Severe Chronic Neutropenia Research Articles

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.

TCIRG1 Mutations As a Cause for Chronic Neutropenia

Background: Heterozygous mutations in TCIRG1 are recently associated with congenital neutropenia. [Makaryan et al, Human Mutation, 2014] Recent large scale population study revealed statistically significant association between absolute neutrophil count and variations in TCIRG1 gene. [Rosenthal et al, Genetic Epidemiology, 2016]Aims: To investigate frequency and reveal possible asymptomatic cases of TCIRG1 associated neutropenia.Methods: The Severe Chronic Neutropenia International Registry (SCNIR) enrolled and followed 2 multigenerational families with genetic neutropenia of unknown etiology with a clear autosome dominant inheritance. Exome sequencing of selected affected members from these families revealed two previously reported heterozygous single point mutations in TCIRG1. Confirmatory Sanger sequencing ratifies exome sequencing results.Results:Family 1: P161L (c.482C>T) single point substitution detected in 3 members of this family. This variant is very rare in population and has relatively low minor allele frequency (MAF) score: 0.08. Proline residue at this position is highly conserved and has a high genomic evolutionary rate profiling (GERP) score: 3.46. PolyPhen-2 analysis suggests this substitution as possibly damaging.Family 2: I721N (c.2162T>A) single point substitution detected in 3 members of this family. This variant is also very rare in population and has MAF score: 0.07. Isoleucine at this position is relatively conserved and has GERP score: 2.73. PolyPhen-2 analysis suggests this substitution as probably damaging.Summary/Conclusion: Understanding the genetic, epidemiological and biological basis of TCIRG1 associated neutropenia is critical for improving the diagnosis and treatment of this form of hereditary neutropenia. The pathological mechanisms for TCIRG1-associated neutropenia are not yet known. In our original report [Makaryan et al, Human Mutation, 2014] of the large family with TCIRG1-associated neutropenia, all carriers of the pathogenic variant had neutropenia, with variable severity. Some members have severe neutropenia and associated infections whereas others are mildly affected. The report of these two new variants in these families suggests that there may be other patients and families with neutropenia caused by variants in TCIRG1. DisclosuresDale:Amgen: Consultancy, Honoraria, Research Funding.

Tbo-Filgrastim: A Review in Neutropenic Conditions.

Tbo-filgrastim (filgrastim XM02; Biograstim(®), Ratiograstim(®), Tevagrastim(®)) is approved in the EU as a biosimilar of filgrastim (Neupogen(®)) for use in all indications for which reference filgrastim is approved, including chemotherapy-induced neutropenia, neutropenia in patients undergoing myeloablative therapy followed by bone marrow transplantation, mobilization of peripheral blood stem cells (PBSCs), severe chronic neutropenia, and neutropenia in HIV infection. Tbo-filgrastim (Granix(®)) is also approved as a biologic in the USA for neutropenia associated with chemotherapy. Tbo-filgrastim has demonstrated bioequivalence to reference filgrastim in terms of its pharmacokinetic and pharmacodynamic profiles. In phase III trials, tbo-filgrastim was equivalent to reference filgrastim in ameliorating severe neutropenia in patients receiving chemotherapy for breast cancer, lung cancer, or non-Hodgkin lymphoma. In addition, the efficacy of tbo-filgrastim for PBSC mobilization in the allogeneic and autologous settings has been demonstrated in several small studies. Tbo-filgrastim was generally well tolerated, with a similar safety profile to that of reference filgrastim. Most adverse events were of mild or moderate severity. Biosimilars such as tbo-filgrastim have the potential to reduce healthcare costs compared with those of reference filgrastim; this may provide patients with more cost-effective treatment options. Current evidence indicates that tbo-filgrastim is a useful alternative to reference filgrastim in patients requiring filgrastim therapy for various neutropenic conditions.

Gamma heavy chain disease associated with large granular lymphocytic leukemia: A report of two cases and review of the literature

