the patient was started on filgrastim with significant improvement. Over the course of years the frequency and duration of filgrastim was optimized to her current regimen of weekly filgrastim injections for 3 weeks followed by one week off which allowed her to maintain an adequate ANC and avoid oral ulcers to this day. Despite the feared complication of kickstarting the transformation of MDS into AML with CSF therapy, patients such as the one mentioned here can benefit greatly from judicious use of CSF when blood work can be monitored for complications. The use of G-CSF in severe chronic neutropenia to raise levels from below 500/µL to the ideal target ANC is 1,500 to 2,000 cells per microliter (µL) for adequate protection is in line with our current understanding that even long-term use of G-CSF does not appear to increase the risk of AML (Dale et al., 2022). G-CSF should continue to be considered in the management of severe chronic neutropenia as the risk for transformation may be insignificant compared to the clinical benefit of its use when properly monitored.

A Single-Center Large Cohort of Chronic Neutropenia Patients and a Model for Estimation of Congenital Neutropenias

Chronic neutropenia (CrN) is defined as neutropenia lasting longer than 3 months and has various underlying etiologies, including congenital neutropenia (CN). We aimed to determine the underlying etiologies of patients with CrN and define the characteristics at diagnosis suggestive for CN as a final diagnosis. The study included 197 pediatric and adolescent patients who were diagnosed with CrN between 2010 and 2022 in a single-center. Patients with transient neutropenia, splenomegaly, alloimmune, secondary autoimmune, myelodysplastic syndrome and hematological malignancies, other bone marrow failure syndromes such as Fanconi anemia, and aplastic anemia due to CrN were excluded from the study. The patients included in the study were sub-classified into 5 groups according to their final diagnosis. Group 1, patients with CN (n=64, %32.5) included all genetically verified CrN patients. Group 2, patients with primary autoimmune neutropenia (pIN) group (n=10, %5.1) with anti-granulocyte antibody (AGA) positivity. Group 3, patients with unclassified chronic idiopathic neutropenia (UCIN) (n=73, %37.1). Group 3 included patients who had a clinical severity resembling immune neutropenia, but whose AGA testing was either negative or not available; but the CrN resolved during follow-up. Group 4, included the patients with unclassified chronic benign neutropenia (UCBN) (n=34, %17.3) and included the patients with ongoing neutropenia, who do not have a history of serious infections, no underlying genetic cause for neutropenia, no AGA positivity. Group 5, unspecified congenital neutropenia (UCN) group (n=16, %8,1), was defined as the group of patients whose genetic tests did not detect a mutation in genes related to CN, but history reveales severe infections and a possible CN as an underlying cause. Genetic testing of patients varies from single gene evaluation with Sanger sequencing analysis to Whole Exome Sequencing (WES). The median age at diagnosis of neutropenia of the patients included in the study was 12 (0-168) months 53.8% of the patients were male (n=106). There was consanguinity between parents in 34.5% and at least 1 family member was diagnosed with neutropenia in the in 18.8%. Among the patients in Group 1, HAX-1 mutation was detected in 19 (29.7%) patients and it was found to be the most common genetic defect causing CN. Other common genetic disorders include ELANE mutations (18.8%), Shwachman Diamond Syndrome (10.9%), Hermansky Pudlak Syndrome (7.8%) and glycogen storage type 1 b (6.3%). G6PC3 deficiency, VPS45 deficiency, Barth syndrome and Kabuki syndrome in 2 each (3.1%), ADA2 deficiency, Cohen Syndrome, Poikiloderma with neutropenia, Prolidase deficiency, CLPB deficiency, Majeed syndrome, GINS4 deficiency, reticular dysgenesis, germline RUNX1 mutation were detected in 1 patient. There are 3 patients with acute myeloid leukemia and all of them died before hematopoietic stem cell transplantation (HSCT). Two of these patients were diagnosed with SDS, and one had ELANE mutation. One patient with the HAX-1 mutation developed non-Hodgkin lymphoma and was treated with HSCT and is still alive. There were 7 patients (3.6%) who underwent HSCT. 9 patients (4.6%) died. Except for 3 patients who died from AML and 1 who died after HSCT; 1 patient with Barth syndrome deceased related to associated dilated cardiomyopathy, Another patient with G6PC3 mutation died after complications of cardiac surgery due to associated cardiac anomaly and 3 patients (2 VPS45, 1 CLPB) died due to infections. Patients in Group1 and Group 2+Group3 were compared, in order to answer the question of “Can we create a model that predicts CN?”. Many parameters were compared and a multivariate model was created with statistically significant risk factors. CN was seen 6,3 times more in those with parental consanguinity, 8,9 times in those with a family history of neutropenia, 4,8 times in those with more than 2 hospitalizations due to infection, and 4,1 times more in those with recurrent oral involvement (p<0.05). Patients diagnosed with CN were estimated based on the results of the multifactor logistic regression model. The rate of correct identification of the patients by the model is 88.9%. In conclusion, our study is one of the largest single-center studies and suggests a novel classification of CrN patients.

