ALBERT

Description: Background: In 1994 Severe Chronic Neutropenia International Registry (SCNIR) opened for enrollment of patients with at least 3 absolute neutrophil counts (ANC) less than 0.5 x 109/L during a three month period. At that time severe chronic neutropenia (SCN) was categorized as cyclic, congenital, autoimmune or idiopathic based largely on clinical criteria. A randomized trial had established effectiveness of treatment with granulocyte colony-stimulating factor (G-CSF), but long-term consequences of such treatment were unknown. Hypothesis: We began the SCNIR based on the hypothesis that underlying pathophysiology, natural history of patients with chronic neutropenia and benefits and risk of G-CSF therapy could only be accurately established through an international registry with long term follow-up of patients with these rare hematological disorders. Methods: SCNIR enrollment requires informed consent, ANC90%) of severe outcomes (e.g. MDS/AML, failure to respond to G-CSF, death from infections, need for stem cell transplant) often many years after SCNIR enrollment and beginning G-CSF therapy. GSD1 patients improve with G-CSF treatment, but experience splenomegaly and continued problems with infections or complications. The SCNIR through a SDS sub-registry is redefining Shwachman-Diamond syndrome; only about one-half of enrollees have “classic” presentation and a substantial number with “classic presentation” lack mutations in SBDS. The SCNIR is participating in an NIH trial of a CXCR4 antagonist for treatment of WHIM syndrome, as an example of molecularly targeted treatment for this rare disease. The SCNIR is also the key resource for discovery of genetic causes for congenital neutropenia, e.g., G6PC3, HAX1, and TCIRG1 and others, recognition of differences in frequency of autosomal dominant and recessive SCN in populations of Europe and North America and identifying congenital neutropenia cases of unknown cause. Genetic testing has also broadened the clinical spectrum of these disorders. Conclusions: Through the efforts of patients, families, physicians, nurses and investigators, and with support from the NIH, industry, and private philanthropy, chronic neutropenia is now far better understood at the genetic, molecular and cellular level than 20 years ago. Treatment responses to G-CSF are well characterized; novel therapies are emerging; and the prognosis for patients with SCN appears to be improving. The knowledge gained through the SCNIR and availability of G-CSF has redefined clinicians’ approach to chronic neutropenia. The SCNIR is a model of international research collaboration to understand rare diseases in hematology and other areas of medicine. Broad enrollment criteria, physician, patient and family participation, a dedicated staff, and continuing cooperation underlie success of the SCNIR and this model to understanding rare diseases. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding. Boxer:Amgen: Equity Ownership. Morrow:Amgen: Employment.

Description: Inherited bone marrow failure syndromes (IBMFs) are a group of rare genetic disorders characterized by deficient hematopoiesis and extra-hematologic traits. Most known entities are related to a specific gene or group of genes, but others still remain unclassified. In many cases, the involved proteins are required for critical processes involved in cell survival, such as ribosome biogenesis, maintenance of telomere length, and DNA repair. Importantly, patients with IBMFs have higher risks of developing a variety of cancers from leukemia to solid tumours of the head and neck. In 2015, we published four patients from three different families with mutations in the Poly(A)-specific ribonuclease (PARN) gene. This gene encodes a ribonuclease which is involved in degradation of the poly(A) tails, which regulate mRNA turnover, and thus gene expression. Three of the patients presented with several degrees of mental illness and/or developmental delay. The fourth patient, harbored both a monoallelic deletion and a point mutation at the catalytic domain of the protein, and presented with bone marrow failure and hypomyelination, similar to a severe form of dyskeratosis congenital (DC) known as Hoyeraal-Hreidarsson syndrome. In the last two decades, zebrafish (Danio rerio) has emerged as an excellent animal model for human disease, and is especially relevant in hematology, since many of the transcription factors and cell types are highly conserved. Zebrafish have a single PARN ortholog,with 64% sequence identity to the human gene. Using CRISPR-Cas9 genome editing and a combination of six sgRNAs, we generated a 1.2 kb deletion in the zebrafish parn ortholog extending from exon 5 to 13, causing a premature stop codon. Homozygous fish were generated by incrossing to replicate the complete loss-of-function observed in the patient with the DC-like phenotype. Using whole-mount in situ hybridization (WISH) at 48 hours post-fertilization (hpf), we observed a decrease in the number of several mature myeloid cell lineages including neutrophils (labeled with mpx; p