Objective and importance: Gamma heavy chain diseases (γHCD) and large granular lymphocyte (LGL) leukemia are two rare lymphoproliferative diseases, respectively with B and T phenotype. Both γHCD and LGL leukemia share some similar clinical features, such as cytopenias, splenomegaly, and recurrent infections. Association of these two diseases is exceptional and suggest pathogenic link. We report two cases of γHCD associated with T-LGL leukemia.Clinical presentation: Patient 1 was a 70-year-old woman, with lymphoplasmacytic lymphoma, refractory to chlorambucil-rituximab treatment. She developed during the follow up a γHCD with T-LGL leukemia, unresponsive to melphalan, thalidomide, and steroids, requiring supportive care. Patient 2 was a 40-year-old man with chronic severe asymptomatic neutropenia, revealing both γHCD and T-LGL leukemia. He is still well without any treatment nor complications, with 7 years follow up.Conclusion: Several types of B lymphoproliferative disease are associated with LGL leukemia. Although exceptional, this association of two rare lymphoproliferative disorders, with a different phenotype, does not seem fortuitous.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Editor and the Section Editor for this issue. EDITOR Thomas P. StosselThomas P. StosselThomas P. Stossel graduated from Princeton University, USA, (Summa Cum Laude) and Harvard Medical School, USA, (Cum Laude) and trained in internal medicine, hematology and research at Boston's Massachusetts General and Children's Hospitals, USA, and at the National Institutes of Health. He served as Head of Hematology-Oncology at the Massachusetts General Hospital, of Experimental Medicine and Hematology at Brigham & Women's Hospital, USA, President of the American Society for Clinical Investigation and of the American Society of Hematology, and as Editor-in-Chief of the Journal of Clinical Investigation. He is currently Senior Physician in the Brigham and Women's Hospital Hematology Division. He is also Editor-in-Chief of Current Opinion in Hematology and on the editorial advisory board of CollabRx. For research on mechanisms of how cells move about the body he was elected to The National Academy of Sciences, The American Academy of Arts and Sciences, and The Institute of Medicine and received honorary degrees from Linköping University, Sweden, and the University of Geneva, Switzerland, an NIH MERIT award, and the Dameshek and Thomas Awards of the American Society of Hematology. He has authored around 300 publications and two textbooks and is an inventor on 15 issued patents. He was a scientific advisor to Biogen and Dyax Corporations and currently is a Director of Velico Corporation and Founding Scientist of BioAegis Therapeutics, companies undertaking clinical development of his discoveries concerning blood transfusion and inflammation control respectively. For his policy work concerning the medicine-industry interface, he received the Academy of Pharmaceutical Physicians’ Sherwood and American Medical Writers Association's McGovern Awards. With his wife, Kerry Maguire, DDS, MSPH and others, Stossel co-founded a 5013c charity, Options for Children in Zambia that provides voluntary dental and medical care in that Sub-Saharan African country. For this effort he received the 2012 Humanitarian Award of the Brigham & Women's Hospital Hippocrates Society. SECTION EDITOR David C. DaleDavid C. DaleDr David C. Dale, Professor of Medicine at the University of Washington, USA, is a native of Knoxville, Tennessee, and a graduate of the Harvard Medical School, USA. He trained in internal medicine at Massachusetts General Hospital Boston, USA, and the University of Washington Medical Center, USA, and received research training at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, USA. His research interests are in inflammation, neutrophils, and the clinical problem of neutropenia, interests which link the specialties of hematology, oncology and infectious diseases. Dr Dale is actively involved in teaching, research and patient care at the University of Washington, where he served as Dean of the School of Medicine. He is a member of the American Society for Clinical Investigation, Association of American Physicians, American Clinical and Climatological Association, American Society of Hematology, Infectious Disease Society of America, and the American Society for Clinical Oncology. He has served as President of the American College of Physicians (ACP) and Chair of the Board of Governors and as member of the Board of Regents of ACP, President of the Society of General Internal Medicine, President of the Western Association of Physicians, President of the Medical Honor Society-Alpha Omega Alpha, and is currently President-elect of the American Clinical and Climatological Association. He is Editor-in-Chief Emeritus of the medical textbook Scientific American Medicine and serves on the National Board of Sponsors for Physicians for Social Responsibility, and is Co-Director of the Severe Chronic Neutropenia International Registry.

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations

GATA2 deficiency formerly described as MonoMAC syndrome; dendritic a cells, monocytes, B cells, and natural killer cell deficiency; familial a myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome a encompasses a range of hematologic and nonhematologic anomalies, mainly a characterized by monocytopenia, B lymphopenia, natural killer cell a cytopenia, neutropenia, immunodeficiency, and a high risk of developing a acute myeloid leukemia. Herein, we present 7 patients with GATA2 a deficiency recruited into the French Severe Chronic Neutropenia a Registry, which enrolls patients with all kinds of congenital a neutropenia. We performed extended immunophenotyping of their whole a blood lymphocyte populations, together with the analysis of their a chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells a in 6 patients. Although only 3 patients displayed natural killer cell a cytopenia, the CD56(bright) natural killer subpopulation was nearly a absent in all 7 patients. Natural killer cells from 6 patients showed a decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, a and most significantly B lymphocytes, displayed enhanced CXCL12induced a chemotaxis compared with healthy volunteers. Surface expression of CXCR4 a was significantly diminished in the patients' natural killer cells, a although the total expression of the receptor was found to be equivalent a to that of natural killer cells from healthy individual controls. a Together, these data reveal that GATA2 deficiency is associated with a impaired membrane expression and chemotactic dysfunctions of CXCR4. a These dysfunctions may contribute to the physiopathology of this a deficiency by affecting the normal distribution of lymphocytes and thus a potentially affecting the susceptibility of patients to associated a infections.

How I diagnose and treat neutropenia.

Neutropenia absolute neutrophil count (ANC) less than 1.5 × 10(9)/l is a common hematological finding, and severe neutropenia, that is, ANC less than 0.5 × 10(9)/l is a well known risk factor for susceptibility to bacterial infections. This review provides a succinct clinical approach to the diagnosis and treatment of neutropenia with specific recommendations on the treatment of severe chronic neutropenia with the myeloid growth factor, granulocyte colony-stimulating factor (G-CSF). Experts agree that patients with acute febrile neutropenia should be treated with antibiotics and that patients at high risk of severe neutropenia (>20% risk) after myelosuppressive chemotherapy should be treated prophylactically with a myeloid growth factor, usually G-CSF. The diversity of causes and consequences of chronic neutropenia make the diagnosis and management of these patients more complicated. The review provides a stepwise approach to neutropenia focusing first on reaching a provisional diagnosis and treatment plan then steps to a final diagnosis. It also provides specific recommendations on the treatment of severe chronic neutropenia with G-CSF.