Mavorixafor for Patients with Chronic Neutropenic Disorders Treated with G-CSF: Preliminary Response Data and G-CSF Dose Reduction in an Ongoing Phase 2, Open-Label, Multicenter Study Support Reduction in G-CSF Dosing

Introduction: Chronic neutropenia (CN) encompasses multiple disorders characterized by an absolute neutrophil count (ANC) <1500 cells/µL for >3 months, leading to increased susceptibility to recurrent and/or severe infections. CN can be sub-classified as congenital neutropenia defined by genetic variants ( ELANE variants are the most common), cyclic neutropenia (CyN) supported by documentation of ANC cycling with reciprocal monocytosis most often associated with certain ELANE variants, or chronic idiopathic neutropenia (CIN) defined as acquired neutropenia without an identifiable cause. The current treatment approach for CN involves long-term use of injectable granulocyte colony-stimulating factor (G-CSF), which can have associated adverse events (AEs) [Donadieu J. Expert Rev Hematol. 2021; Fioredda F. Hemasphere. 2023; Newburger PE. Semin Hematol. 2013]. Therefore, efforts are under way to discover other therapies for CN. In a phase 3 trial (NCT03995108), mavorixafor, an investigational oral CXCR4 antagonist, showed durable increases in ANC and reduction in total infection rate and severity in people with WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome [Badolato R. Clin Immunol. 2023]. Mavorixafor is being studied in a phase 2 trial for chronic neutropenic disorders (NCT04154488) [Warren JT. Blood. 2022] to evaluate the durability, safety, and tolerability of chronic dosing of mavorixafor with or without concurrent G-CSF. Here, we describe preliminary results for 3 participants in the ongoing phase 2 trial. Methods: Individuals (aged ≥12 years) with diagnosis of congenital neutropenia, CyN, or CIN ≥6 months prior and ANC <1000/µL (or no lower limit but <10,000/µL if on G-CSF) at screening visits were enrolled in the phase 2 trial. Eligible adult participants received once-daily 400 mg of mavorixafor while adolescents received weight-based dosing (>50 kg, 400mg; ≤50 kg, 200 mg) for 6 months with or without G-CSF. Participants already on G-CSF continued their individualized G-CSF dosing for ≥8 weeks, followed by potential dose and/or frequency reduction based on monthly ANC assessments. Hematologic parameters, including ANC, were assessed over 8 hours on day 1 and at months 1 and 3, with continued assessment at month 6. At months 2 and 4 (assessment to continue at month 5), hematologic parameters were assessed within 2 to 4 hours of dosing to reduce trial burden. Results: Data are reported as of July 17, 2023. Preliminary data from the first 3 participants on G-CSF receiving chronic once-daily oral mavorixafor for at least 2 months showed increases in mean ANC compared with baseline (BL) at all time points assessed in all participants. ANCs reached normal ranges for all participants. Increases in ANC supported physicians' decisions to reduce G-CSF dosing. Two participants (P1 and P2) with CIN achieved increases in ANC that enabled initiation of G-CSF tapering at month 2. For P1, G-CSF dose was tapered to 50% at month 2, and further G-CSF tapering occurred at month 3. ANC remained within normal ranges during this period. At month 4, G-CSF dosing was fully withdrawn, and the participant continued daily mavorixafor monotherapy. For P2, ANC increased ~3x baseline by month 2. G-CSF dose was tapered to 50% after month 2. ANC remained within normal ranges during this period. At month 3, G-CSF dosing was fully withdrawn, and the participant continued daily mavorixafor monotherapy with an ANC ~1000 cells/µL. A third participant (P3) with CyN receiving mavorixafor daily with concurrent G-CSF showed increases in ANC over BL for 4 months. All 3 participants remain on study, and have not experienced any serious AEs Conclusions: Our report shows that chronic treatment with oral, once-daily mavorixafor in combination with G-CSF resulted in durable increases in ANC compared with BL for ≥3 months in 3 study participants. Reduction of injectable G-CSF dosing was implemented in 2 study participants with CIN following robust ANC increase observed after the first 2 months of combination treatment. These preliminary data also support the safety of concomitant treatment with G-CSF and mavorixafor. Additional data from the ongoing phase 2 study will further elucidate the potential of daily, oral mavorixafor as a treatment for CN to reduce the burden and associated AEs of injectable G-CSF. A phase 3 trial of people with CN is being planned.

Dynamin-2 deficiency causes age- and sex-dependent neutropenia and myelodysplasia in mice.

The dynamins are a family of ubiquitously expressed GTPase proteins, best known for their role in membrane remodeling. Their contribution to hematopoiesis is incompletely recognized. Individuals with Charcot-Marie-Tooth disease with dynamin-2 (DNM2) mutations often develop neutropenia. We previously reported that dynamin (DNM) inhibition impairs SDF1a-mediated migration in megakaryocytes. Here, we report on conditionally Dnm2 deleted mice in hematopoietic tissues using the Vav-Cre murine strain. Homozygous Dnm2 deletion in blood tissues is embryonic lethal. Dnm2het male mice only developed a slightly decreased hemoglobin level. Dnm2het female mice developed leukopenia by 40 weeks of age and neutropenia by 65 weeks of age. Flow cytometry revealed decreased lineage-negative cells and granulocyte-monocyte progenitors in Dnm2het female mice. Immunohistochemical staining of bone marrow (BM) for mature neutrophils with Ly6G was decreased and myelodysplastic features were present in the BM of Dnm2het female mice. A linear distribution of Ly6G+ BM cells along blood vessels was observed in fewer Dnm2het mice than in controls, suggesting that the migration pattern in the marrow is altered. Marrow neutrophils treated with dynamin inhibitor, dynasore, showed increased cell surface CXCR4, suggesting that abnormal migration results in marrow neutrophil retention. Dnm2het female mice also developed splenomegaly secondary to germinal center hyperplasia at younger ages, suggesting perturbed immunity. In summary, female mice with BM Dnm2 haploinsufficiency developed neutropenia as they aged with decreased granulocyte progenitor production and migration defects. Our studies indicate a potential mechanism for the development of chronic idiopathic neutropenia, a disease that predominantly presents in middle-aged women.

