ALBERT

Description: Key Points The natural inhibitor of neutrophil elastase, SLPI, is severely reduced in severe congenital neutropenia patients. SLPI controls myeloid differentiation by regulation of NFκB, ERK1/2:LEF-1, and c-myc activation.

Description: Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood (

Description: Congenital neutropenia (CN) is a rare inherited disorder of hematopoiesis with a 20% risk of evolving into acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Using next-generation sequencing in 31 CN patients who developed leukemia we found that 20 of the 31 patients (64.5%) had mutations in RUNX1 (runt-related transcription factor 1). Of these 20 patients, 19 had inherited mutations associated with CN. Intriguingly, the majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R (colony stimulating factor 3 receptor) mutations. Other leukemia-associated mutations (EP300, FLT3-ITD, CBL, and SUZ12) were less frequent. In eight patients, we detected two distinct heterozygous RUNX1 mutations. These mutations were localized to the splice-acceptor site of intron 4, affecting splicing of exons 3 and 4, which encode the Runt homology/DNA binding domain (RHD) of RUNX1, or solely in the RHD or were present in both RHD and trans-activation domain (TAD). In two patients, we were able to perform allele-specific analysis of RUNX1 mutations. Patient #10 had an Phe13TrpfsX14 deletion on one allele of RUNX1 and an Arg139ProfsX47 deletion on the other allele. In Patient #14, two RUNX1 mutations were on the same allele; one of the mutations (Met240Ile) was inherited from the mother and was localized two amino acids before the TAD, and the second acquired mutation (Arg139Gly) was in the RHD of RUNX1. Ten patients with RUNX1 mutations developed monosomy 7 and six patients developed trisomy 21 at diagnosis of leukemia. In contrast to their high frequency in CN evolving into AML, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric AML. RUNX1 mutations were mainly found in pediatric AML patients with an adverse prognosis. A sequential analysis at stages prior to overt leukemia in ten CN/AML patients showed that RUNX1 mutation is a late event in leukemogenic transformation. In 6 of 10 patients, a CSF3R mutation occurred prior to RUNX1 mutations (24-192 months prior to CN/AML for CSF3R mutations vs. 1-36 months prior to CN/AML for RUNX1 mutations). Interestingly, monosomy 7 or trisomy 21 appeared after acquisition of RUNX1 mutations and no additional chromosomal aberrations were detected by array-CGH. Single-cell analyses in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Moreover, functional studies demonstrated elevated G-CSF-induced proliferation with diminished myeloid differentiation of hematopoietic CD34+ cells after co-transduction with mutated RUNX1 and CSF3R, in comparison to cells transduced with mutated RUNX1 or mutated CSF3R only. The importance of RUNX1 mutations in leukemogenic transformation was substantially strengthened by the analysis of a unique family with two siblings suffering from CN that subsequently transformed to AML. In both children, cooperating RUNX1 and CSF3Rmutations were detected that were not present in healthy family members. Taken together, the high frequency and the time course of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia suggests a unique molecular pathway of leukemogenesis similar as has been reported in the Gilliland-Griffin two-hit hypothesis for AML development. The concomitant detection of RUNX1 and CSF3Rmutations represents a useful biomarker for identifying CN patients with a high risk of progressing to leukemia or MDS. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: Key Points CN/AML patients have a high frequency of CSF3R and RUNX1 mutations. CSF3R and RUNX1 mutations induce elevated proliferation of CD34+ cells.

Description: Congenital neutropenia (CN) and Cyclic neutropenia (CyN) are rare hematological conditions in which ELANE mutations have been found. The discrimination of Cyn from CN is based on the cycling neutrophil counts which decrease to

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 11 Cyclic 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.

Description: Abstract 12 To 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.

Description: Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a high risk of evolving into leukemia or myelodysplastic syndrome (MDS). Recently we demonstrated a very high frequency of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia or MDS (Skokowa, et al. Blood 2014). We proposed a novel molecular pathway of leukemogenesis: mutations in the cytokine receptor (G-CSFR) in combination with the second mutations in the hematopoietic transcription fator (RUNX1). In the majority of CN patients, CSF3R mutations were acquired prior to RUNX1 mutations. CSF3R mutations alone are unable to induce leukemia in CN patients or in mice expressing a transgenic d715 G-CSFR. Co-acquisition of RUNX1 mutations is an essential step in the leukemogenic transformation in CN. To characterize the expression signature of hematopoietic cells of CN/AML patients carrying CSF3R mutations prior to and after acquisition of RUNX1 mutations, we analyzed expression profiles of CD34+ hematopoietic cells of CN patient who developed AML. This patient acquired CSF3R mutation (p. Q718*) five years and RUNX1 mutation (p. R139G) 16 months prior to leukemia. We compared expression profiles of CD34+ cells harbouring CSF3R mutation only, or both CSF3R and RUNX1 mutations. Co-acquisition of RUNX1 and CSF3R mutations led to marked reduction of the expression of hematopoietic growth factors such as IL6 and NAMPT, inhibitors of cytokine signaling SOCS3, as well as of components of neutrophil granules OLFM4, DEFA4, MMP8, SLPI, CRISP3 and CTSG. At the same time expression levels of pro-proliferative downstream effectors of G-CSF such as STAT5A, STAT5B, SMAD1 and cyclin A1 (CCNA1) were dramatically elevated. Moreover, genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD109, HPGDS, PDLIM1, MLLT11 and FLT3 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations. Intriguingly, elevated expression of DNTT, BAALC, CD109 and FLT3 was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests rapid transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after acquisition of RUNX1mutation. To elucidate the role of cooperative CSF3R and RUNX1 mutations on the clonogenic capacity and myeloid differentiation of hematopoietic progenitors, we performed functional studies in mice. We transduced lineage negative (lin-) bone marrow hematopoietic progenitor cells of WT or transgenic d715 G-CSFR mice with lentiviral expression constructs containing either WT or mutated forms of RUNX1 cDNA. We used two different mutants of RUNX1 by introduction of mutations at amino acid positions 139 and 174. Acquired RUNX1 mutations in these amino acids were presented with high frequency in our cohort of CN/AML patients and in most of the cases were associated with acquired CSF3R mutations. We found that similar to the effect of CSF3R mutations, lin-hematopoietic cells of WT mice transduced with mutated RUNX1 alone did not show elevated clonogenic capacity in replating experiments. Interestingly, transduction of WT cells with RUNX1 mutants resulted in severely reduced numbers of CFU-G colonies but unaffected CFU-M and BFU-E colonies. Intriguingly, transduction of lin- hematopoietic cells from transgenic d715 G-CSFR mice with RUNX1 mutants resulted in a markedly elevated clonogenic capacity in replating experiments, as compared to cells transduced with WT RUNX1 or control vector: numbers of colonies after second replating were 7 and 8 times higher in RUNX1-R139G and RUNX1-R174X mutants, respectively, in comparison to RUNX1 WT transduced cells. Moreover, granulocytic differentiation of lin- cells from d715 G-CSFR mice transduced with RUNX1-R139G mutant was severely diminished, in comparison to cells transduced with WT RUNX1, as revealed by 5-fold reduction of CFU-G colonies. Taken together, co-acquisition of RUNX1 and CSF3R mutations shifted the hematopoietic differentiation program towards more primitive hematopoietic progenitors with elevated proliferative capacity and reduced myeloid differentiation, which ultimately lead to leukemia. Disclosures No relevant conflicts of interest to declare.