Cyclic Neutropenia Patients Research Articles

Differential transcriptional control of hematopoiesis in congenital and cyclic neutropenia patients harboring ELANE mutations.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most cyclic neutropenia (CyN) cases and approximately 25% of congenital neutropenia (CN) cases. In CN and in CyN, a median of 2.8% of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to granulocyte colony-stimulating factor (G-CSF) with a significant upregulation of the hematopoietic stem cell-specific transcription factors, C/EBPα, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBPα expression in CN restored granulopoiesis.

Spectrum of Pathogenic Genetic Variants in a Large Cohort of North American Congenital and Cyclic Neutropenia Patients: A Report from the Severe Chronic Neutropenia International Registry

▪Severe congenital neutropenia (SCN) is characterized by persistent neutropenia and risk of invasive, life-threatening infection as well as transformation to hematopoietic malignancy. The closely related syndrome cyclic neutropenia is characterized by recurrent episodic neutropenia accompanied by symptoms including infection. Understanding the genetic etiology of congenital neutropenia can help to direct therapy, guide surveillance and health maintenance strategies, and contribute to our understanding of basic neutrophil biology. Although mutations in ELANE are the most frequent cause of congenital neutropenia, there is a wide and ever-growing list of additional causative variants. Additionally, there appears to be regional genetic variability. For example, mutations in HAX1 are rarely if ever observed in North America while they are more common in Europe. We undertook exome sequencing of a large cohort from the Severe Chronic Neutropenia International Registry (SCNIR) of North America in an effort to define the genetic spectrum of congenital neutropenia and aid in the discovery of new pathogenic variants.We expanded our previously reported study of whole exome sequencing to include 152 cases of chronic neutropenia, comprised of 94 cases of SCN and 58 cases of cyclic neutropenia. We selected cases in which ELANE testing was negative, or in a small minority of cases, where ELANE testing had not yet been performed. Indeed, exome sequencing only identified 7 cases (5 SCN and 2 cyclic) carrying pathogenic ELANE mutations in this cohort. In the remaining 145 cases, we analyzed exomes for the presence of variants in genes previously associated with congenital neutropenia including AK2, AP3B1, CD40LG, CLPB, CSF3R, CXCR2, CXCR4, DNAJC21, DNM2, DOCK2, EFL1, EIF2AK3, ELANE, G6PC3, GATA1, GATA2, GFI1, GINS1, HAX1, IRAK4, JAGN1, KAT6A, KRAS, LAMTOR2, LYST, MYD88, PGM3, PSTPIP1, RAB27A, RAC2, SBDS, SEC61A1, SLC37A4, SMARCD2, SRP54, STK4, TAZ, TCIRG1, TCN2, TLR8, USB1, VPS13B, VPS45, WAS, WDR1 and WIPF1. Pathogenic heterozygous mutations of CLPB that localize to the ATP-binding pocket were identified in 7 cases, making it the second most common cause of congenital neutropenia in North America. We additionally identified 4 cases with G6PC3 pathogenic variants, and one case each with pathogenic variants in JAGN1, CXCR4 (the cause of WHIM syndrome), germline homozygous CSF3R, and GFI1.Interestingly, we identify 2 unrelated individuals (one with SCN and one with cyclic) and 2 siblings with SCN all of whom possess a recently described heterozygous variant in SRP54 (p.T117del). We collected genomic DNA from the affected mother of the 2 siblings, an additional unaffected sibling, and the unaffected grandparents. Through this kinship, we can confirm the de novo appearance of this variant in the second generation and demonstrate that it tracks with disease status (Figure 1). We also identified 2 unrelated individuals with SRP54 variants affecting residue 175 also located within the GTPase domain (p.G175E or p.G175del). Both variants are absent from the gnomAD database, and studies are underway to demonstrate de novo acquisition.In summary, we have defined the spectrum of mutations present in ELANE-wildtype chronic neutropenia cases in North America. Pathogenic or likely pathogenic variants were identified in 26 out of 145 (18%) cases. The most frequently mutated genes were of CLPB, SRP54, and G6PC3, while mutations in HAX1 were not seen. Importantly, some of these mutations are associated with genetic syndromes with extra-hematopoietic findings (for example, CLPB and SRP54) that would warrant additional evaluations and targeted health maintenance. These findings emphasize the importance of sending large panels for genotyping, rather than targeted ELANE testing. [Display omitted] DisclosuresBolyard: X4 Pharmaceuticals: Research Funding. Makaryan: Emendo Biotherapeutic: Research Funding. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Cyclic Neutropenia, Congenital and Idiopathic Neutropenia