Prevalence of Chronic Neutropenic Disorders in the United States: A Retrospective Analysis of a Large Claims Database

Introduction Chronic neutropenia encompasses different blood disorders characterized by low levels of peripheral blood neutrophils, with absolute neutrophil count ≤1500/uL for >3 months [Donadieu J. Exp RevHematol. 2021]. The most common chronic neutropenic disorders (CNDs) are congenital, resulting from genetic variants; cyclic, consisting of recurrent neutropenia with episodes repeating every 3 weeks; and chronic idiopathic neutropenia (CIN), which is characterized by persistent neutropenia of unknown underlying cause and may overlap with autoimmune neutropenia [Skokowa J. Nat Rev Dis Primers. 2018; Donadieu J. Exp Rev Hematol. 2021; Dale DC. Curr Opin Hematol. 2017]. Moderate and severe CND are associated with increased risk of recurrent and/or severe infections and significant deterioration of quality of life in affected individuals [James RM. Arch Dis Child. 2006; Dale DC. Curr Opin Hematol 2017; Territo M. Merck Manual; Professional Version; 2021; Donadieu J. Orphanet J Rare Dis. 2011]. There are limited data available for prevalence of CND in the US. This study aimed to examine the prevalence of CND in the US using retrospective analysis of a large US claims database. Methods This is a retrospective analysis of CND prevalence based on US medical claims data for people diagnosed with neutropenia during the calendar years 2018, 2019, and 2021. The analysis used longitudinal prescription data (LRx) and office-based claims data (Dx), with a 2-year lookback period for each individual. The year 2020 was excluded from this analysis owing to anticipated reduced claims during the coronavirus 2019 pandemic. People diagnosed with congenital, cyclic, and CIN were identified using the earliest relevant diagnosis claim based on International Classification of Disease 10th Edition (ICD-10) codes in the calendar year of interest as index date. A 13- to 24-month lookback period prior to index date was used to confirm chronic status. For people who had multiple ICD-10 codes, a hierarchical order (cyclic>congenital>CIN) was established to avoid double counting. People with neutropenia resulting from secondary causes including chemotherapy, drug exposure, solid organ transplantation, myelodysplastic syndrome, infection, and end-stage renal disease within 24-month period prior to selection were excluded. Demographic data including age and sex were captured for all individuals based on diagnostic claims. Results Of the projected number of people with CND identified during the years 2018, 2019, and 2021, the prevalence of chronic congenital (D70.0), cyclic neutropenia (D70.4), and CIN (D70.8, D70.9) was 1787, 4991, and 33,965, respectively, in 2018 (N=40,743); 2081, 5426, and 39,257, respectively, in 2019 (N=46,764); and 2102, 4839, and 40,636, respectively, in 2021 (N=47,576) (Table). People with CND were more likely to be female (65%), be aged ≥18 years (91%), and have been seen by a hematologist (36%), as compared to other specialties. Among people aged <12 years, a greater proportion was diagnosed with congenital neutropenia (13%-20%) compared with cyclic neutropenia (5%-8%) or CIN (5%-6%) (Table). Conclusions This analysis demonstrates a prevalence of CND in the United States approaching 50,000 people. The most common type of CND is CIN, followed by cyclic, and the majority of affected patients are adults. Further research is needed to characterize the number and severity of infections and overall quality of life in people diagnosed with CND in the United States. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigation of Neutrophil Extracellular Trap (NET) Induction in Patients with Chronic Idiopathic Neutropenia Bearing Mutations in the Mediterranean Fever (MEFV) Gene

Introduction: Chronic idiopathic neutropenia (CIN) is a disorder characterized by the prolonged and unexplained reduction in the number of circulating neutrophils. The pathophysiology of CIN has been associated with an inflammatory bone marrow microenvironment consisting of activated T-cells with an oligoclonal/monoclonal profile and intermediate CD14bright/CD16+ monocytes that induce the apoptotic death of the granulocytic progenitor cells. To probe further the basis of immune cell activation in CIN we have recently investigated by next generation sequencing (NGS) the frequency of mutations in the MEFV gene encoding pyrin, in a cohort of 50 CIN patients without any signs/symptoms of Familial Mediterranean Fever (FMF). We have found that 9/50 patients displayed variants associated with typical or atypical FMF phenotype in the Greek population implicating exon 10 and/or exon 2 (Skendros P et al. Blood, 138, Sup.1, p3124;2021). Aim:MEFV mutations have been associated with increased neutrophil activation in FMF and other inflammatory diseases, neutrophil entry in autophagy and extrusion of Neutrophil Extracellular Traps (NETs) decorated with the pro-inflammatory cytokine IL-1β. The aim of the current study is to investigate the association between MEFV mutations and NET formation in neutrophils of CIN patients. Patients-Methods: We studied 20 CIN patients (17 females and 3 males) aged 27 to 78 years. Six CIN patients harbored at least one MEFV mutation with potential clinical significance (A744S homo/R202Q homo, I720M hetero, R202Q homo, K965R hetero, A744S hetero, M694V hetero) and 14 were negative for MEFV pathogenic mutations. The MEFV R202Q mutation is located in exon 2 whereas all the rest mutations are located in exon 10. We isolated neutrophils from healthy subjects (n=5), age- and sex- matched with the patients, and co-cultured them with serum from CIN patients. We used the supernatants of the cultures to quantify NETs with Sytox green staining for extracellular DNA and/or ELISA for dsDNA/MPO complexes. We also used the serum-treated neutrophils for the evaluation of NETosis by immunostaining using confocal microscopy. In addition, neutrophils isolated from CIN patients were used to detect their direct response to spontaneous NETosis induction. Statistical analysis was performed with one-way ANOVA adjusting for multiple comparisons. Results: By co-culturing patients' sera with healthy neutrophils obtained from five different healthy donors, we did not identify statistically significant differences in NETosis evaluation between the three study groups i.e. CIN patients with MEFV mutations, CIN patients negative for pathogenic MEFV mutations, and healthy individuals. On an individual basis, the patient harboring the double homozygous MEFV mutations (A744S/R202Q) displayed increased NETosis in immunostaining, Sytox and ELISA evaluation, as compared to MEFV unmutated CIN patients and healthy individuals. In addition, ex vivo culture of patients' neutrophils and microscopic observation gave us the indication that CIN patients, regardless of their MEFV gene variants, present increased NETosis compared to healthy individuals, suggesting an overactivation status of neutrophils in CIN. Conclusions: The neutrophils of CIN patients display increased NETosis irrespectively of the MEFV mutation status and may thus contribute to the inflammatory processes associated with the disease, a field that has not been investigated so far. Although in our study MEFV mutations did not appear to contribute significantly to inflammatory processes through the NETosis mechanism in CIN patients, their involvement in other aspects of immune deregulation such as through the mTOR/autophagy pathway remains to be further explored.