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations.

GATA2 deficiency-formerly described as MonoMAC syndrome; dendritic cells, monocytes, B cells, and natural killer cell deficiency; familial myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome-encompasses a range of hematologic and nonhematologic anomalies, mainly characterized by monocytopenia, B lymphopenia, natural killer cell cytopenia, neutropenia, immunodeficiency, and a high risk of developing acute myeloid leukemia. Herein, we present 7 patients with GATA2 deficiency recruited into the French Severe Chronic Neutropenia Registry, which enrolls patients with all kinds of congenital neutropenia. We performed extended immunophenotyping of their whole blood lymphocyte populations, together with the analysis of their chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells in 6 patients. Although only 3 patients displayed natural killer cell cytopenia, the CD56(bright) natural killer subpopulation was nearly absent in all 7 patients. Natural killer cells from 6 patients showed decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, and most significantly B lymphocytes, displayed enhanced CXCL12-induced chemotaxis compared with healthy volunteers. Surface expression of CXCR4 was significantly diminished in the patients' natural killer cells, although the total expression of the receptor was found to be equivalent to that of natural killer cells from healthy individual controls. Together, these data reveal that GATA2 deficiency is associated with impaired membrane expression and chemotactic dysfunctions of CXCR4. These dysfunctions may contribute to the physiopathology of this deficiency by affecting the normal distribution of lymphocytes and thus potentially affecting the susceptibility of patients to associated infections.

Application of Spectral Density/Periodogram Analysis to Serial Neutrophil Counts to Diagnose Cyclic Neutropenia

▪Background: Cyclic neutropenia is characterized by oscillatory fluctuations in blood neutrophil counts, usually with nadirs <0.2 x 109/L at approximately 3 week intervals. Visual inspection of graphs of serial counts is usually the basis for diagnosis. Detection of mutations in ELANE is helpful but not diagnostic because of the overlap of the specific mutation patterns with those associated with severe congenital neutropenia. Making the correct diagnosis of cyclic neutropenia is important because these patients are not thought to be at risk of developing myelodysplasia or acute myeloid leukemia (MDS/AML). In contrast, patients with severe congenital neutropenia, whose counts are usually lower, are at risk of developing MDS/AML.Methods: We have implemented a website application for easy and direct data entry of serial blood counts to detect statistically significant periodicities using the Lomb periodogram. Physicians, nurses, other healthcare providers or patients can directly enter the blood count data for analysis on a website to allow immediate visualization of the serial counts and calculation of the probability of statistically significant cycling and the period, i.e., length of the cycle.Results: We have analyzed the counts from 42 patients (21 ELANE positive, 8 ELANE negative, 13 ELANE unknown) enrolled in the Severe Chronic Neutropenia International Registry with a clinical diagnosis of cyclic neutropenia to determine the accuracy of clinical diagnoses based on this form of statistical analysis.Our preliminary results showed that it is easy to learn how to use this program. We estimate that at least 20 counts obtained at 2-3 day intervals for 6 weeks are the minimum needed to detect cyclic neutropenia on a statistically sound basis, while 20-40 counts obtained at 2-3 day intervals over an 8-10 week period was more likely to yield statistical and clinical certainty about the diagnosis. The figure below shows readouts for the periodogram analysis for one patient. It shows the influence of 17 counts versus 31 counts for a patient with the clinical diagnosis of cyclic neutropenia and a mutation in ELANE. The confidence intervals (95%) and (99%) are exceeded for the series of 31 counts but not for the shorter series. The peak, approximate cycle length is 22 days for this series of counts.As of yet, we do not have the sufficient daily count data to determine if more frequent testing (e.g. daily testing) is better than testing every 2-3 days. We are currently testing the patterns of neutrophil fluctuations in patients on G-CSF to see if cyclic neutropenia can be diagnosed in patients that are on (or during) treatment. We have learned that many patients with the clinical diagnosis of CyN do not have sufficient serial blood cell count data to confirm this diagnosis on a statistical basis.Conclusion: We have developed a simple method for making periodogram analysis much more widely available to clinicians and patients on a world-wide basis. Statistical analysis of carefully collected serial data will help to secure the diagnosis of cyclic neutropenia and provide patients with important prognostic information. [Display omitted] DisclosuresDale:Amgen: Consultancy, Honoraria, Research Funding.

Long Term Outcomes for Patients with Cyclic Neutropenia Treated with Granulocyte Colony-Stimulating Factor (G-CSF)