Description: Shwachman-Diamond syndrome (SD), an inherited disorder with varying cytopenias and a marked tendency for malignant myeloid transformation, is an important model for understanding genetic determinants in hematopoiesis. To define the basis for the faulty hematopoietic function, 13 patients with SD (2 of whom had myelodysplasia with a clonal cytogenetic abnormality) and 11 healthy marrow donors were studied. Patients with SD had significantly lower numbers of CD34+ cells on bone marrow aspirates. SD CD34+ cells plated directly in standard clonogenic assays showed markedly impaired colony production potential, underscoring an intrinsically aberrant progenitor population. To assess marrow stromal function, long-term marrow stromal cell cultures (LTCs) were established. Normal marrow CD34+ cells were plated over either SD stroma (N/SD) or normal stroma (N/N); SD CD34+cells were plated over either SD stroma (SD/SD) or normal stroma (SD/N). Nonadherent cells harvested weekly from N/SD LTCs were strikingly reduced compared with N/N LTCs; numbers of granulocyte-monocyte colony-forming units (CFU-GM) derived from N/SD nonadherent cells were also lower. SD/N showed improved production of nonadherent cells and CFU-GM colonies compared with SD/SD, but much less than N/N. Stem-cell and stromal properties from the 2 patients with SD and myelodysplasia did not differ discernibly from SD patients without myelodysplasia. We conclude that in addition to a stem-cell defect, patients with SD have also a serious, generalized marrow dysfunction with an abnormal bone marrow stroma in terms of its ability to support and maintain hematopoiesis. This dual defect exists in SD with and without myelodysplasia.

Description: Abstract 549 Barth syndrome (BTHS) is a severe X-linked stem cell disorder characterized by neutropenia, cardio- and skeletal myopathies, and growth retardation. Barth patients have a high rate of mortality due to progressive cardiomyopathy and/or overwhelming bacterial infections. Majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene that appear to truncate the tafazzin protein resulting in the loss of TAZ function. Based on protein homology, tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin (CL), the main lipid of the inner mitochondrial membrane. Therefore, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. However, the function of TAZ protein and how these metabolic defects are triggered by TAZ mutations in BTHS remain largely unknown. The cellular or mouse models of this disorder are not availabel yet, and thus, the link between TAZ mutations and severe neutropenia in Barth syndrome remains elusive. Earlier study reported increased annexin V staining but absence of apoptosis in peripheral blood neutrophils. However, the patients' bone marrow stem and myeloid progenitor cells have not been examined. We hypothesized that TAZ mutations trigger accelerated apoptosis of bone marrow stem/progenitor cells, which in turn leads to reduced production of neutrophils in the bone marrow and severe neutropenia in Barth patients. To test this hypothesis, we used TAZ-specific shRNA to knock down the expression of the tafazzin gene in human myeloid progenitor HL60 cells and examined its effect on cell survival. Transfection of human myeloid progenitor cells with two different TAZ-specific but not control scrambled shRNA results in substantial down-regulation in the tafazzin expression level as determined by Western blot and confirmed by RT-PCR using TAZ and GAPDH-specific primers. Human myeloid progenitor cells with knocked down TAZ expression exhibit significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA as evidenced by flow cytometry analysis of DIOC6-labeled cells ((p

Description: Severe congenital neutropenia (SCN) is a heterogeneous disorder of myelopoiesis characterized by an absolute neutrophil count (ANC) persistently below 0.50 x 109/L (500/[um]L), with maturation arrest of neutrophil precursors of the bone marrow at the promyelocyte/myelocyte stage. G-CSF treated and untreated SCN patients are at risk of developing MDS/AML. Clinicians caring for these patients must vigilantly observe for evidence of evolution to malignancy and continually consider hematopoietic transplantation as an alternative therapy. We have previously reported that SCN patients requiring higher daily doses of G-CSF treatment (〉 8 mcg/kg/day) are at an increased risk of MDS/AML, and that this risk is not predicted by pre-treatment bone marrow examinations or the pre-treatment ANC. We have reviewed clinical data for 46 patients over a 15-year period who were referred to the Severe Chronic Neutropenia International Registry (SCNIR) and developed MDS/AML. Four patients referred with a history of SCN had increased blasts on the bone marrow evaluation prior to G-CSF treatment or enrollment in the SCNIR. Blood counts were not available for two patients. For the other 40 patients, all treated with G-CSF for a median of 79 months (range 0.8 to 182 months), comparison of blood counts for the periods 12–24 months and 0–3 months before the diagnosis of MDS/AML showed: Changes in blood counts for the patient population may suggest a potential for malignant transformation. In these 40 cases, the principal clinical findings leading to the diagnosis of MDS/AML were: routine bone marrow surveillance 38%, decreased peripheral blood counts 26%, increased infections 10%, increased blasts in the blood 5%, decreased ANC with requirement for increased G-CSF dosage 2%, hepatosplenomegaly 2%, other (1 septal panniculitis and 1 leukemia cutis) 5%, and unknown 12%. The finding of mutations in the ELA2 gene in 10 of 15 cases (67%) did not influence the likelihood of MDS/AML. Previously we found that the duration and dose of G-CSF and alterations of the ANC on G-CSF therapy, suggest a high risk of transformation over time. Based on these data, the SCNIR recommends that patients with SCN have regular surveillance of blood counts every 1 to 2 months and annual bone marrow examinations with cytogenetic studies. Our findings suggest that a decline or change in blood counts of individual patients over a 3-month period may be informative in recognizing transformation to MDS/AML.