Mavorixafor for Patients with Chronic Neutropenic Disorders: Results from a Phase 1b, Open-Label, Multicenter Study

Introduction Chronic neutropenia (CN) encompasses different blood disorders all characterized by low levels of neutrophils (absolute neutrophil count [ANC] <1500/µl for >3 months) and is associated with increased risk of recurrent and/or severe infections [Donadieu J. Exp RevHematol. 2021]. The most common chronic neutropenic disorders (CNDs) are chronic idiopathic neutropenia (CIN), which is not attributable to drugs or a specific infection or inflammatory, malignant, or genetic cause [Dale DC. Curr Opin Hematol. 2017]; cyclic neutropenia (CYN), characterized by recurrent episodes of neutropenia approximately every 21 days [Donadieu J. Exp Rev Hematol. 2021]; and severe congenital neutropenia (SCN), caused by genetic variants [Skokowa J. Nat Rev Dis Primers. 2018]. The current management strategy for CN is long-term treatment with injectable granulocyte colony-stimulating factor (G-CSF). Although typically effective in the short-term, G-CSF comes with many adverse effects such as bone pain and, especially for patients with SCN, an increased risk for hematologic malignancy [Newburger PE. Semin Hematol. 2013; Dale DC. Blood Advances. 2022]. Mavorixafor is an investigational oral antagonist of CXCR4 in clinical development for treatment of patients with multiple CNDs, including Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome [Dale DC. Blood. 2020]. Herein we report results of phase 1b, open-label, multicenter trial (NCT04154488) evaluating the safety, tolerability, and serving as a proof of concept for efficacy, of mavorixafor alone or with concurrent G-CSF use across several CNDs. Methods Patients aged ≥12 years with diagnosis of CIN, congenital neutropenia, or CYN ≥6 months prior and ANC <1000/µL at screening visits were included. Patients on G-CSF with ANC >1000/µL were also included. All patients with congenital neutropenia without documented genetic variants underwent genetic screening via a 642-gene comprehensive immune and cytopenia panel. Patients received ≥1 dose of mavorixafor (>50 kg, 400 mg; ≤50 kg, 200 mg), regardless of current treatment with or without G-CSF. Patients were monitored for hematologic parameters such as total white blood cell (WBC) and subset counts, including ANC, absolute lymphocyte count (ALC), and absolute monocyte count (AMC), over the course of 6 to 8 hours 1 day before receiving mavorixafor (day -1) and on the first day of mavorixafor treatment (day 1) and were followed for 30 days for safety and tolerability. Fold change in total peripheral WBC count, ANC, ALC, and AMC from day -1 levels was assessed. Results As of June 2022, 15 patients (CIN, n=11; congenital neutropenia, n=3; CYN, n=1) were enrolled and dosed. The mean (SD) age in years was 39.7 (17.2); 40% were male, and 60% were on G-CSF. All patients received ≥1 dose of mavorixafor once daily. The mean (SD) maximum fold change in ANC from day -1 to day 1 across all included patients was 4.72 (2.74) at peak over the course of 6 to 8 hours after dose 1. Table 1 shows fold change in ANC for individual participants, and Figure 1, by type of neutropenia. The mean (SD) maximum fold change in other hematologic parameters, including total WBC count, ALC, and AMC, from day -1 to day 1 was 3.14 (0.65), 3.05 (0.62), and 2.64 (0.80), respectively. For the subgroup of patients on G-CSF at day -1, the mean (SD) maximum fold change in total WBC count, ANC, ALC, and AMC from day -1 to day 1 was 2.87 (0.58), 3.67 (1.29), 3.07 (0.73), and 2.44 (0.59), respectively. For patients not on G-CSF, the mean (SD) maximum fold change in total WBC count, ANC, ALC, and AMC from day -1 to day 1 was 3.54 (0.52), 6.28 (3.45), 3.01 (0.47), and 2.95 (0.95), respectively. Treatment-emergent adverse events (AEs; n=11) were all grade 1; no serious AEs were reported. Conclusions Meaningful increases in ANC and all other hematologic parameters evaluated were observed on day 1 in all patients treated with ≥1 dose of mavorixafor. Responses were observed regardless of concurrent G-CSF use or type of CN. Mavorixafor treatment was well tolerated overall, with only mild AEs reported. This study supports the potential therapeutic use of mavorixafor in the treatment of CNDs beyond WHIM syndrome and underscores the possibility for mavorixafor to become the first oral treatment for CN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessment of the Frequency of Variants of Undetermined Significance (VUS) in Adult Patients with Chronic Idiopathic Neutropenia Studied for Myeloid Gene Mutations By Next Generation Sequencing