Cyclic neutropenia (CyN) is a rare hematological condition with neutrophil counts decreasing to &lt;0.2 x 109/L, usually at 21 day intervals, accompanied by fever, mouth ulcers and the risk of severe sepsis during the neutropenic periods. Twenty-six years ago we reported results of treating six patients with CyN on G-CSF (N Engl J Med. 1989;320: 1306-1311). We have now followed these patients and many other CyN on G-CSF for up to 28 years through the Severe Chronic Neutropenia International Registry (SCNIR). The original six patients (2 male, 4 female, ages 8.8 to 65) are all living, now ages 36 to 92 years. All of the original six had documented fever and recurring infections prior to treatment (i.e. mouth ulcers, gingivitis, lymphadenopathy, cellulitis, abscesses, pharyngitis, otitis, and pneumonia). Five were found to have mutations in ELANE after the discovery of mutations in this gene as the cause for CyN. The sixth patient, in retrospect, probably more appropriately should have be given the diagnosis of severe idiopathic neutropenia, is now age 92, off G-CSF and residing at home with skilled nursing care. The other five have maintained good health on G-CSF treatment and are now employed as teachers, working in a business, or retired. None have developed notable infections, hematological or other sequelae except for one patient with decreased bone density and one patient with idiopathic thrombocytopenia purpura (ITP) responding to splenectomy. In aggregate these six patients have received approximately 6.6 grams of G-CSF over 143 patient-years (median dose of G-CSF per patient per year is 0.035 gm, range 0.018 to 0.075 gm, treatment period: 7-28 years). Our broader experience with 308 patients with the diagnosis of CyN based on serial blood counts and/or genotyping and treated with G-CSF is similar to these original six patients. However, we are aware of at least 40 cases of severe sepsis and 17 deaths in patients with CyN not on G-CSF. We are not aware of any such severe infections in CyN patients consistently receiving G-CSF (i.e. at least two or three times per week). We have recorded one case on chronic myeloid leukemia in a CyN patient never on G-CSF. For patients receiving G-CSF we have recorded one case of non-Hodgkin's lymphoma, one case of AML in patient with probable CyN having 2 ELANE mutations and a secondary CSF3R mutation, and one case of AML after chronic immunosuppressive therapy with cyclophosphamide. Conclusion: Observations in 308 patients for 2993 years demonstrate that G-CSF is a very beneficial treatment for patients with CyN. There is good evidence that this treatment prevents severe infections and death from infections and there is a very low risk myeloid malignancies, and specifically conversion to MDS or AML. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Boxer:Amgen: Equity Ownership.

Is There a Role for Anti-Neutrophil Antibody Testing in Predicting Spontaneous Resolution of Neutropenia in Young Children

▪Immune neutropenia is the most common referral to pediatric hematologists. Controversy exists as to whether anti-neutrophil antibody testing is necessary or helpful in establishing a diagnosis, predicting outcomes or establishing the necessity of treatment with G-CSF. It is also unclear if clinicians can separate immune neutropenia and hereditary/congenital neutropenia based on patients' clinical presentations, blood counts and anti-neutrophil antibody testing.We reviewed the records of 60 patients with the clinical diagnosis of autoimmune or idiopathic neutropenia, enrolled in the Severe Chronic Neutropenia International Registry (SCNIR) who had the onset of neutropenia before age 2 and who were tested for anti-neutrophil antibodies (positive or negative) to see if they resolved neutropenia by age 7 years. We identified 36 antibody positive and 24 antibody negative patients. Neutropenia resolved by age 7 in 27/36 (75%) antibody positive and in 15/24 (62.5%) antibody negative patients (p = 0.3910, Fisher's exact test). The median age at recovery for those with resolving neutropenia was 3.10 years for the antibody positive and 3.60 years for antibody negative patients (mean ages 3.39 and 3.52 years, respectively; p = 0.7614, unpaired t-test). All 60 patients had severe neutropenia (mean ANC 0.376 x 109/L ± 0.620, median 0.193 x 109/L) and history of recurrent fevers and infections, and were treated with G-CSF (mean dose antibody positive 3.12 ± 7.22 mcg/kg/day, mean dose antibody negative 2.57 ± 3.21 mcg/kg/day, p = 0.7311, unpaired t-test; median doses 1.42 and 1.53 mcg/kg/day, respectively). The ANC responses were quite similar, with antibody positive patients' mean ANC on treatment 3.36 x 109/L ± 1.48; antibody negative mean ANC on treatment 4.10 x 109/L ± 2.06 (p = 0.107, unpaired t-test; median ANCs 1.83 and 2.42 x 109/L, respectively). There were no apparent differences in demographic or hematological characteristics of the patients that resolved or failed to resolve.We also identified 8 children with ELANE mutations who had a positive test for anti-neutrophil antibodies. A positive anti-neutrophil antibody result is generally thought to predict a benign course and favor withholding treatment with G-CSF. Six of eight of these children did not receive G-CSF until after the development of severe infections, which included pneumonia, liver abscess, and cellulitis, including one who developed severe cellulitis with the loss of a lower extremity. Importantly, neutropenia did not resolve in any of the ELANE mutated patients.This retrospective review demonstrates that anti-neutrophil antibody testing in young children is of very limited or perhaps no value in predicting prognosis or response to G-CSF treatment. Furthermore, the results of the antibody testing may lead to withholding G-CSF therapy from children who are at risk of severe infectious complications. We believe it is prudent to treat young children with severe neutropenia who have recurrent fevers and infections independent of the anti-neutrophil antibody test results. We also advocate genetic testing to detect congenital neutropenia and predict outcomes in these patients. DisclosuresBoxer:Amgen: Equity Ownership. Dale:Amgen: Consultancy, Honoraria, Research Funding.