Background: Cyclic neutropenia (CyN) is an autosomal dominant disease usually attributable to mutations in the gene for neutrophil elastase, ELANE . The diagnosis depends upon serial blood counts (at the minimum every other day or three days a week for 6 weeks) showing periods of severe neutropenia (absolute neutrophil count [ANC] < 0.2 x 109/L) recurring at approximately 21 day intervals. The diagnosis can be confirmed by Lomb periodogram analysis. Genetic testing is helpful but not diagnostic because of the overlap of the mutational patterns in ELANE for CyN and severe congenital neutropenia (SCN).Methods: We reviewed clinical and laboratory data for 87 patients enrolled in the Severe Chronic Neutropenia International Registry (SCNIR) with a diagnosis of CyN in 41 kindreds. Their family histories indicated that there were 117 other neutropenic family members. For this review we focused on 53 CyN patients in 15 kindreds who met the following criteria: two or more patients within the kindred had 10 or more CBCs available for analysis. Using data collected before treatment with G-CSF by the patients' primary physicians, we examined graphically displayed serial ANC's for each affected family member and performed periodogram analysis. We compared patterns of ANC fluctuations within families and for patients of similar ages within these families.Findings and implications: •Within families, if 1 patient has a mutation in ELANE, all other neutropenic members will have the same mutation in ELANE.•Within families if the index case has typical and diagnostic oscillations in blood neutrophils, other affected family members often have neutropenia without clear or diagnostic oscillations in their counts. If the patient has a mutation in ELANE, the diagnosis of SCN is sometimes assigned.•In familial CyN the oscillations tend to dampen with lower peaks and somewhat less severe neutropenia in older adults (> 30 years of age). Unless these patients have genetic testing showing a mutation in ELANE or another gene associated with neutropenia, they may be given the diagnosis of chronic idiopathic neutropenia.•The Lomb periodogram is very useful for diagnosing CyN. It is a web-based tool readily accessible on the SCNIR web site, https://depts.washington.edu/registry/. However, it is necessary to collect sufficient number of counts (usually at least 20 counts spaced at regular intervals) to use this method to make the diagnosis of CyN. Although visually there was cycling in at least one member of the 15 families, only 5 families had a member who had CyN with a 95 or 99 percent confidence level based on the Lomb periodogram.Conclusions:Careful family history and serial measurements of ANCs are the foundations for the diagnosis of CyN. Genetic sequencing and identification of specific mutations in ELANE are helpful for prognosis. The Lomb periodogram is helpful, but requires a robust series of counts to assure that cycling is present with a high (95%) confidence level. DisclosuresDale:Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Genkyotex: Other: DSMB (work completed 6/2015); Hospira: Consultancy; Prolong: Consultancy; Boheringer-Ingelheim: Consultancy; Coherus: Consultancy; Omeros: Other: DSMB; Shire: Other: Independent Review Board; X4Pharma: Research Funding; Philips: Research Funding; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding; University of Washington: Employment, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding.

Management of acute cholecystitis in patient with cyclic neutropenia: a case report

BackgroundCyclic neutropenia is a disease that causes a neutropenic decrease in peripheral blood in a cycle of about 21 days. It is a rare hereditary disorder with an estimated incidence of 0.5–1 cases per million population. The absolute neutrophil count can drop to zero, and neutropenic nadir may last for 3–5 days. This is a rare disease, and there are few reports of abdominal surgery in cyclic neutropenia patients; thus, we report this case of neutrophil count fluctuation and perioperative management.Case presentationA 31-year-old man with cyclic neutropenia was transferred to our hospital complaining of right season rib pain, but no rebound tenderness. His C-reactive protein was elevated (4.37 mg/L) and computed tomography revealed a large number of small stones in the gallbladder body and an incarceration in the gallbladder neck. He was diagnosed with acute cholecystitis. Ideally, surgical intervention should have been performed immediately, but because his neutrophil count was 300/μL, endoscopic naso-gallbladder drainage was performed and he was provided antibiotics until his neutrophil count increased to acceptable levels. Three days after admission, his neutrophil count had increased and laparoscopic cholecystectomy was performed. For one week after the operation, antibiotics were administered; he had an uneventful postoperative recovery. He was discharged on the seventh postoperative day and provided an oral antibiotic.ConclusionsInfection can be serious in patients with cyclic neutropenia, and it is therefore, important to determine the timing of surgery and to apply appropriate perioperative management with drainage and antibiotic administration.

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Game of clones: A case of ELANE gene mutation-related neutropenia

Mutations in the ELANE gene, which encodes the neutrophil elastase (NE) protein in neutrophils, result in ELANE-related neutropenia. ELANE-related neutropenia encompasses both cyclic neutropenia (CN) and severe congenital neutropenia (SCN), with ELANE mutations seen in a majority of SCN and almost all of CN patients. Clinical history in affected individuals typically reveals recurrent episodes of fever, oral ulcerations and bacterial as well as fungal infections, correlating with periodic oscillations or chronically low levels in the absolute neutrophil count (ANC). ELANE-related neutropenia is characterised by severely low neutrophil counts where ANCs may drop as low as zero. A two-year-old child presented at our hospital with a longstanding history of recurrent bacterial infections and previous admissions at another centre during some of these infectious episodes. Her full blood count demonstrated a markedly low ANC, which was chronic on assessment of her previous results. Decreased granulopoiesis with maturation arrest was seen on her bone marrow and genetic testing for the ELANE gene demonstrated a pathogenic variant of the mutation. Treatment with granulocyte colony-stimulating factor (G-CSF) was initiated.