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnosis of CIN is based on exclusion criteria, namely the absence of clinical/laboratory evidence of any underlying disease or use of drugs that might be associated with neutropenia, absence of history of exposure to irradiation or use of chemical compounds, negative antineutrophil antibodies, and normal bone marrow (BM) morphology and karyotype. We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients and we found that 11.35% of the patients display clonal hematopoiesis in term of presence of pathogenic/likely pathogenic variants with VAF higher than 2%. Variants of undetermined significance (VUS) were ruled out from the analysis and their potential significance was not assessed. Aims: In the current study we have assessed the presence, frequency and potential clinical significance of VUS detected in our cohort of CIN patients previously studied for clonal hematopoiesis. Methods: Genomic DNA was extracted from CIN patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies (Tsaknakis G et al. Blood. 2021;138:1249-1257). Results: NGS analysis of myeloid genes has been performed in 139 CIN patients (106 female subjects and 33 male subjects, aged 21 to 89 years with a median age of 59 years). Re-analysis of patients' NGS data and assessment of VUS revealed that 35 out of 139 patients (25.18%) carried one or more VUS (a total of 39 VUS were detected, all with an allelic frequency of 50% and all missense variants). Genetic variants were evaluated according to the ACMG guidelines and classified as VUS. The majority of VUSs were observed in ATM (n=7) and SETBP1 (n=6) genes followed by TET2, NOTCH1 (4 occurrences respectively) and PLCG2 (n=3). Two different patients shared the same variant in TET2 gene whereas two siblings shared the same variant in SETBP1 gene. Pathogenicity score calculation based on the combined evidence from multiple other in-silico predictors identified 3 variants in 3 different genes as having the highest probability to be pathogenic. Namely these were PLCG2K456E,CXCR4R235C and SF3B1T56I. PLCG2 variants have been implicated in familial cold autoinflammatory syndrome 3 (PLCG2 associated antibody deficiency and immune dysregulation, PLAID) and in autoinflammation, antibody deficiency, and immune dysregulation syndrome (APLAID) whereas CXCR4 variants are known to be associated with Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. SF3B1 mutations have not yet been shown to be involved in hereditary disease. Conclusions: Our study of VUS assessment in a cohort of 139 well characterized adult CIN patients identified 39 VUS, all heterozygous, in a total of 35 patients. Further steps include paired analysis with constitutional reference tissue to determine the germline nature of the mutations. Moreover, these variants should be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to disease onset. This study of VUS analysis in CIN patients is anticipated to contribute to the better understanding of the pathogenetic causes of this rare disease.

New Approaches to Chronic Idiopathic Neutropenia in the Era of Clonal Hematopoiesis

New Approaches to Chronic Idiopathic Neutropenia in the Era of Clonal Hematopoiesis

Increased Frequency of Mutations in the Gene Responsible for Familial Mediterranean Fever (MEFV) in a Cohort of Patients with Chronic Idiopathic Neutropenia

▪Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients.Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms.Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or “atypical” FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (P<0.0001, Fisher's exact test). NGS analysis confirmed the mutational pattern of NIRCA and specifically showed: (a) one patient with heterozygous I720M (ATC>ATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels.Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. DisclosuresNo relevant conflicts of interest to declare.

Late Onset and Long Lasting Idiopathic and Autoimmune Neutropenia As Epiphenomena of Immune Dysregulation: Preliminary Data Study from the Italian Neutropenia Registry

▪INTRODUCTION: Idiopathic neutropenia (IN) of childhood is a benign, self-limiting disorder usually occurring in the first 3 years of life that differs from primary autoimmune neutropenia (AIN) because anti neutrophil antibodies are not detected on repeated indirect testing over time(1). Chronic idiopathic neutropenia (CIN) is a well characterized disorder of elderly with a peculiar immunological pattern(2). Pediatric AIN patients, with atypical features represented by longer disease duration (Long Lasting) or diagnosed later (Late Onset), were recently shown by our group to display a different immune-hematological profile vs typical primary AIN(3). Atypical pediatric IN subjects, i.e. diagnosed later or with longer disease duration, though occasionally reported, were never systematically investigated. In the present study we analyzed a cohort of young patients with atypical IN and AIN, diagnosed after the age of 3 years or with longer disease duration, aiming to identify an immunological signature that might predict their different outcome from classical AIN and IN of infancy.PURPOSE OF THE STUDY: to analyze a cohort of patients affected with AIN and IN rising > 3 years of age and with duration >12 months or rising <3 years of age but persisting over 36 months.MATERIALS AND METHODS: Clinical, immunological and genetic data (NGS panel of 160 immunodeficiency/disimmunity genes) of eligible patients were collected from the database of the Italian Neutropenia Registry.RESULTS: From 2005 to 2020, data from 46 patients (24F, 24 autoimmune and 22 Idiopathic Neutropenia) were retrieved. Median age at onset was 11.2 years (IQR13.2-16.7), median follow-up was 4.3 years (IQR 3.2-6.8). Neither autoimmunity nor additional cytopenias to neutropenia were present at onset. Cumulative incidence at 5 years of autoimmune manifestations (thyroiditis, arthritis, vitiligo, recurrent skin rash) was 11.8% (CI 95% 4.3-30). Throughout the follow up, infections occurred in 32/46 (70%) subjects and in only 16% were severe (meningitis, recurrent pneumonia, sepsis and pericarditis). Infections sites were upper respiratory (in 84% of subjects mouth and gums (47%), and skin (40%) ear (25%), lung (19%) and urinary tract (12%). Fever of unknown origin was detected in 40% of patients. Recurrent infections (more than 3/y) involved mouth (85%) and ear (88%). White Blood Cell, Absolute Neutrophil and Lymphocyte counts retrieved at the beginning of the study, in the middle and at the end of follow up are shown in Table 1. Leucocytes and Lymphocytes at the end of follow up were significantly lower than values seen at diagnosis (p < 0.001) whereas neutrophil count remained stable (p=ns). One third of the cohort had lower values than normal of CD19+ and CD3-CD56+CD16+ cells. B switched memory CD27+/IgD-/IgM- (in 94%) were lower, while marginal zone B lymphocytes CD27+/IgD+/IgM+ (62%) and Tγδ cells (70%) were increased than the normal population according to a pattern comparable to that described in chronic idiopathic neutropenia (CIN). Immunoglobulin serum levels (IgA, IgG and IgM) were below the normal value in 7.5% of the population, but specularly a small subset (7.5%) of IN showed an increase of IgM was seen, similar to what already descibed in adult CIN. The genetic study carried out in 32 patients showed in 5 (16%) pathogenic variants: of immunodeficit/dysregulation (2 TACI, 1 TINF2, 1 CARD11) and in 9 cases (28%) VUS in genes of the same groups (1CARD11, 4CASP10,1DDX41/SOCS1, 1TSR2/DCLRE1c, 1PIK3D, 1TERT/TACI).CONCLUSIONS: Atypical IN and AIN (of longer duration or occurring in advanced pediatric age) are different from those appearing in early infancy. They seem to display dysimmune/autoimmune features that is some case is proven to rely on a genetic dysimmune background. They might represent an anticipation of more complex autoimmune disorders which may fully manifest later in life. Interstingly these patients seem to share some features with CIN of adults. Definitive conclusions will be drawn by applying more comprehensive genetic nalysis (WES and WGS) on larger group of subjects.