Severe chronic primary neutropenia in adults: report on a series of 108 patients

AbstractSevere chronic primary neutropenia (CPN) is a rare entity, and long-term outcome and risk factors for infections in severe CPN adults have not been described to date. We report the characteristics and outcomes of 108 severe adult CPN patients enrolled in a multi-institutional observational study. Severe CPN adults were mostly female (78%), and median age at diagnosis was 28.3 years. Diagnosis was fortuitous in 62% of cases. The median absolute neutrophil count (ANC) at diagnosis was 0.4 × 109/L, and median ANC without granulocyte colony-stimulating factor (G-CSF) during follow-up was 0.5 × 109/L. Twenty-three of 66 (34.8%) evaluable patients had neutrophil autoantibodies, and 6 of 47 (12.8%) a T-cell clone. The presence of neutrophil autoantibodies or T-cell clone was not associated with any specific clinical or biological characteristics. No death or hematologic malignancies occurred, and 44 severe bacterial infections were reported in 27 patients with a median follow-up of 8.3 years. Fifty patients received G-CSF either sporadically (n = 24) or continuously (n = 26) and responded (96%). Nineteen patients received immunosuppressive therapies: overall response (OR) was 41%, and median duration of response was 3 months. At diagnosis, the only predictive factor for the occurrence of severe bacterial infections was an ANC count below 0.2 × 109/L (OR, 0.76). Severe CPN in adults is characterized by a female predominance and a benign outcome with a low rate of severe bacterial infections and no secondary malignancies. G-CSF is efficient and well tolerated but is not required in a majority of patients.

Neutrophil disorders: diagnosis and hematopoietic stem cell transplantation

Neutrophil disorders are classified into abnormal neutrophil function and granulopoiesis. The identification of genetic defects causing neutropenia and neutrophil dysfunction has revealed the mechanisms controlling myeloid differentiation and their functions. The International Union of Immunological Societies of Primary Immunodeficiencies represents the most current catalog of approximately 30 neutrophil disorders. In this report, we show the progress made in studies of the pathophysiology and treatment of these disorders, focusing on chronic granulomatous disease (CGD) and severe congenital neutropenia (SCN). Hematopoietic stem cell transplantation (HSCT) is the only available curative therapy for CGD and SCN. However, the use of HSCT as treatment for both diseases is limited by transplant-related mortality (TRM) because of active infections and intractable inflammatory complications. Recently, reduced-intensity conditioning regimens have been introduced to minimize the TRM and the late adverse effects of HSCT for both diseases. The results of HSCT using the RIC regimen for 40 patients with CGD and SCN in Hiroshima University Hospital are summarized herein. Determining the optimal line of treatment will require further accumulation to cases to refine HSCT for both diseases.

The diversity of mutations and clinical outcomes for ELANE-associated neutropenia

Mutations in the gene for neutrophil elastase, ELANE, cause cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). This study summarized data from the Severe Chronic Neutropenia International Registry (SCNIR) on genotype-phenotype relationships of ELANE mutations to important clinical outcomes. We also summarize findings for ELANE mutations not observed in SCNIR patients. There were 307 SCNIR patients with 104 distinctive ELANE mutations who were followed longitudinally for up to 27 years. The ELANE mutations were diverse; there were 65 single amino acid substitutions; 61 of these mutations (94%) were 'probably' or 'possibly damaging' by PolyPhen-2 analysis, and one of the 'benign' mutations was associated with two cases of acute myeloid leukemia (AML). All frame-shift mutations (19/19) were associated with the SCN. The pattern of mutations in the SCN versus CyN was significantly different (P < 10), but some mutations were observed in both groups (overlapping mutations). The cumulative incidence of severe adverse events, that is, myelodysplasia, AML, stem cell transplantation, or deaths was significantly greater for patients with SCN versus those with CyN or overlapping mutations. Specific mutations (i.e. G214R or C151Y) had a high risk for evolution to AML. Sequencing is useful for predicting outcomes of ELANE-associated neutropenia.

Reduction of Granulocyte-Colony Stimulating Factor Requirement in the Course of Long-Term Treatment for More Than 5 Years in Congenital Neutropenia Patients Harbouring ELANE Mutations