Nicotinamide (NA) Treatment Improves Response to G-CSF in Severe Congenital Neutropenia (SCN) Patients

Background: SCN is an inherited bone marrow failure syndrome with markedly reduced granulocyte numbers and functions that often manifests with life-threatening bacterial infections. G-CSF treatment is the primary therapeutic approach, yet some SCN patients require high doses of G-CSF which increases the risk of acute myeloid leukemia development, or do not respond to GCSF at al. We have demonstrated that nicotinamide (NA) treatment increases granulocyte numbers in healthy volunteers. Aim: To asses the effect of NA treatment in SCN and cyclic neutropenia (CyN) patients. Methods: 15 SCN and three CyN patients ages 0.5 to 31 (M-5.47) years were orally treated with 20 mg/kg/day of NA in addition to G-CSF therapy. G-CSF dose varied between 0.6 and 50.8 µg/kg/day before start of NA treatment. Results: After three months of NA treatment, absolute neutrophil counts (ANC) increased from median 0.80 x 109/l (Q1 0.61, Q3 1.29 x 109/l) to 1.77 x 109/l (Q1 1.49, Q3 2.33 x 109/l) (p &amp;lt; 0.001). This allowed G-CSF dose reduction from 15 µg/kg/day (Q1 5, Q3 25 µg/kg/day) to 10 µg/kg/day (Q1 1.77, Q3 15.16 µg/kg/day) in 17 patients, and complete G-CSF tapering in one patient. After one year of observations, ANC were 1.50 x 109/l (Q1 0.99, Q3 2.86 x 109/l), median increase of ANC from start of therapy was 0.89 x 109/l fold (Q1 0.39, Q3 1.57; р = 0.004). NA was well tolerated, without severe adverse events. We also observed a marked reduction of the frequency and severity of bacterial infections upon combination of NA and G-CSF. Conclusions: In our group of SCN/CyN patients, NA treatment led to increased neutrophil counts and decreased required G-CSF doses with continuous clinical and laboratory responses. We also observed a reduction of the frequency and severity of bacterial infections. Therefore, the use of NA in combination with a reduced dose of G-CSF for the treatment of SCN and CyN patients is promising and should be further investigated in a larger cohort of patients. Disclosures No relevant conflicts of interest to declare.

Homozygous HAX1 mutation in cyclic neutropenia patient: A novel mutation

Homozygous HAX1 mutation in cyclic neutropenia patient: A novel mutation

New insights into the pathomechanism of cyclic neutropenia.

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral blood absolute neutrophil counts (ANCs) show cycles of approximately 21-day intervals. The majority of CyN patients harbor ELANE mutations, but the mechanism of ANC cycling is unclear. We performed analysis of bone marrow (BM) subpopulations in CyN patients at the peak and the nadir of the ANC cycle and detected high proportions of BM hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) at the nadir of the ANC cycle, as compared with the peak. BM HSPCs produced fewer granulocyte colony-forming unit colonies at the ANC peak. To investigate the mechanism of cycling, we found that mRNA expression levels of ELANE and unfolded protein response (UPR)-related genes (ATF6, BiP (HSPA5), CHOP (DDIT3), and PERK (EIF2AK3)) were elevated, but antiapoptotic genes (Bcl-2 (BCL2) and bcl-xL (BCL2L1)) were reduced in CD34+ cells tested at the ANC nadir. Moreover, HSPCs revealed increased levels of reactive oxygen species and gH2AX at the ANC nadir. We suggest that in CyN patients, some HSPCs escape the UPR-induced endoplasmic reticulum (ER) stress and proliferate in response to granulocyte colony-stimulating factor (G-CSF) to a certain threshold at which UPR again affects the majority of HSPCs. There is a cyclic balance between ER stress-induced apoptosis of HSPCs and compensatory G-CSF-stimulated HSPC proliferation followed by granulocytic differentiation.

Myelodysplasia, Leukemia, Lymphoid Malignancies, and Other Cancers in Patients with Severe Chronic Neutropenia