Description: Abstract 3203 Poster Board III-140 Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. We hypothesize that SBDS protects early erythroid progenitors from apoptosis by promoting ribosome biosynthesis and translation. During early erythroid differentiation of human K562 cells and primary CD133+ cells, a prominent upregulation of SBDS by RT-qPCR was found. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and a mild reduction in proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. Both cell expansion and translation defects were rescued upon re-introduction of SBDS in K562 cells. Interestingly, leucine partly corrected the cell expansion and translational defects of non-differentiating SBDS-deficient K562 cells, while differentiating SBDS-deficient K562 cells showed improved cell expansion in the presence of additional translation stimulators such as IGF-1. SBDS-knockdown CD133+ cells showed increased BFU-E colony formation under conditions with leucine and a combination of leucine and IGF-1 treatment. Although the erythroid cell expansion defect in K562 cells is independent of p53 as these cells do not express the gene, an upregulation of TAp73, was found in resting SBDS deficient K562 cells. However expression of TAp73 was lost during differentiation. DNp63 was also not upregulated in SBDS-deficient K562 erythroid cells. These results demonstrate that the role of SBDS in non-differentiated cells versus differentiated cells represents two dynamic scenarios and that SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation through a pathway independent of p53 family members. Disclosures No relevant conflicts of interest to declare.

Description: Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations. Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes. Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (p=0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p〉0.1). However, patients with RPL5 mutations had significantly more hand malformations (p=0.02), and patients with RPS26 mutations had more genitourinary malformations (p=0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (p=0.58). Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. Disclosures Lipton: Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Description: Background: Over the last decade major progress has been made in developing new diagnostic methods and in phenotypic and molecular classification of inherited bone marrow failure syndromes (IBMFSs). Nevertheless, data from the Canadian Inherited Marrow Failure Registry (CIMFR) indicates that 28% of patients with inherited bone marrow failure syndromes (IBMFS) cannot be assigned a specific syndromic diagnosis. These unclassified IBMFS (UIBMFS) cases may represent either novel syndromes or atypical presentations of previously described disorders. Hematopoietic stem cell transplantation (HSCT) is the only curative option for bone marrow failure and malignant myeloid transformation in IBMFSs. However, it is unknown whether the application of this treatment to UIBMFS patients without an ability to modify the procedure according to the underlying genetic and syndromic diagnosis affects outcome. To our knowledge, there are no published transplant data on cohorts of patients with UIBMFSs. The aims of this study were to evaluate the outcome and prognostic factors of HSCT in a cohort of patients with UIBMFSs and to determine whether the knowledge of the syndromic/genetic diagnosis before HSCT has an impact on transplant outcome. Methods: Patients were enrolled on the CIMFR if they were diagnosed with a specific IBMFSs (e.g. Fanconi anemia), and/or they had bone marrow failure and either a family history of bone marrow, or physical malformations or a diagnosis before the age of one year. Patients were considered as having an UIBMFS if they fulfilled the above criteria, but could not be assigned a specific syndromic diagnosis since they did not meet the diagnostic criteria for any known IBMFS. HSCT data were extracted from the CIMFR database and analyzed. Descriptive statistics were used to compare between groups. Cox proportional hazards model was used for univariate analysis to identify risk factors for worse overall survival post HSCT in patients with UIBMFSs. Results: Among the patients enrolled in the CIMFR, 22 with UIBMFSs and 68 with classified IBMFSs (CIBMFSs) underwent HSCT between January 2001 and December 31, 2017. Transplanted patients with UIBMFSs were hematologically characterized by multilineage cytopenia (n=13), single-lineage cytopenia (n=1), myelodysplastic syndrome (MDS) (n=5) or acute myeloid leukemia (AML) (n=3). Patients with CIBMFSs had Fanconi anemia (n=30), dyskeratosis congenita (n=7), Shwachman-Diamond syndrome (n=9), Kostmann syndrome (n=6), Diamond-Blackfan anemia (n=4) or others (n= 11). Median age at diagnosis of patients with UIBMFSs was 4.18 years (range; 0 to 32.0 years) and median age at HSCT for UIBMFSs was 5.74 years (range; 0.17-66.67 years). Median time between diagnosis of UIBMFS and HSCT was 0.48 years (range; 0.12 - 34.67), this was significantly shorter than that of CIBMFS (1.77 years, range; 0.17 - 15 years, P=0.014). Six patients (27.3%) of UIBMFS and 9 patients (19.7%) with CIBMFS underwent HSCT for MDS-RCEB or AML (P=0.15). The overall 5-year survival of UIBMFS patients was significantly inferior to that of CIBMFS patients: 56±11.4% vs. 76±5.5%, respectively (P=0.047). 5-year overall survival of patients with UIBMFSs was significantly worse among those whose stem cell source was cord blood (15±13.3%) vs. those who received other stem cell sources (91±8.7%, P=0.04), while stem cell source did not affect prognosis of patients with CIBMFSs. Engraftment failure among UIBMFS patients who received cord blood was significantly higher than engraftment failure among those who received bone marrow (55.6% vs. 9.1%, P=0.024). No other factors reached statistical significance when the impact of stem cell source on overall survival was analyzed, including transfusion load, transplant indications, intensity of conditioning regimens, related/non-related donor, degree of human leukocyte antigen (HLA) matching or identifying a diagnosis after HSCT. Conclusion: Identifying the syndromic diagnosis of IBMFSs is critically important when considering HSCT. The worse HSCT outcome of UIBMFSs in this study might be related to an inability to tailor the transplant approach to the patient specific phenotype and genotype. Our data suggest that cord blood should be avoided as a stem cell source in patients with UIBMFSs. Disclosures No relevant conflicts of interest to declare.