Congenital neutropenias include a heterogeneous group of diseases characterized by a decrease in circulating neutrophils and different underlying germ-line gene mutations. Since 1988 recombinant human G-CSF is available for the treatment of severe chronic neutropenia patients. In phase I/II/III studies in patients with severe congenital and cyclic neutropenia, treatment with recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) resulted in a rise in the absolute neutrophil counts (ANC) and a reduction in infections. The Severe Chronic Neutropenia International Registry collects clinical information on patients suffering from severe chronic neutropenia since 1994. 312 of 379 CN and 65 of 79 CyN patients receive long-term G-CSF treatment for a median duration of 8,26 years in CN and 9,37 in CyN. Median G-CSF doses vary by neutropenia subtype and gene mutation. Patients with congenital neutropenia revealing ELANE mutations require the highest G-CSF doses compared to other subtypes (median G-CSF dose 5 µg/kg/day in 88 patients). SCNIR follow-up data suggest that pediatric ELANE-CN patients were maintained at a particular G-CSF dose per kg body weight for longer than expected by the gain of body weight. We therefore analysed the reported yearly G-CSF doses in all treated ELANE-CN patients to evaluate the dose trend:From 88 G-CSF treated patients with ELANE-CN we excluded 16 patients with nonresponse (ANC remained below 0.5 x 109 /L) and partial response (ANC remained between 0.5 and 0.99 x 109 /L). Since the gain of body weight is highest during the first 5 years of life with and 10-fold increase we divided G-CSF good responders by age at G-CSF initiation (0-5 years vs above 6 years) and compared G-CSF doses at the end of the dose finding period with the last dose reported. 51 of the remaining 72 patients started G-CSF treatment between the first and 5th year of life with a median G-CSF treatment duration of 9.76 years. 37 of the 51 patients were treated for at least 5 years. In 21 of the 72 patients G-CSF treatment was initiated after their 10thbirthday with a median follow up of 20.45 years. 19 of the 21 patients were treated for at least 5 years. All patients with a treatment duration of less than 5 years were excluded from further analysis.In ELANE-CN patients with treatment start at an age of 0-5 years the mean G-CSF dose decreased significantly from 13.47 µg/kg/day at the time of ANC-response to 7.96 µg/kg/day at the last report (median G-CSF dose decreased from 6.85 µg/kg/day to 4.42 µg/kg/day) during a treatment duration of at least 5 years.In summary, a significant decrease in the individual G-CSF doses could be observed in ELANE-CN patients who started G-CSF treatment during the first five years of their lives suggesting an age dependent alleviation of the severity of the disease as judged by the response to G-CSF. DisclosuresNo relevant conflicts of interest to declare.

Understanding Neutropenia: The 20 Year Experience of the Severe Chronic Neutropenia International Registry (SCNIR)

Background: In 1994 Severe Chronic Neutropenia International Registry (SCNIR) opened for enrollment of patients with at least 3 absolute neutrophil counts (ANC) less than 0.5 x 109/L during a three month period. At that time severe chronic neutropenia (SCN) was categorized as cyclic, congenital, autoimmune or idiopathic based largely on clinical criteria. A randomized trial had established effectiveness of treatment with granulocyte colony-stimulating factor (G-CSF), but long-term consequences of such treatment were unknown.Hypothesis: We began the SCNIR based on the hypothesis that underlying pathophysiology, natural history of patients with chronic neutropenia and benefits and risk of G-CSF therapy could only be accurately established through an international registry with long term follow-up of patients with these rare hematological disorders.Methods: SCNIR enrollment requires informed consent, ANC<0.5 x 109/L at least 3 times over a 3 month period, neutropenia not due to known systemic autoimmune disease (e.g., lupus, rheumatoid arthritis), cancer or cancer chemotherapy. There is a centralized enrollment process directed through offices in the US (Seattle) and Germany (Hannover). Continued enrollment requires annual follow-up information, i.e., clinical status, treatments and blood counts, and bone marrow reports for some categories of patients. Data on pregnancies, stem cell transplantation (SCT), non-hematological features and complications are also collected on standardized forms for subsets of patients. Comprehensive immunological assessments and genetic testing are encouraged but not required for enrollment. For some patient groups, e.g., Shwachman-Diamond syndrome (SDS) and Barth syndrome, the SCNIR now enrolls patients without severe neutropenia to gain perspectives on long-term outcomes for these disorders.Since 1994 the SCNIR has enrolled more than 2000 patients; 174 died, 193 resolved neutropenia, 543 withdrew or were lost to follow-up and almost all others continue in this long term observation study. The median follow-up for enrolled patients is now almost 10 years. The most common patient categories are idiopathic, cyclic (CyN) and congenital neutropenia (CN); 68% of CyN and 65% of CN patients having sequencing studies have mutations in ELANE. Some specific mutations are associated with high frequency (>90%) of severe outcomes (e.g. MDS/AML, failure to respond to G-CSF, death from infections, need for stem cell transplant) often many years after SCNIR enrollment and beginning G-CSF therapy. GSD1 patients improve with G-CSF treatment, but experience splenomegaly and continued problems with infections or complications. The SCNIR through a SDS sub-registry is redefining Shwachman-Diamond syndrome; only about one-half of enrollees have “classic” presentation and a substantial number with “classic presentation” lack mutations in SBDS. The SCNIR is participating in an NIH trial of a CXCR4 antagonist for treatment of WHIM syndrome, as an example of molecularly targeted treatment for this rare disease. The SCNIR is also the key resource for discovery of genetic causes for congenital neutropenia, e.g., G6PC3, HAX1, and TCIRG1 and others, recognition of differences in frequency of autosomal dominant and recessive SCN in populations of Europe and North America and identifying congenital neutropenia cases of unknown cause. Genetic testing has also broadened the clinical spectrum of these disorders.Conclusions: Through the efforts of patients, families, physicians, nurses and investigators, and with support from the NIH, industry, and private philanthropy, chronic neutropenia is now far better understood at the genetic, molecular and cellular level than 20 years ago. Treatment responses to G-CSF are well characterized; novel therapies are emerging; and the prognosis for patients with SCN appears to be improving. The knowledge gained through the SCNIR and availability of G-CSF has redefined clinicians’ approach to chronic neutropenia. The SCNIR is a model of international research collaboration to understand rare diseases in hematology and other areas of medicine. Broad enrollment criteria, physician, patient and family participation, a dedicated staff, and continuing cooperation underlie success of the SCNIR and this model to understanding rare diseases. DisclosuresDale:Amgen: Consultancy, Honoraria, Research Funding. Boxer:Amgen: Equity Ownership. Morrow:Amgen: Employment.