Background: Since 1994 the Severe Chronic Neutropenia International Registry (SCNIR) has enrolled children and adults with > 3 absolute neutrophil counts (ANCs) < 0.5 x 109/L during a 3 month period to understand the pathobiology, natural history and treatment responses for severe chronic neutropenia. We have previously reported on the frequency and risk of myelodysplasia (MDS) and acute myeloid leukemia (AML) in patients with congenital neutropenia. For this report we reviewed patterns of hematological complications and malignancies occurring in all patients enrolled through the North American office of the SCNIR.Methods: Enrollment required informed consent, and patients and their physicians provided demographic, clinical and laboratory data including bone marrow results. Genetic testing was not required. Patients were followed with annual reports on blood counts, infections, malignancies and hospitalizations.Results: From 1994 to 2018 the Seattle SCNIR office has enrolled 1672 patients in the following categories: congenital 637 (38%), cyclic 259 (15%), and idiopathic / autoimmune 776 (47%), and many have been followed now for more than 15 years. There are approximately 17,577 person years of the observational data in this Registry. The congenital category now includes patients with mutations in ELANE, SBDS, TAZ, COH1, CXCR4, SLC37A4, G6PC3, WAS, CSF3R, SRP54, GFI1, VPS45, JAGN1, HAX1 and also patients with severe neutropenia from an early date in childhood without a genetic diagnosis. Cyclic neutropenia patients have demonstrated oscillations in ANC. The idiopathic and autoimmune category includes children and adults including some with large granular lymphocytes (LGL) syndrome without recognized features of a lymphoproliferative disorder. Most patients in all categories have been treated with granulocyte colony-stimulating factor (G-CSF).Findings: MDS or AML has occurred in 70 of the 1672 patients; 99% have clinical diagnosis of a hereditary type of neutropenia: severe congenital neutropenia (55), glycogen storage disease 1b (3), congenital immunodeficiency (2), Shwachman-Diamond syndrome (SDS) (5), WHIM syndrome (1),Wiskott-Aldrich syndrome (2), cyclic neutropenia (1) and idiopathic neutropenia (1). The median age at diagnosis of AML/MDS was 15.3 years (mean 18.3, +/- 1.79 SEM [range 0.40 - 70.6]); 69 of 70 were treated with G-CSF, median dose = 7.1 mcg/kg/day (mean 7.3, +/- 1.3 SEM )(range 0.18 - 100). One Shwachman-Diamond patient never received G-CSF. Outcomes for AML/MDS patients receiving chemotherapy with HSCT before 2000 were poor with 3/17 (18%) survivors. Since 2000 there were 35/53 (66%) survivors. Five patients developed myelofibrosis (4 congenital and 1 idiopathic). Two of the congenital patients later developed AML (1 living after treatment with a HSCT, 1 deceased). The clinical diagnosis of cyclic neutropenia has a favorable prognosis with G-CSF treatment, with only one probable case in 3,833 person years of clinical observation. 1Twelve patients developed T-cell lymphoproliferative disorder (1 autoimmune neutropenia, 3 congenital neutropenia, 8 idiopathic neutropenia (4 with LGL features)). Five of these patients are living, all in the idiopathic group, 3 of 5 living patients have features of the LGL syndrome.Ten patients have reported other hematological malignancies; CML in a congenital patient after treatment with HSCT (living), CLL in a cyclic patient (living), CMML in an idiopathic patient after treatment with a HSCT (deceased). Six of the 10 patients have developed lymphoma; cyclic neutropenia (1), idiopathic/autoimmune neutropenia (5). Only one SDS patient has developed aplastic anemia.Other cancers/non-hematological malignancies have occured mostly in older patients: breast cancer (15) colon cancer (6), dermatological malignancies (13), hepatoma (1), lung cancer (1), prostate cancer (1), thyroid cancer (1).Conclusions: The hematological consequences of severe chronic neutropenia depend on the underlying etiology. MDS and AML occur largely in patients with the congenital or hereditary neutropenias. The diagnosis of cyclic neutropenia and chronic idiopathic / autoimmune neutropenia portends a favorable prognosis, based on a total of 10482 person years of observation. Marrow failure and aplastic anemia are not expected consequences of severe chronic neutropenia. DisclosuresDale:Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy. Newburger:X4 Pharmaceutics: Consultancy, Honoraria; TransCytos LLC: Consultancy; Janssen Research & Development, LLC: Consultancy, Honoraria.

Optimization of CSF3R Mutation Detection in Severe Congenital Neutropenia and Cyclic Neutropenia Patients for Routine Diagnostics Using Next Generation Sequencing