Follow-up Next Generation Sequencing (NGS) Analyses on Clonal Chronic Idiopathic Neutropenia (CIN) Patients: Insights in the Natural History of the Disease

▪Background: We have previously performed NGS analysis of genes that are recurrently mutated in myeloid malignancies in a cohort of patients with the diagnosis of chronic idiopathic neutropenia (CIN) according to previously reported criteria that largely overlap with those proposed for idiopathic cytopenia/neutropenia of undetermined significance (ICUS-N). We have thus estimated for the first time the frequency of clonal hematopoiesis in patients with CIN/ICUS-N (11.54%) and found that clonal CIN patients have a significantly higher risk of developing a myeloid neoplasm than those with no evidence of clonality (non-clonal). 1 However more longitudinal follow-up NGS studies are required for the tracking of clonal evolution and delineation of clonal CIN natural history.Aims: To conduct longitudinal follow-up NGS analyses in order to assess clonal evolution and associate the clinical significance of detected clonal aberrations with the risk of transforming to myeloid malignancy in clonal CIN clinical outcome.Methods: Genomic DNA was extracted from patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted next generation sequencing (NGS) on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies.Results: Follow-up analysis by NGS was performed in 16 clonal CIN patients (Figure 1). (Out of these 16 patients, follow-up NGS data has already been published in 9 patients, however additional timepoints were tested in 3 of them). 1 The median time between the first and subsequent analysis was 28.5 months (range 8-164 months). Ten of these patients carried the initial somatic mutations with only subtle changes in the size of clone as estimated by the variant allele frequency (VAF); the patients displayed absence of additional mutations and did not develop myeloid malignancy (Figure 1A-C, E-G, I, K, L, P). Two patients acquired a second mutation at follow-up. One of them still displayed stable disease course (Figure 1D) whereas the second eventually progressed to CMML (Figure 1H). The analysis also revealed that one patient lost the initial detected mutation at follow-up after 98 months (Figure 1J). Two patients who progressed to MDS/MPN and AML respectively, displayed a notable clonal expansion with additional mutations at the time of progression (Figure 1M and Figure 1N, respectively). Specifically, the patient who progressed to MDS/MPN acquired a mutation in JAK2 and ASXL1 while the patient who progressed to AML acquired the typical NPM1 p.L287fs mutation. The patient who developed MDS with multilineage dysplasia, carrying three mutations in DNMT3A and IDH1, showed a moderate increase in the VAF of these mutations at first follow-up (Figure 1O). The patient progressed to acute lymphoblastic leukemia (2 nd follow-up) with acquisition of additional truncating mutation in ETV6. Following treatment (3 rd and 4 th follow-up) mutation in ETV6 was lost, however the three mutations in DNMT3A and IDH1 persisted and their clone size increased.Conclusions: In the majority of patients tested for clonal evolution over time, most mutant clones appeared to be remarkably stable, with minimal VAF change, no acquisition of new molecular alterations and no progression to overt myeloid malignancy. Two CIN patients who transformed to a myeloid malignancy displayed a clonal expansion as was reflected by the increase of VAF and the development of additional mutations whereas in the third patient only a modest VAF increase was identified before malignant transformation. Finally in the patient bearing 4 mutations no progression to overt malignancy was observed after 12 months of follow-up. This ongoing study of sequential NGS analysis of CIN patients is anticipated to contribute to the better understanding and enrich further the knowledge on the natural history of this rare disease.