Successful Retransplantation of Bone Marrow Cells Following Failure of Initial Engraftment in 4 Patients with SCN

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia with a absolute neutrophil count of less than 0.5×109/L, maturation arrest of myeloid precursors at the promyelocyts/myelocyte stage, and the recurrent bacterial infections from early infancy. The induction of G-CSF therapy dramatically improved the prognosis of most patients with SCN. Analysis on patients on the Severe Chronic Neutropenia International Registry has demonstrated that the patients with SCN on long-term G-CSF treatment are at a risk of developing myelodysplastic syndrome or acute myeloid leukemia (MDS/AML). Recently, hematopoietic stem cell transplantation (HSCT) has been applied for a curative treatment in SCN patients without malignant transformation. However, the optimal conditions of HSCT for SCN have not been established. The failure of engraftment has been reported in some of SCN patients receiving HSCT with reduced intensity conditioning (RIC) or using cord blood as a donor source. Here, we tried to undergo retransplantation of bone marrow cells for 4 SCN patients with ELANE mutation who referred to our hospital because of the engraftment failure of initial HSCT. The summary of initial HSCT is shown in Table. The source of stem cells and the conditioning regimen were heterogenous in four patients. In this study we used bone marrow cells as stem cell source for all patients to transfuse the sufficiently total nucleated cells. Bone marrow cells were obtained from an HLA-matched related, an HLA-matched unrelated, and two HLA-mismatched unrelated (7/8) donors, respectively. Conditioning regimen consisted of fludarabine (125 mg/m2), cyclophosphamide (140 mg/kg), anti-thymocyte globulin (ATG, 10-12 mg/kg), melpharan (90 mg/m2), and TLI (3.6 Gy). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft versus host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 21 days in all patients. Three of 4 patients have achieved complete engraftment without any signs of GVHD during their course. One patient required additional donor-lymphocyte infusion to sustain the stable engraftment. All patients are alive for 1 to 5 years after HSCT with no signs of infections or transplantation-related morbidity. Furthermore, we observed successful BMT using similar conditioning regimen in other 4 SCN patients without prior transplantation. In our cohort, sufficient administration of ATG is thought to be critical for preventing both engraftment failure and acute GVHD. These results suggest that bone marrow transplantation using fludarabine-based conditioning regimen with a sufficient dose of ATG may be a feasible and effective treatment for SCN patients irrespective of initial engraftment failure. Indications for proceeding to HSCT have been under discussion in patients with SCN. Accumulation of cases and further analysis are necessary to assess the efficacy and safety of this regimen including late complication related to HSCT.TableHSCT characterictecs at the initial transplantationCase1234EtiologyELANE G185RELANE V72MELANE 4501-4503 delELANE V72MAge at the time of HSCT10 months2 years3 years 9 months15 monthsStem cell sourcecord bloodbone marrowcord bloodbone marrowHLA disparitymismatched unrelated donor (5/6)matched unrelated donor (6/6)matched related donor (6/6)mismatched unrelated donor (6/8)Transfused NCC (108/kg)0.71.90.491Transfused CD34 cells (106/kg)0.6991.90.071.07Conditioning regimenBU 16, CY 120, Flu 120CY 140, Flu 125, L-PAM 90 TBI 3, ATG 2.5CY 100, Flu 120 TBI 3, ALG 40Flu 125, L-PAM 140 TBI 2, ATG 5Posttransplant day of rejection72 days55 days after 4 times of DLI14 daysone year after mixed chimerismDLI (times)0430Busulfan (BU, mg/kg), Cyclophophamide (CY, mg/kg), Fludarabine (Flu, mg/m2), Melpharan (L-PAM, mg/m2), Total body irradiation(TBI, Gy), Anti-thymoglobulin (ATG, mg/kg), Anti-lymphoglobulin (ALG, mg/kg), donor lymphocyte infusion (DLI) DisclosuresNo relevant conflicts of interest to declare.

Clinical and Laboratory Features of Severe Neutropenia Developed in Childhood

Background: Neutropenia is not uncommon in children. We performed this study to in- vestigate the etiology, clinical course and laboratory characteristics for prediction of re- covery in children with severe neutropenia. Methods: In this study, we studied the clinical course and hematological features of 107 patients with severe neutropenia who were diagnosed and treated at the Department of Pediatrics, The Catholic University of Korea from April 2009 to July 2014. Patients with hematologic disorders and malignant disease were excluded. Chronic severe neu- tropenia (CSN) was defined as an absolute neutrophil count of 0.5×10 9 /L or less for at least 3 months. Acute severe neutropenia (ASN) were defined who recovered within 3 months from diagnosis. Results: Among 107 patients, 72 patients showed ASN and 35 patients were CSN. Median age of ASN (15.2 months) was higher than that of CSN (10.7 months). The median dura- tion of recovery from neutropenia was 7 days (range: 2-35) in ASN. ASN was commonly related to infectious diseases and 24 cases (33.3%) had documented etiologic agents. Median duration of recovery from neutropenia was 18 months in CSN. Three of 35 pa- tients in CSN had ELANE gene mutation. The number of white blood cells, platelets, monocytes, and eosinophils were significantly higher in group of CSN compared with ASN (P<0.05). Conclusion: We confirmed the great etiological heterogeneity of severe neutropenia in this study. The kinetics of recovery from neutropenia was different between the ASN and CSN group and the complete blood counts may be useful indices for discriminating ASN from CSN.