Severe congenital neutropenia (CN) is a group of bone marrow failure syndromes characterized by absolute neutrophil counts below 0.5x109/L, susceptibility to bacterial infections and frequently associated with maturation arrest at promyelocyte stage in the bone marrow (BM).There is a high incidence of malignant transformation among CN patients with a cumulative rate of MDS/AML 22 % after 15 years of G-CSF treatment. The acquisition of G-CSFR truncating mutations is a risk factor for leukemic transformation in CN patients. Therefore, annual monitoring of CSF3Rmutations by means of next generation sequencing (NGS) is required for identification of CN patients with high risk of MDS/AML development.Since CSF3R mutations usually occur at low frequency without additional clinical features, it is important to carefully select suitable clinical sample type and methods for mutation detection. Next, it remains to be evaluated which CN genetic subgroups should be considered for annual screening of CSF3Rmutations.We performed CSF3R mutational screening in DNA and/or cDNA in 101 patients (ELANE, n = 42; HAX1, n = 16; G6PC3, n = 7; JAGN1, n = 2; WASP, n = 1; digenic ELANE, HAX1, n =1; digenic HAX1 and G6PC3, n =1; inherited mutations in CSF3R, n = 2; genetically unclassified CN, n = 9; cyclic neutropenia (CyN), n = 20) from the European Branch of the Severe Congenital Neutropenia Registry (SCNIR).Using DNA deep sequencing we screened 63 of 81 CN-patients and 20 CyN patients. Using this method, we identified CSF3R mutations in 22.2% (14/63) of CN patients and 10% (2/20) of CyN patients. The frequency of CSF3R mutations in CN patients with known inherited mutations was 20% (11/55): 30 % (3/10) in CN-HAX1 patients, and 22.9 % (8/35) in CN-ELANE patients. Interestingly, 3/8 (37.5 %) patients harbouring CSF3R mutations were observed in genetically unclassified CN. We did not detect any acquired CSF3R mutations in the small groups of CN patients (n=10) harbouring inherited G6PC3, JAGN1, CSF3Ror digenic mutations.In order to increase the sensitivity of mutation detection we performed cDNA deep sequencing of the critical region of G-CSFR. We sequenced 38 CN patients (ELANE, n = 15; HAX1, n = 11; JAGN1, n = 2; G6PC3, n = 2; WASP, n = 1; germline CSF3R, n = 1; genetically unclassified, n = 6). We found 13% (2/15) CN-ELANE, 27% (3/11) CN-HAX1 and 33% (2/6) genetically unclassified CN patients to be positive for acquired mutations in the critical region of G-CSFR. One CN patient with WASP mutation also acquired CSF3R mutation. Based on our sequencing data we would suggest CSF3Rmutation sequencing in all studied groups of patients regardless of mutations in ELANE and HAX1 genes.Intriguingly, 3 out of 5 CN patients with CSF3R mutations detected by cDNA deep sequencing were negative based on results of previous DNA deep sequencing. All of them were found to acquire low frequency CSF3R mutant clones (ELANE pos. patient with 0.3% of p.Q739* clone; genetically unclassified CN patient with 2% of p.Q749*clone; HAX1pos. patient with 0.9% of p.Q749* clone) in cDNA deep sequencing.In 2 patients (one CyN-ELANE and one CN-HAX1) with multiple acquired CSF3R mutations we compared mutant clone enrichment in different cell types (BM MNC; BM PMN; PB MNC and PB PMN) by means of cDNA and DNA deep sequencing. In the CyN-ELANE patient with 2 CSF3R mutant clones, the highest mutant allele frequency (MAF) was detected in the cDNA sample of PB PMN (11% of p.Q749* clone and 0.44% of p.Q739* clone), whereas in the PB MNC cDNA sample clone p.Q749* had only 2.5% MAF and clone P.Q739 was not detectable. Similar to that, in the CN-HAX1 patient the highest MAFs for all 3 CSF3R mutant clones were in PB PMN cDNA and the lowest in PB MNC DNA sample. Frequency of mutated CSF3Rclones in BM PMNs of both patients was comparable to PB PMN samples.Taken together, sequencing of cDNA extracted from peripheral blood or bone marrow PMN samples may provide better results than from MNC in terms of frequency of CSF3R mutation detection in CN and CyN patients. Sequencing of cDNA extracted from BM or PB samples allows enrichment of G-CSFR expressing mutant cells, but due to intrinsic low fidelity of reverse transcriptase the threshold level for positive calls could not be improved significantly (current threshold for candidate calls is 0.2-0.5%).We would suggest CSF3R mutation screening using deep-sequencing of cDNA from peripheral blood PMN in all patient groups (CN and CyN) for routine diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Time Course of Acquisition of a CSF3R Mutation and Subsequent Development of AML in a Patient with Cyclic Neutropenia

Acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations have been detected with a high frequency (17-34%) in severe congenital neutropenia (CN) patients. Moreover, in CN patients who developed overt acute myeloid leukemia (AML) the frequency of CSF3R mutations in the intracellular critical region reaches more than 80%. Up to now there was no report on the detection of acquired mutations in CSF3R or development of AML/MDS in cyclic neutropenia patients (CyN). This group of patients reveals the same genotype with mutations in the ELANE gene as CN patients. In contrast, both groups of patients have a clearly distinct disease penetration. Using ultra-deep sequencing approach we screened 22 CyN patients for the presence of genetic abnormalities in the critical region of the CSF3R gene. Interestingly, we found only in one CyN patient a clone with acquired mutation in G-CSF receptor (NP_000751.3, p.Gln741X) at 3% of allele frequency. The cycling neutrophils and platelets were well documented and verified the diagnosis of CyN. Within two years after the first detection, the mutant clone harboring the CSF3R mutation expanded and was detected in 16% of patient's bone marrow (BM) MNCs. The sequencing of the ELANE gene in the CyN patient with CSF3R mutation revealed the presence of two mutations p.Ala233Pro and p.Val235TrpfsX (NP_001963.1). Intriguingly, both ELANE mutations were absent in DNA isolated from each of parents indicating their de novo origin. Additionally, the patient was found to be heterozygous for synonymous SNP rs17216649 (dbSNP build 138) on the same ELANE exon permitting determination of the parental origin of mutations. The sub-clone analysis in E.coli showed that both mutations were always found on the paternally-derived allele. The missense mutation p.Ala233Pro (NP_001963.1) is well known to be associated with CN phenotype, whereas the single-base deletion p.Val235TrpfsX (NP_001963.1) was reported as cause of CN only once. There are no reports about detection of these mutations in CyN patients. Three years after the first detection of the CSF3R mutation the patient presented with 14% blast cells in bone marrow acquiring monosomy 7 and trisomy 21. Based on results of cDNA sequencing all CSF3R expressing cells in the sample were positive for p.Gln741X (NP_000751.3) mutation. Recently, we demonstrated acquisition of cooperative of RUNX1 and CSF3R mutations in more than 60% of CN-AML/MDS patients. We also found RUNX1 mutation p.Asp171Asn (NP_001001890.1) at 10% allele frequency in BM MNCs of CyN-AML patient. To evaluate at which stage of hematopoietic differentiation acquisition of CSF3R mutations appeared leading to the leukemogenic transformation, we performed colony forming unit (CFU) assays using patients BM MNCs. We observed the appearance of abnormal «CFU-blast» colonies after cultivation of BM MNCs in MethoCult H4434 Enriched medium supplemented with rh SCF, rh G-CSF, rh IL-3, rh IL-6 and rh EPO (72,2% of CFU-blasts (n=232), 21,8% of CFU-G (n=70), 5% of CFU-GM (n=16) and 1% of BFU-E (n=3)). Interestingly, we found that all sequenced CFU-G (n=4), CFU-GM (n=6) and «CFU-blast» colonies (n=20) were positive for p.Q741X CSF3R mutation. At the same time, no CSF3R mutations were identified in BFU-E colonies. In summary, we report a first case of acquisition of CSF3R mutation and subsequent development of AML in a CyN patient underlying the complex nature of genotype:phenotype relations in CN and CyN patients. This case also represents an example of an extremely rare situation of double de novo ELANE mutations and provides a unique opportunity to investigate pathogenic molecular mechanisms of leukemia development in CyN patients. The ultra-deep next generation sequencing (NGS) for identification of CSF3R mutations is a useful tool to study the frequency of CSF3R mutations not only in CN but also in CyN patients and is clinically important for the identification of patients with the risk of progression to leukemia. Disclosures No relevant conflicts of interest to declare.