Dynamin-2 Deficiency Causes Neutropenia and Dysplastic Bone Marrow Changes in an Age and Sex Dependent Manner in Mice

▪The dynamins are a family of ubiquitously expressed proteins with GTPase activity and are known for their role in membrane remodeling and intracellular trafficking. However, their exact role within various hematopoietic lineages is incompletely understood. In humans, most of the clinical cases with cytopenia in Charcot-Marie-Tooth (CMT) disease due to dynamin-2 mutations are associated with neutropenia, and CMT patients may suffer from impaired wound healing. Of interest, pregnancy notably worsens CMT disease, possibly due to hormonal changes. Antiprogesterone treatment was successfully given in a CMT rat model, and similar treatment is being considered for human CMT. We previously reported that inhibiting dynamin (DNM) activity impairs migration capability in mature megakaryocytes.We obtained the conditional deletion of Dnm2 and targeted its deletion in hematopoietic tissues with the vav-cre murine strain. Homozygous deletion of Dnm2 in blood tissues appears embryonic lethal. None of the pups born showed a Vav-cre/Dnm-2 fl/fl genotype, whereas a third of the pups born had a Vav-cre/Dnm-2 fl/wt (Dnm2 het) genotype. Bone marrow cells from the heterozygous female mice (Dnm2 het) had 35% to 50% decreased Dnm2 expression in comparison with age-matched controls (CTRL). Evaluation of the complete blood counts demonstrated that Dnm2 het female mice developed leukopenia which was detected from 40 weeks of age (average granulocyte-monocyte counts: CTRL 532/mm3 vs. Dnm2 het 300/mm3; p=0.0164). Neutropenia was unequivocal at 65 weeks of age (average neutrophil counts, CTRL 700/mm3 vs. Dnm2 het 343/mm3, p=0.016). Dnm2 het showed a trend for higher platelet counts than controls, but this was non-statistically significant. Further analysis of hematopoietic lineage maturation by flow cytometry indicated that lineage-negative cells and granulocyte-monocyte progenitors were decreased in Dnm2 het mice (average bone marrow lineage-negative cells: CTRL 2.8x10E6 vs. Dnm2 het 1.97x10E6, p =0.0056; average granulocyte-monocyte progenitors: CTRL 1.35x10E6 vs. Dnm2 het 0.85x10e6, p=0.01), along with a relative increase of common lymphoid progenitors and of megakaryocyte/erythrocyte progenitors in the bone marrow. Immunohistochemical staining for mature neutrophils with Ly6G showed an overall decreased number of mature granulocytes in the bone marrow of Dnm2 het mice (average Ly6G-positive cells: CTRL 20% vs. Dnm2 het 29%, p=0.0026). A linear pattern of distribution of Ly6G positive bone marrow cells along blood vessels was observed in fewer mice in the Dnm2 het group than in the CTRL group (25% vs. 59%, p=0.02), indicating that the migration pattern within the bone marrow is altered in the Dnm2 het group (see Figure). In addition, Dnm2 het mice developed splenomegaly (average spleen weight: Dnm2 het 146 mg vs. CTRL 99 mg, p=0.006), which was secondary to a marked florid reactive germinal center hyperplasia. Some of the Dnm2 het mice, including 5 mice whose pregnancy occurred in middle-age (p=0.005 when comparing with CTRL or young Dnm2 het mice) and 2 non-pregnant older mice, showed physical signs of distress with markedly reduced activity, poor grooming, ruffled furs, and hunched posture. Both non-pregnant sick mice showed a marked decrease in Ly6G positive mature neutrophils at 0.3% of total marrow cells (Figure), and the bone marrow from one mouse was completely effaced by immature myeloid precursors, consistent with the development of acute myeloid leukemia. In addition, a third of Dnm2 hetmice showed no distress but displayed morphological bone marrow abnormalities including megakaryocytic dysmorphology.In summary, female mice with loss of Dnm2 in the hematopoietic compartment develop persistent neutropenia as they age, with decreased granulocyte progenitor production and with migration defects. These abnormalities are associated with a risk for developing megakaryocytic dysplasia, and acute myeloid leukemia. These findings might also suggest a mechanism for chronic idiopathic neutropenia, which has a predominance in middle-aged women. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy and Safety of Cyclosporine Treatment in Autoimmune Cytopenias: The Experience of Two Italian Reference Centers