Outcome and management of pregnancies in severe chronic neutropenia patients by the European Branch of the Severe Chronic Neutropenia International Registry.

Long-term granulocyte-colony stimulating factor treatment has been shown to be safe and effective in severe chronic neutropenia patients. However, data on its use during pregnancy are limited. To address this issue, we analyzed all pregnancies reported to the European branch of the Severe Chronic Neutropenia International Registry since 1994. A total of 38 pregnancies in 21 women with chronic neutropenia (16 pregnancies in 10 women with congenital, 10 in 6 women with cyclic, 12 in 5 women with idiopathic neutropenia) were reported. Granulocyte-colony stimulating factor was administered throughout pregnancy in 16 women and for at least one trimester in a further 5 women. No major differences were seen between treated and untreated women with respect to pregnancy outcome, newborn complications and infections. In addition, we evaluated the genetic transmission of known or suspected genetic defects in 16 mothers having 22 newborns as well as in 8 men fathering 15 children. As a proof of inheritance, neutropenia was passed on to the newborn in 58% from female and in 62% from male patients with ELANE mutations, but also to some newborns from parents with unknown gene mutation. Based on our results, granulocyte-colony stimulating factor therapy has been shown to be safe for mothers throughout pregnancies and for newborns without any signs of teratogenicity. With an increasing number of adult patients, genetic counseling prior to conception and supportive care of mothers during pregnancy are crucial. The acceptance of having affected children may reflect the high quality of life obtained due to this treatment.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Editor and the Section Editor for this issue. EDITOR Thomas P. StosselThomas P. StosselThomas P. Stossel was educated at Princeton University, USA and Harvard Medical School, USA. He trained in internal medicine at the Massachusetts General Hospital, USA and in hematology at Boston Children's and Peter Bent Brigham Hospitals, USA. He was Head of Hematology and Oncology at Massachusetts General Hospital from 1976–1991, Co-Director of the Hematology Division at Brigham & Women's Hospital, USA through 2006, and is currently Director of the Division of Translational Medicine at that hospital and American Cancer Society Professor of Medicine at Harvard Medical School. In these positions he trained students who have gone on to leading academic positions in science and medicine around the world. Dr Stossel's basic research concerns fundamental mechanisms of cell crawling movements, such as how white blood cells defend against infection and cause inflammation. This research led to discoveries that may reduce critical care complications of major injury and impact platelet transfusion therapy. His policy interests concern physician and researcher interactions with private industry. To promote research and education concerning this topic, he has established a Center for Medical Innovation at Brigham & Women's Hospital funded by the Searle Freedom Trust. He has also co-founded The Association of Clinical Researchers and Educators to advocate for the value of such interactions and has established a Center for Biomedical Innovation to provide research and education concerning these relationships. He was President of the American Society for Clinical Investigation and Editor in Chief of its Journal of Clinical Investigation, served as President of the American Society of Hematology and received its Dameshek and Thomas Awards. He has been elected to Membership in the National Academy of Sciences, the American Academy of Arts and Sciences, and the Institute of Medicine. He is currently Editor in Chief of Current Opinion in Hematology, and on the Board of Directors of the BioAegis and Velico (formerly Zymequest) Corporations. He previously was a member of the Scientific Advisory Board of Biogen and has also participated on Advisory Boards for Merck. He has been awarded honorary MD degrees from the Universities of Linköping (Sweden) and Geneva (Switzerland). Dr Stossel has assisted his wife, Kerry Maguire DDS MSPH, in doing dental prevention and treatment in orphanages and a remote rural village in Zambia, and they have established a 5013c non-profit corporation, Options for Children in Zambia. Dr Stossel also has faculty appointments at University Teaching Hospital where he is working with Zambian physicians to improve management of sickle cell disease and at the Zambian Dental Training School, Lusaka, Zambia. SECTION EDITOR David C. DaleDavid C. DaleDr David C. Dale, Professor of Medicine at the University of Washington, USA, is a native of Knoxville, Tennessee, and a graduate of the Harvard Medical School, USA. He trained in internal medicine at Massachusetts General Hospital Boston, USA, and the University of Washington Medical Center, USA and received research training at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, USA. His research interests are in inflammation, neutrophils, and the clinical problem of neutropenia, interests which link the specialties of hematology, oncology and infectious diseases. Dr Dale is actively involved in teaching, research and patient care at the University of Washington, where he served as Dean of the school of medicine. He is a member of the American Society for Clinical Investigation, Association of American Physicians, American Clinical and Climatological Association, American Society of Hematology, Infectious Disease Society of America, American Society for Clinical Oncology. He has served as President of the American College of Physicians (ACP) and Chair of the Board of Governors and as member of the Board of Regents of ACP, President of the Society of General Internal Medicine, President of the Western Association of Physicians, and President of the Medical Honor Society-Alpha Omega Alpha. He is Editor-in-Chief Emeritus of the medical textbook, ACP Medicine and serves on the National Board of Sponsors for Physicians for Social Responsibility, and is Co-Director of the Severe Chronic Neutropenia International Registry.