Differential Expression of Neutrophil Granule Protein Genes in Bone Marrow Myeloid Cells at the Peak and Nadir of Neutrophil Counts in Cyclic Neutropenia

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral-blood neutrophil counts show cycles at approx. 21-days intervals. The majority of CyN patients (ca. 90 %) harbor inherited mutations in the ELANE gene. The mechanism of cycling hematopoiesis downstream of ELANE mutations is unclear. In the present study we aimed to identify if there is a geterogeniety of bone marrow (BM) myeloid progenitors and granulocytic cells at the peak and nadir of the cycle of neutrophil counts.We performed FACS analysis of BM populations in CyN patient at the peak and nadir of the cycle and revealed reduced number of CD33high promyelocytes at the peak, as compared to the nadir neutrophil counts (6% vs 47%). Morphological examination of BM smears confirmed this observation. These data suggest differences in myeloid differentiation potential of hematopoietic cells of CyN patient during cycle. To compare the myeloid differentiation of BM cells at the peak and nadir, we performed CFU assay using BM cells isolated at these two different time points. Indeed, we found diminished capacity to produce CFU-G colonies at the peak of cycle, in comparison to the nadir (50 vs 68). This difference might be explained by the presence of different sub-populations of myeloid cells during the cycle. It was shown that the neutrophil populations can be distinguished by membrane expression of CD177, which is GPI-linked neutrophil antigen, localized primarily to the membrane of specific granules and to the plasma membrane. The proportion of CD177+ cells increased during neutrophil maturation in BM. Interestingly, in healthy individuals the fraction of CD177+ cells appeared to be constant in each individual. We evaluated the differences of CD177+ cell populations in CyN patients at the peak and nadir of cycle by FACS. We found that numbers of CD33+ CD177+ and CD16+ CD177+ populations were different during the cycle. At the peak we measured 7,1% of CD33+ CD177+ cells and 83% of CD16+ CD177+ cells. At the nadir 3,78% of cells were CD33+ CD177+ and 69% were CD16+ CD177+.We further performed mRNA expression analysis of CD33+ BM cells isolated from CyN patient at the peak and nadir of cycle and compared it to healthy individuals. We found lower mRNA expression (more than 10-fold) of CRISP3, ELANE, OLFM4, CEACAM6, MMP8, DEFA4 and LCN2 in CD33+ cellsat the peak of the cycle comparing to the nadir. These genes encode for neutrophil granule proteins, playing an important role in the developement and function of mature neutrophils. We further confirmed differential expression of these factors in CFU colonies using BM of CyN patient isolated at the peak and nadir of the cycle: CFU-G colonies grown from cells taken at the peak of the cycle expressed less mRNA levels of granula proteins than CFU-G colonies grown from cells taken at the nadir of the cycle.In summary, we hypothesize that the differential expression of the granule proteins is involved in the regulation of the cycle in myeloid cells in CyN. At the peak and nadir of neutrophil counts different populations (based on CD177 expression) of myeloid progenitors and neutrophils are present in the CyN BM. DisclosuresNo relevant conflicts of interest to declare.

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.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.