Description: Diamond Blackfan Anemia (DBA) is a rare inherited anemia caused by disruption of erythroid progenitor cell development at the burst-forming and colony-forming units. Approximately 50% of DBA patients have additional non-hematopoietic abnormalities including heart defects and short stature. Sixteen genes encoding specific ribosomal proteins (RPs) and the transcription factor GATA1 have been found mutated in approximately 65% of DBA patients, with the RPS19 gene being mutated in 25% of patients. Because RPs are components of the ribosome that is critical for protein synthesis, the identification of RP mutations suggest a translation deficiency as a mechanism for erythroid progenitor failure. However, erythropoietic failure in DBA may arise by causes other non-translational functions of the DBA genes. We have previously reported that inhibiting the heme exporter protein FLVCR1 in human hematopoietic stem cells and progenitors (HSC/Ps), is sufficient to specifically block erythropoiesis, which mimics the hematological features observed in DBA. Moreover, we reported aberrant splicing of exons (E) 2 and 3 of the FLVCR1 transcript in DBA erythroid cells that resulted in disruption of FLVCR1 protein expression and function. We observed aberrant FLVCR1 splicing in erythroid cells from patients with RPS19 and RPL5 mutations and from typical DBA patients whose genetic background has not been known at that time; thus, suggesting FLVCR1 dysfunction as central for the DBA erythropoietic failure. In the present work, we obtained evidence suggesting that DBA pathogenesis is caused by abnormality in the cellular splicing machinery. We performed In silico analysis of the binding motifs of seven SR proteins on each of the FLVCR1 exons. We found binding motifs for TRA2-β, SRSF1 and 9G8 on E2 and/or E3, the two FLVCR1 exons that are skipped in DBA erythroid cells and in RPS19-disrupted K562 cells. We focused on expression analysis of TRA2B and SRSF1 in RPS19-reduced K562 cells. Expression of both TRA2B and SRSF1 mRNA were significantly disrupted in RPS19-disrupted K562 cells, which correlated with disrupted expression of their respective proteins. To test the specificity of disrupted SR splicing regulators and aberrant FLVCR1 splicing, we analyzed TRA2-β, and SRSF1 protein expression, and FLVCR1 aberrant splicing, in K562 cells disrupted in SBDS gene. The SBDS gene product is critical for ribosome biogenesis and the gene is mutated in 90% of patients with Shwachman-Diamond Syndrome. Interestingly, K562 cells down-regulated in SBDS showed normal levels of TRA2-β and SRSF1 expression, and no FLVCR1 aberrant splicing was observed. Importantly, erythroid differentiation potential of the DBA cell model can be rescued by expression of exogenous Tra2-β cDNA or FLVCR1 cDNA. Altogether our data suggests that mutations in the ribosome protein genes in DBA result in down-regulation of the splicing factors, TRA2-β and SRSF1, which impair normal FLVCR1 splicing and consequently erythropoiesis failure. Disclosures No relevant conflicts of interest to declare.