ALBERT

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

Description: Background: Primary Autoimmune Neutropenia (AIN) is the most frequent type of neutropenia in children with a prevalence of 1/100,000 between infancy and 10 years of age. Primary AIN is caused by anti-neutrophil antibodies binding to neutrophil-specific antigens, resulting in a decrease of circulating neutrophils in the blood, but normal numbers of mature neutrophils in the bone marrow. Typically, primary AIN is present from infancy on until spontaneous remission in early childhood, when anti-neutrophil antibodies disappear. Patients with primary AIN show severe to moderate neutropenia but only rarely suffer from serious infections. Patients remain in the registry for follow up after normalization of blood counts to evaluate late secondary events. Aims: Here we describe the cohort of AIN pts with positive anti-neutrophil antibody testing documented by the European Branch of the SCNIR. We analyzed the course of neutropenia, the frequency of G-CSF treatment for AIN, the incidence of severe bacterial infections and administration of AB prophylaxis. Methods: We identified 102 primary AIN patients within the neutropenia cohort documented by the European Branch of the SCNIR since 1994. We classified primary AIN by positive anti-neutrophil antibody testing (95 pts) or severe neutropenia in peripheral blood with normal bone marrow morphology in patients with age under 5 years (7 pts). Results: Primary AIN has been identified in 102 (61 female; 41 male) pts. The median age of the cohort is 5.18 years (range 1.37-22.71 years), with 630.28 pt years under observation. Median age at diagnosis was 12.07 months (range 0.9-70 months). All pts are currently alive, 40 patients already resolved from primary AIN at a median age of 3.02 years (range 0.83-9.08 years). Median follow-up time after neutropenia had resolved was 2.25 years (range 0-9.27 years). Sixteen of 102 pts (15.7%) received intermittent G-CSF treatment with a median dose of 4.5 µg/kg/day compared to 4.77 µg/kg/day for the congenital neutropenia cohort of the SCNIR Europe. Analysis of infections (tab.1) showed less minor and severe infections comparing to congenital neutropenia (CN) pts. Life-threatening infections like liver abscesses were not seen in primary AIN patients but in 1.8% of CN pts. Twelve AIN pts (11.7%) have received antibiotic prophylaxis for prevention of infection, 6 pts intermittent and 6 pts continuously. However, antibiotic prophylaxis was usually stopped before termination of AIN. Due to the milder course of infections most AIN pts were able to go to Kindergarten and to live a normal life. In 3 pts additional auto-immune related diseases were identified (autoimmune thrombocytopenia, allergic colitis and Kawasaki syndrome) during AIN. Sixteen of 102 AIN patients received genetic analysis, with no mutation being detected. In addition to the 102 AIN pts we identified another 31 CN pts who have initially been classified as AIN due to positive anti-neutrophil antibodies, but who were later genetically confirmed as CN (15 ELANE+, 8 HAX1+, 2 SBDS+, 2 CXCR4+, 2 CSF3R+, 1 G6PC3+). This proportion of pts showed more and more severe infections compared to primary AIN. Genetic testing has been performed in these pts due to ongoing infections and prolonged neutropenia until school age. Conclusions: Pts suffering from primary AIN present with severe to moderate neutropenia. A minority of pts might require G-CSF treatment on demand/interventionally due to antibiotic resistant infections, but long-term G-CSF treatment is regularly not indicated. Primary AIN is a self-limiting condition and in most pts neutropenia resolves until early childhood. Accumulation of secondary diagnoses like autoimmune related diseases, though postulated, has not been confirmed for AIN pts by our data. In AIN pts with severe infections, or prolonged neutropenia CN should be ruled out by genetic analysis and/or bone marrow morphology. Registries are needed to document long-term data on primary AIN pts to analyse potential additional features of primary AIN, possibly other accompanying autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