High Insta High Instability Of The CSF3R Gene In Severe Congenital Neutropenia Patients As Judged By Multiple CSF3R Mutations In The Majority Of Patients

▪It has been reported by us and others that approx. 30 % of all patients with congenital neutropenia (CN) acquire CSF3R mutations in their life time. More than 80 % of the CN patients who develop myeloid leukemia (CN/AML) harbour CSF3R mutations. This suggests that they are the first hit in leukemogenesis. However, detecting sequence changes e.g. by Sanger sequencing reveals only mutations presented in more than 20 % of the cells due to its technical detection limit. Therefore, we asked whether there is a systematic underestimation of cell clones harbouring CSF3R mutations, which might have been traditionally overlooked. We applied the deep-sequencing technology (SOLID 5500xl) to identify CSF3R mutations in myeloid cells from 158 patients with different types of neutropenia (86 severe congenital neutropenia (CN) patients with known inherited mutations (ELANE, HAX1, G6PC3), 21 cyclic neutropenia (CyN) patients, 28 patients with severe chronic neutropenia with so far unknown inherited mutations, 11 patients with SBDS-associated neutropenia) as well as a group of 12 healthy individuals. All neutropenia patients were treated with G-CSF and notably 21 CN patients developed leukemia or MDS. Deep sequencing data were processed according to our custom NGS pipeline (annotation of sequences and prediction of damaging effects on the coding sequence by Polyphen2, removal of known dbSNP variants, and accepting significant Phred-scores at the variant calling stage). Overall the read numbers ranged between 18 and 128069 (median 716), while only variants with at least two percent of the reads were accepted for further consideration (the statistically significant limit is between one and two percent of all reads). All together, we detected 92 CSF3R mutations in 42 CN patients leading to 49 distinct amino acid exchanges (38 missense and 11 stop-codon mutations). The frequency of the mutant alleles ranged from 2 to 96 %. In contrast, in CyN only five out of 21 patients harbour CSF3R mutations; interestingly, two of them in isoform IV of CSF3R (p.P752T). Most notably, whereas 18 patients displayed only one CSF3R mutation, 24 individuals had more than one CSF3R mutation (2-10 mutations, in total 74 mutations). During follow up of some patients, we could demonstrate that the number of mutations increased over time. The majority of mutations were located in the cytoplasmatic region (aa 651-831) of CSF3R, while 15 patients presented mutations within the extracellular region of CSF3R. Intriguingly, in 16 patients we detected 23 non-sense mutations, where 20 of these are stop-codon mutations affecting glutamine (Q) 768, 770, 776, and 781. This suggests that this part of CSF3R is highly instable. In two patients who did not respond to Filgrastim treatment, we detected a stop codon at aa 546 and 547, respectively, affecting the Fibronectin type-III like part of the CSF3R. Twelve patients who developed leukemia (CN/AML) had more than one CSF3R mutations (two to ten) , whereas eight with CN/AML harbored only one mutation.None of the healthy controls, only three neutropenia patients with unknown inheritance, and only one SBDS patient revealed mutations in CSF3R. Taken together, this data suggests that CSF3R is highly prone to genetic instability in severe congenital neutropenia, because more than one mutation in half of the patients was observed and various CSF3R mutations during the course of life accumulated. Once a cell clone harboring CSF3R mutation obtains a second hit (e.g. RUNX1 mutation), they are prone to undergo leukemic transformation. Disclosures:No relevant conflicts of interest to declare.

The Spectrum ofELANEMutations and their Implications in Severe Congenital and Cyclic Neutropenia

Neutrophil elastase gene (ELANE) mutations are responsible for the majority of cases of severe congenital neutropenia (CN) and cyclic neutropenia (CyN). We screened CN (n=395) or CyN (n=92) patients for ELANE mutations and investigated the impact of mutations on mRNA expression, protein expression, and activity. We found 116 different mutations in 162 (41%) CN patients and 26 in 51 (55%) CyN patients, 69 of them were novel. CyN-associated mutations were predicted to be more benign than CN-associated mutations, but the mutation severity largely overlapped. The frequency of acquired CSF3R mutations, malignant transformation, and the need for hematopoietic stem cell transplantation was significantly higher in CN patients with ELANE mutation than in ELANE mutation negative patients. Cellular elastase activity was reduced in neutrophils from CN/CyN patients, irrespective of the mutation status. In CN, enzymatic activity was significantly lower in patients with ELANE mutations compared with those with wild-type ELANE. Despite differences in the spectrum of mutations in CN or CyN, type or localization of mutation only partially determine the clinical phenotype. Specific ELANE mutations have limited predictive value for leukemogenesis; the risk for leukemia was correlated with disease severity rather than with occurrence of an ELANE mutation.

Identification of New Leukemia Associated Gene Mutations by Whole Genome Sequencing of DNA From Two Siblings with Congenital Neutropenia and Secondary AML

