ALBERT

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: Background: In Ontario, Canada's largest province, population-based health administrative data represents an accessible and useful tool for population surveillance of people with chronic diseases. While hemoglobinopathies can be identified using data from universal hemoglobinopathy screening, which was implemented in November 2006, these data would not contain information on affected immigrants (21.9% of the population). We validated algorithms using provincial health administrative data and newborn screening data to identify children with hemoglobinopathies whether or not they were born in Ontario, thereby creating a population-based surveillance cohort. Objectives: (1) Validate algorithms to identify children with sickle cell disease, thalassemia and other hemoglobinopathies from within health administrative data; and (2) Determine incidence and prevalence of hemoglobinopathies in Ontario children. Methods: For the validation study, a positive reference cohort was established using lists of known hemoglobinopathy patients who were followed at five pediatric hemoglobinopathy treatment sites in Ontario and born between November 24, 2006 and March 31, 2013. Health card numbers of these patients were linked deterministically to unique identification numbers in administrative data, which included data on hospitalizations, physician claims, sociodemographic characteristics, immigration records and cause of death. The negative reference cohort included all children residing in Ontario cities who had never been seen at a hemoglobinopathy centre, and therefore assumed not to have disease. Various combinations of administrative data codes were tested for their ability to identify children 80%. Using two validated algorithms, we identified all children with hemoglobinopathies born between April 1, 1991 and March 31, 2013. We described the crude incidence and prevalence per 100,000 patient-years (PYs). Results: Two algorithms functioned best to identify incident and prevalent hemoglobinopathy cases (see Table). Among children born between April 1, 1991 to March 31, 2013, 1526 incident hemoglobinopathy patients were identified using Algorithm 1 (crude incidence of 4.85 per 100,000 PYs) and 1660 new hemoglobinopathy patients were identified using Algorithm 2 (crude incidence 5.28 per 100,000 PYs, 95% CI 3.51 to 3.92). In 2013, the overall prevalence of children

Description: Introduction Inherited bone marrow failure syndromes (IBMFSs) are rare genetic disorders characterized by abnormal hematopoiesis resulting in cytopenias and increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Once patients develop MDS the only curative therapy is hematopoietic stem cell transplant (HSCT). The rate of progression from early MDS to advanced MDS and AML is variable and risk factors for progression in IBMFS patients are poorly defined. We hypothesized that certain variables could predict the likelihood of progression from early stages of IBMFS-associated MDS/clonal hematopoiesis to advanced MDS or AML, and that the type of disease progression may impact overall survival (OS). Methods Data were collected from patients prospectively enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR), a collaboration of 1 adult and 16 pediatric hospitals in Canada that care for 〉95% of pediatric IBMFS patients. IBMFS patients were diagnosed as having a specific syndrome or unclassified IBMFS (UCIBMFS) based on published criteria from our lab and others'. Diagnostic criteria for pediatric MDS defined by Hasle et al. were used. Progression of MDS was defined as 1 or more of: (1) a new cytogenetic abnormality, (2) progression in cytopathology from refractory cytopenia (RC) or refractory cytopenia with ringed sideroblasts (RCRS) to refractory cytopenia with dysplasia (RCD), refractory cytopenia with excess blasts (RCEB) or AML, or (3) increased degree of cytopenia severity. Time to progression was described by Kaplan-Meier analysis and risk factors were evaluated using the Cox proportional hazards model. Results Of 601 patients enrolled in CIMFR, 59 (9.8%) developed cytogenetic clones/MDS. Thirteen (22%) had Fanconi Anemia (FA), 13 (22%) had Shwachman-Diamond Syndrome (SDS), 10 (16.9%) had UCIBMFS, and 23 (39%) had other marrow failure syndromes (i.e. Dyskeratosis Congenita, Severe Congenital Neutropenia, Diamond Blackfan Anemia, GATA2-related disorders). The majority presented with cytogenetic clones/RC (n=45, 76%), 9 (15%) had RCEB, 3 (5%) RCD and 1 (1.7%) RCRS. The most common cytogenetic abnormalities at presentation were -7/-7q (n=18, 30%) and isochromosome 7q10 (n=7, 12%). Four patients had complex cytogenetics (6.8%). Of the patients who developed MDS, 32 (54%) went to HSCT. Patients who developed MDS had significantly worse OS (HR 3, 95% CI 2 to 6, p