▪Background: Autoimmune cytopenias (immune thrombocytopenia ITP, autoimmune hemolytic anemia AIHA, and chronic idiopathic neutropenia CIN) are a heterogeneous group of disorders characterized by the presence of autoantibodies directed against platelets (PLT), erythrocytes, and neutrophils (ANC). Frontline steroids are the mainstay of treatment, although most patients relapse and require 2 nd line therapies which slightly differ according to disease subtype. Rituximab is mainly effective in AIHA, although only a fraction of cases would experience long-term relapse-free. Splenectomy may be contraindicated due to age and risk of thrombotic and infectious complications and thrombopoietin-receptor agonists (TPO-RA), effective in more than 70% of ITP patients, may result in great PLT fluctuations and increased bone marrow reticulin fibrosis.Cyclosporine (CyA) is an immunosuppressant used for over 30 years in the post-transplant setting and in the treatment of aplastic anemia. It is a manageable oral drug with known toxicities and its plasma concentrations may be monitored to optimize treatment. However, few data exist about its efficacy in autoimmune cytopenias either used as single drug or in combination with other treatments.Aim: The aim of this study was to evaluate the efficacy and safety of CyA in a cohort of patients with ITP, AIHA, and CIN, followed at two reference hematology centers in Milan, Italy.Methods: Medical charts of consecutive patients treated with CyA 3-5 mg/ kg day in the last 20 years were evaluated. Responses were assessed at 3, 6 and 12 months, and divided into partial (PR, for Hb> 10 g/dL; PLT> 30x 109/L and ANC> 0.8 x 109/L) and complete (CR, for Hb> 12 g/dL; PLT> 100x109/L; ANC>1 x 109/L). Adverse events were recorded according to CTCAE criteria.Results: 41 patients, 27 ITP (66%), 11 AIHA (27%) and 3 CIN (7%), were included, 16 men (39%) and 25 women (61%), with a median age of 60 year (21-81). The median time from diagnosis to CyA start was of 10 years (5-15), with a median of 3 (1-8) previous therapy lines. Most patients were receiving concomitant treatment including steroids (N=13), IVIG (3), or TPO-RA (14). Reasons to start CyA included no response to previous treatments (N=27), platelets fluctuations (N=5) or bone marrow fibrosis (N=5) on TPO-RA, and contraindication for splenectomy (N=4). Median duration of CyA therapy was 5 years (1-9) and 34 patients (83%) responded: 34% CR, 44% PR at month+3; 39% CR and 39% PR at month+6; and 26%CR and 43%PR at month+12 (Figure 1). Specifically, median PLTs increased by 32 x10^9/L at month+3, by 121 x10^9/L at month+6, and by 43 x10^9/L at month+12. Median Hb improved by 0.5 g/dL at month+3, by 1 g/dL at month+6, and by 2,6 g/dL at month +12. Median ANC augmented by 0.7 x10^9/L at month +3, by 1.7 x10^9/L at month +6, and by 0.07 x10^9/L at month +12. Importantly, 9 patients could stop steroids, and 10 subjects discontinued or tapered TPO-RA (5 each). Better responses were observed in ITP patients with baseline bone marrow hypocellularity (p = 0.01), absence of reticulin fibrosis (p=0.02), and in those not previously splenectomized (p=0.04). Among AIHA cases, those with IgG+C direct antiglobulin test positivity showed higher percentage of non-response (67 versus 22% in IgG+). Adverse events were mainly G1-2, occurring in 52% of patients, and included asthenia, dyspnea, myalgia, nausea, vomiting, diarrhea and abdominal pain, epistaxis, petechiae and an Escherichia Coli cystitis. Three patients on concomitant long-term steroids developed a G3 event (1 pulmonary embolism, 1 Aspergillus lung infection and 1 bronchitis), and 1 died for Pneumocystis jirovecii pneumonia.Conclusion: Cyclosporine was effective in about 80% of highly pretreated patients and allowed tapering/discontinuation of concomitant treatments in 63% of cases. Responses were higher in those with ITP, and hypocellular bone marrow without reticulin fibrosis. The occurrence of infectious episodes, including a fatal pneumonia, warrants careful surveillance in this heavily pretreated patient population. [Display omitted] DisclosuresFattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Barcellini: Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Alexion Pharmaceuticals: Honoraria.

MANAGEMENT AND OUTCOMES OF NEUTROPENIA IN PREVIOUSLY HEALTHY CHILDREN

Immun-component children with moderate neutropenia do not have an increased risk for severe bacterial infections. However, there is limited data for the management of benign neutropenia in children. Therefore, we aimed to determine the most common etiology and outcomes in children with neutropenia. In addition, we compare the laboratory findings of different severity levels (neutrophil levels <0.2 × 10³/mL, 0.2 × 10³-0.5 × 10³/mL, 0.5 × 10³- 1 × 10³/mL, 1 × 10³- 1.5 × 10³/mL). This retrospective study included children with neutropenia (neutrophil < 1.5 × 10³/mL) diagnosed between December 2019-November 2020 in a tertiary hospital. The patients aged between one month-eighteen year had no history of chronic disease, immunosuppressive therapy, malignancy, or drug administration. Ministry of Health's ethics committee approved the study. We evaluated the etiologies and compared age, sex, time of follow-up, duration of neutropenia, thrombocyte, monocyte and immunglobulin levels of neutrophil levels (<0.2 × 10³/mL, 0.2 × 10³-0.5 × 10³/mL, 0.5 × 10³- 1 × 10³/mL, 1 × 10³- 1.5 × 10³/mL). The most common etiology was acute neutropenia (81.5%) and infections (66%). Five (2.5%) had coronavirus disease. Chronic and autoimmune neutropenia are the most common in chronic neutropenia. Lower neutrophils are associated with prolonged neutropenia (p=0.003), higher monocyte (0.03), higher IgM levels (0.038), younger ages (p<0.001), higher IgG (p=0.002) levels. Sex, time of follow-up, thrombocyte levels, total IgE levels are similar in children with different neutrophil counts. Our study demonstrates the etiology in children with neutropenia. The most common etiology is acute neutropenia with infections. In SARS-CoV2 diseases, neutropenia is less common than other hematologic findings. However, we detected in two point five percent of all. Unknown etiologies are also seen in the acute setting. Immun neutropenia and chronic idiopathic neutropenia are the leading causes of chronic cases. IgM levels were higher than the standard ranges in the agranulocytosis group, with a mean age of 1,05 ± 0,80. Therefore, children with ages of one-two should be carefully checked and followed for immunodeficiencies.

Incidence and prognosis of clonal hematopoiesis in patients with chronic idiopathic neutropenia

AbstractThe incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. The current study sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next-generation sequencing in patients with CIN (n = 185) with a long follow-up. We found that 21 (11.35%) of 185 patients carried a total of 25 somatic mutations in 6 genes with a median variant allele frequency of 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving >80% of patients, followed by IDH1/2, SRSF2, and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5 of 185 patients [2.70%]). However, from the transformed patients, 4 belonged to the clonal group (4 of 21 [19.05%]) and 1 to the nonclonal group (1 of 164 [0.61%]), indicating that the presence of mutation(s) confers a relative risk for transformation of 31.24 (P = .0017). The variant allele frequency of the mutant clones in the transformed patients was >10% in all cases, and the genes most frequently associated with malignant transformation were SRSF2 and IDH1. No significant differences were identified between the clonal and nonclonal groups in the severity of neutropenia. Patients with clonal disease were older compared with nonclonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.