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: 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: The mechanism of maturation arrest of bone marrow myeloid progenitors in severe congenital neutropenia (CN) patients is not fully elucidated. We found, that treatment of healthy individuals with G-CSF induces mRNA expression of GADD45b (Growth Arrest and DNA-Damage-inducible, beta) in CD33+ bone marrow myeloid progenitors. However, the expression of GADD45b was not activated in CD33+ cells of G-CSF treated CN patients. GADD45b functions as stress sensor downstream of G-CSF signaling and is essential in stress-induced murine myelopoiesis. Less is known about the function of GADD45b in the myeloid differentiation of human HSPCs. We hypothesized, that the inability of G-CSF to induce GADD45b expression might be a cause of diminished granulopoiesis in CN patients. To test this hypothesis, we inhibited GADD45b expression in CD34+ cells and iPSCs of healthy donors by introducing indels in exon 1 of the GADD45B gene using specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). We evaluated G-CSF-triggered myeloid differentiation of GADD45b-deficient iPSCs using embryoid body (EB)-based method and found that iPSCs cells present with severely diminished granulocytic differentiation upon GADD45b knockout, as assessed by FACS, CFU assay and morphological examination of cytospin slides. We also observed reduced G-CSF-mediated granulocytic differentiation of GADD45b-deficient CD34+ cells of healthy individuals in colony-forming units (CFU) assay and liquid culture differentiation followed by FACS analysis on day 7 and day 14. Importantly, rescue of GADD45b in HSPCs of one CN patient by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation, as compared to control transduced cells. These data strongly support the essential role of GADD45b in G-CSF-mediated granulocytic differentiation. GADD45b rescue analysis of additional CN patients cells is ongoing. To study the mechanism of GADD45b activation upon G-CSF stimulation of hematopoietic cells, we performed in silico analysis of GADD45B promoter and found putative binding sites for G-CSF responsive hematopoietic transcription factors, including CEBPA, CEBPB, KLF4, STAT3 and STAT5. Using the dual luciferase reporter assay with 1.6 kb region of the GADD45B gene promoter, we found that KLF4, STAT5, CEBPA and CEBPB activate GADD45b expression in a dose-dependent manner. Intriguingly, CEBPA expression is severely diminished in myeloid cells of CN patients (Skokowa et al., 2006) and we assumed that G-CSF is not able to activate GADD45b expression in CN patients because of defective CEBPA. To study the mechanism by which GADD45b mediates myeloid differentiation, we performed RNA sequencing of WT or GADD45b-deficient CD34+ HSPCs treated or not with G-CSF. Interestingly, in GADD45b-deficient cells, G-CSF failed to induce mRNA expression of several genes essential for granulocytic differentiation and granulocyte functions including GLI1, CAMP/LL37, MMP8, CD16, LCN2, OLFM4, CX3CR1, SIGLEC5, as compared to WT cells. Reactome and Gene Set Enrichment Analysis (GSEA) of RNA-Seq data sets also revealed deregulation of the "myeloid CEBPA network", "GLI proteins pathway" and "neutrophil degranulation pathway" in G-CSF-treated GADD45b-deficient CD34+ cells, as compared to control G-CSF-exposed cells. Of note, severely diminished expression of plasma CAMP/LL37 levels is a unique feature of CN patients (Y. Ye et al. 2015). In summary, our data suggest that GADD45b plays an essential role in granulocytic differentiation of human hematopoietic cells and inability of G-CSF to induce GADD45b expression in myeloid cells of CN patients may be a reason for the defective granulopoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Objectives: Epstein-Barr virus (EBV) is associated with lymphoproliferative disease in immunocompromised hosts and with human B-cell lymphomas and carcinomas. Adoptive transfer of virus-specific T cells is not practical when memory T cells from HLA-matched donors are not available. Hence, we designed T cells expressing EBV-specific chimeric antigen receptors (CARs) to bypass the need of matched memory T cells. The surface-bound glycoprotein 350 (gp350) was used as target, because it is abundantly expressed during lytic EBV replication and it can also be detected in EBV-immortalized cells. Methods: gp350-CARs were constructed by fusion of single-chain variable fragments of two high affinity gp530-specific human mABs (7A1 and 6G4) to CAR-backbones containing the CD28/CD3ζ domains. Transduction of human T cells from PBMC and cord blood with γ-retroviral vectors showed higher expression levels of 7A1-gp350-CAR than 6G4-gp350-CAR. Results: We used 293T cells expressing gp350 and B95-8 immortalized cells from a tamarin monkey (6-10% gp350+) and human B cells immortalized with the EBV laboratory strain M81 (30% gp350+) in order to compare the potency of gp350-CAR T cells in vitro. Both 7A1-gp350-CAR and 6G4-gp350-CAR were activated and proliferated in the presence of gp350+ cells, inducing cytotoxicity of the target cells. Pilot experiments in a preclinical humanized mouse model consisting of Nod.Rag mice transplanted with human cord-blood (CB) stem cells and infected with an EBV/fLUC strain, we could confirm persistence of CB-matched 7A1-gp350-CAR T cells in spleen, bone marrow and lung for up to 6 weeks. In some animals, this was correlated with lower EBV dissemination measured by optical imaging and PCR. Conclusions: We showed that EBV-specific CARs can reprogram naïve or memory T cells from PBMC or CB to react against EBV infected cells in an HLA-independent manner. This approach can be translated in the future for the generation of anti-EBV-CAR T cells for patients in Need. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. We recently reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. To study the mechanism of leukemia development in CN, we established a model for step-wise leukemia progression in CN using iPSC-based hematopoietic differentiation in combination with CRISPR/Cas9-mediated gene editing of iPSCs. Using this model, we confirmed that co-acquisition of CSF3R and RUNX1 mutations is necessary and sufficient to induce leukemia in CN. We also identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event downstream of RUNX1 and CSF3R mutations. BAALC mRNA was upregulated in CN/AML blasts (n = 5) and in CD34+ HSPCs generated from CN/AML iPSCs of two patients. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs reversed defective myeloid differentiation of CN/AML blasts to the levels observed in healthy donor hematopoietic stem cells. We further investigate the mechanism of BAALC up-regulation. In silico analysis of the BAALC gene promoter in combination to publicly available ChIP-Seq data revealed three putative RUNX1 binding sites that were validated using ChIP assay in lysates of NB4 cells. Interestingly, transduction of healthy donor CD34+ cells with lentiviral constructs expressing WT RUNX1 led to inhibition of BAALC mRNA expression, whereas transduction with two RUNX1 RUNT domain mutants resulted in the marked BAALC up-regulation, as compared to the control BFP transduced cells. These data suggest that mutated RUNX1 failed to inhibit BAALC expression in CD34+ HSPCs. To evaluate the mechanism of leukemogenic transformation in CN, we performed RNA-Seq analysis of CD34+ cells derived from CN and CN/AML iPSC clones. GSEA revealed that changes in gene expression between CN- and CN/AML-HSPCs were strongly correlated with gene expression signatures of "Wierenga STAT5 targets" and "reactome ATF4 targets", an observation in line with the markedly elevated levels of STAT5 and ATF4 in CN/AML-HSPCs. Importantly, gene expression differences between CN/AML-HSPCs and CN-HSPCs were correlated to "Valk AML" targets in GSEA, suggesting that HSPCs generated from CN/AML-iPSCs possess characteristics of AML cells. Strong support for the leukemogenic role of upregulated BAALC in CN/AML was provided by further GSEA analysis of the BAALC KO CN/AML-HSPCs. We observed a reversal in the expression of a majority of genes in the studied leukemia-associated pathways in CN/AML-HSPCs after BAALC knockout compared with CN/AML-HSPCs. Since there are no direct inhibitors for BAALC available and protein structure is not solved yet, BAALC effects can be targeted only indirectly. Morita et al., Leukemia, 2015 showed that BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and Klf4. We treated CD45+ cells generated from CN/AML or healthy donor (HD) iPSCs with MEK1/2- or MEK1-specific inhibitors or vehicle control and evaluated cell proliferation and differentiation (CFU assay). We were able to induce ~ 40-60 % cell death of CN/AML cells upon treatment with each of inhibitors, whereas HD CD45+ cells were unaffected. Moreover, treatment of CN/AML cells with MEK1/2 inhibitor led to an increase in CFU-G formation, as compared to vehicle control cells. Using connectivity Map analysis of RNA-Seq data of CD34+ cells generated from CN/AML iPSCs vs CN/AML BAALC KO iPSCs, we identified small molecule p38/MAPK14 inhibitor that could possibly reverse BAALC-mediated leukemogenic gene expression signature. We treated CN/AML iPSC-generated CD34+ cells for 7 days with this inhibitor and subsequently performed CFU assay. We found an increase in CFU-GM formation. In summary, using CN/AML-iPSC-model, we confirmed the major role of BAALC in leukemia development downstream of CSF3R and RUNX1 mutations in CN. Inhibition of MAPK/ERK-pathway downstream of BAALC reduced proliferation and partially induced myeloid differentiation of CN/AML-derived hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Description: Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1(mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation. We first investigated differences in DNA damage susceptibility of CD34+ and CD33+ bone marrow cells from CN-ELANE (n = 3) and CN-HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair (BRCA1 and RAD51), mismatch repair (MSH2 and PCNA) as well ascell cycle regulation (CHEK2 and CDKN2C) in CD33+ of both CN groups as compared to healthy individuals. Interestingly, CN-ELANE and CN-HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B, RAD50 and ATR expression in the CN-HAX1 group only and of MRE11A in the CN-ELANEgroup only. Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation. Disclosures No relevant conflicts of interest to declare.