Abstract 12To identify the pattern of genetic aberrations, which may promote leukemia development in patients with severe congenital neutropenia (CN), we have performed a whole genome sequencing (WGS) of DNA samples from myeloid leukemic cells of two affected siblings suffering from CN. Both children harbored ELANE gene mutations. The father of the children demonstrates somatic mosaicism for the ELANE mutation and has no severe neutropenia. For WGS we used Complete Genomics technology (Complete Genomics. Inc, Mountain View, CA.). More than 90 % of genomes were sequenced at high quality with minimum coverage of at least 20-fold. As an example, 3.355.399 single nucleotide variants (SNVs) were identified in DNA isolated from leukemia blasts of one CN patient. The following filters were used to identify mutations in the leukemic cells from the two patients: 1) Non-synonymous SNVs in coding sequences only (9288 SNVs), 2) 54 healthy individuals sequenced by Complete Genomics (557 SNVs), 4) five members of one family from the same ethnic area (healthy parents, one cyclic neutropenia patient and her healthy brother, 471 SNVs), 5) five family members of the affected two children: parents and three healthy siblings (two healthy sisters and one heatlthy brother). Remaining SNVs presented in the two affected children were subsequently analysed using in silico prediction software Polyphen 2, which predicts possible impact of an amino acid substitution on the structure and function of human proteins. Fourteen SNVs with predicted damaging effects on the protein function were used for further analysis. All candidate SNVs were validated by Sanger sequencing. We detected nine inherited candidate SNVs presented in the two affected children but not in healthy siblings. The SNV in the ELANE gene (c.452G>A p.C151Y, dbsnp.129:rs57246956) was inherited from the father. Novel SNVs inherited from the father were as follows: in the TCTE1, FAM135A, M6PR, C20orf144 and PTPN23 genes. Only three SNVs were inherited from the healthy mother (in BLOC1S1, DUS3L and KIAA1543 genes). All SNVs were heterozygous. We also found 5 sporadic SNVs presented in leukemia sample of one CN patient only, but absent in his DNA sample from an earlier time point of CN diagnosis. These are heterozygous SNVs in the CSF3R, ACAP2, GRM1, LASS3, and RUNX1 gene. All five gene mutations might be involved in leukemogenesis. Interestingly, both affected patients had somatic mutation in the RUNX1 gene at the same nucleotide position (c.415C>G, p.R139G in sick brother and c.415C>T, p.R139* in sick sister). In summary, we identified candidate genes that may be relevant for leukemogenesis in CN patients. Our study also establishes WGS as an unbiased method for discovering leukemia-initiating mutations in previously unidentified genes that may respond to targeted therapies Disclosures:No relevant conflicts of interest to declare.

Mutations in the TNFRSF1A, CEBPE and ELANE genes in a Congenital Cyclic Neutropenia Patient: A New Syndrome?

Abstract 11Cyclic neutropenia (CyN) is a hematologic disorder in which blood cell counts particularly granulocytic neutrophil numbers show cycles at 21 day intervals. The majority of CyN patients (ca 90 %) harbor inherited mutations in the ELANE gene. Intriguingly, same ELANE mutations are present in two different hematologic syndromes: congenital as well as in cyclic neutropenias. It is unclear how mutation in the same gene cause congenital or cyclic neutropenia. We aimed to identify genes which are exclusively mutated in cyclic or in congenital neutropenia additionaly to the ELANE gene mutations. Recently, we found in congenital neutropenia patients additional to ELANE mutations inherited mutations in for example the G6PC3 gene or the HAX1 gene (Germeshausen, M., et al, Haematologica 2010). This suggests cooperating effects of different defective intracellular signaling pathways and excludes that mutated ELANE alone is responsible for the pathogenesis of congenital or cyclic neutropenia.To identify gene mutations causing cyclic neutropenia in association with ELANE mutation, we performed whole genome sequencing using Complete Genomics technology (Complete Genomics. Inc, Mountain View, CA.) of a family with an affected CyN patient. The CyN patient harbors sporadic heterozygous ELANE mutation (c.761C>G p.W241L) and her family consists of a healthy brother and healthy parents. We identified a novel heterozygous point mutation in the tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene (c.664C>T; p.R121Q) in the affected patient and her mother. This mutation was confirmed by Sanger sequencing. The TNFRSF1A gene encodes p55 subunit of the TNFa receptor (TNFR1) and intriguingly this gene is known to be frequently mutated in patients with Tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), a disease clinically similar to cyclic neutropenia. TRAPS is an autosomal dominant disorder characterized by episodes of fever, inflammation and periodical changes in the neutrophil counts. Functional studies in patients with TRAPS described heterogenous effects of different TNFRSF1A mutations on the surface expression and PMA-induced clevage of TNFR1 on the neutrophilic granulocytes and monocytes. In our CyN patient we measured elevated mRNA levels of TNFR1 on neutrophils, in comparison to her healthy family members and unrelated healthy individuals. However, we detected diminished surface expression of the TNFR1 protein, but elevated PMA-induced receptor shedding in the affected CyN patient, in comparison to healthy individuals, as assessed by estimation of soluble TNFR1 in supernatants of PMA-stimulated neutrophils.We also identified in the same patient and her father a second novel heterozygous point mutation in the CEBPE gene (c.636C>A; p.L155M), which was also confirmed by Sanger sequencing. This C>A substitution changes CTG to ATG creating a new start site for translation of a novel isoform of C/EBPε protein. C/EBPε is a myeloid-specific transcription factor playing an important role in granulopoiesis. Mutations in the CEBPE gene have been described in patients with neutrophil-specific granule deficiency (SGD).In summary, we identified additional to the ELANE mutation two novel mutations in a CyN patient, one in the TNFRSF1A gene inherited from the mother, another in the CEBPE gene inherited from the father. Mutations in both genes are already described in patients with TRAPS (periodic fever syndrome) and granulocyte abnormalities, respectively. These mutations in association with the ELANE gene mutation may contribute to the pathogenesis of cyclic neutropenia in this patient. Whether the combination of these three mutations might be responsible for a subgroup of CyN patients remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations

Abstract 14Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI. Disclosures:No relevant conflicts of interest to declare.