Description: Background and Objectives. Phenotypic overlap among the inherited bone marrow failure syndromes (IBMFSs) frequently limits the ability to establish a diagnosis based solely on clinical manifestations. Since a large number of IBMFS genes (〉70) have been identified, genetic testing is often prolonged and costly. Correct diagnosis, care and counseling often depend on identifying the mutated gene. Thus time-efficient and cost-effective strategies for genetic testing are essential. The aims of this study were to develop and evaluate the application of a next generation sequencing (NGS) IBMFS Gene Panel assay for genetic testing of patients with previously characterized categories of IBMFSs (e.g. Fanconi anemia and Diamond Blackfan anemia) but unknown genotype, as well as patients with unclassified IBMFSs. Methods. We designed a NGS assay to test a comprehensive panel of 72 known IBMFS genes. Genomic DNA from patients enrolled on the Canadian Inherited Marrow Failure Registry was analyzed using the Haloplex technology and Illumina Seq2000 platform. The average gene coverage was 99.12%. SureCall program was used to align, map, and identify variants. Polyphen, Sift and MutationTaster were used to predict the effect of variants on the protein. Human Splicing Finder program was used to analyze effect of splicing. The assay was validated by detecting all 50 mutations and polymorphic variants that were previously found by Sanger sequencing in 31 patients. Results. A total of 158 patients with unknown mutations were studied. Among 75 patients with known categories of IBMFSs but unknown genotypes, we found deleterious mutations in 43 patients (57.3%). These categories included Diamond Blackfan anemia, Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, TAR syndrome, familial thrombocytopenia and Kostmann/severe congenital neutropenia. Among 83 patients with unclassified IBMFSs, we found deleterious mutations and established the diagnosis in 16 patients (19.2%). Established diagnoses included dyskeratosis congenita, Diamond-Blackfan anemia, myelokathexis, GATA2-associated familial MDS, WAS-associated severe congenital neutropenia, G6PC3-associated severe congenital neutropenia, MYH9-associated disorder, MASTL-associated disorder and Wiskott-Aldrich syndrome. All identified mutations were validated. The assay allowed identification of mutant genes that had not been previously reported to be associated with the patient phenotypes in two cases. The assay led to amendment of established diagnoses in two other cases. The assay results directed a change in clinical care in multiple cases, including implementation of cancer surveillance program and consideration for prenatal diagnosis. The cost of the NGS was $470/patient compared to $4643/patient among those who underwent genetic testing by Sanger sequencing during the tenure of the study. Conclusion. Our novel assay is a rapid, accurate, and cost saving strategy for genetic investigation of patients with IBMFSs. It can identify mutations in classified and unclassified IBMFSs with high level of sensitivity and precision. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1266 Introduction: Inherited bone marrow failure syndromes (IBMFSs) are a group of rare, genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The clinical characteristics and outcome of IBMFS-related clonal and malignant myeloid transformation are unclear, particularly in cases of early transformation such as isolated clonal marrow cytogenetic abnormalities. Objectives: The aims of this study were to determine the risk and clinical outcome of IBMFS-related clonal and malignant myeloid transformation using data from the Canadian Inherited Marrow Failure Registry (CIMFR). Methods: The CIMFR is a multicenter collaborative study which is intended to enroll all patients with IBMFSs in Canada. The registry was approved by the Institutional Ethics Board of all the participating institutions, and includes 15 of 16 pediatric tertiary care centers across all provinces in Canada. We estimate that these centers care for 〉95% of the eligible pediatric IBMFS population in Canada. The CIMFR is population-based as 〉90% of the patients in this study are from centers who enrolled 〉80% of the patients in their institutions. Clonal and malignant myeloid transformation was defined as having either clonal marrow cytogenetic abnormalities or prominent bi-lineage morphologic dysplasia or increased percentage of marrow blasts (≥5%) or a combination of the above. Results: Among 327 IBMFS patients enrolled on the CIMFR, 45 (13.8%) developed clonal and malignant myeloid transformation. In these 45 patients, the three most common IBMFS diagnoses were Fanconi anemia (31.1%), Shwachman-Diamond syndrome (20.0%) and unclassifiable IBMFSs (28.9%). Clonal marrow cytogenetic abnormalities were identified in 38/45 (84.4%) patients, while 5/45 (11.1%) patients had constitutional cytogenetic changes, 1/45 patients had AML with no cytogenetic abnormalities and 1/45 patients had no cytogenetic abnormalities. Two out of the 5 patients with constitutional cytogenetic abnormalities developed a clonal marrow cytogenetic abnormality later in their disease course. The most common clonal marrow cytogenetic abnormality was monosomy 7, which was found in 14/38 (36.8%) patients. Cytology in the majority of patients 20/45 (44.4%) was consistent with refractory cytopenia. Eight out of the 45 patients developed AML and 2 of these patients had monosomy 7. Twenty-two out of 45 (48.9%) patients with clonal and malignant myeloid transformation underwent hematopoietic stem cell transplantation due to severe cytopenia, excess blasts or leukemia. Fourteen out of the 22 (63.6%) transplanted patients are alive at last follow-up. Out of 8 patients who had AML, 3 received transplant and are alive at last follow-up. The 5 remaining AML patients died; 3 while awaiting transplant, 1 did not achieve remission and 1 refused transplant. Overall mortality in the group of patients with clonal and malignant myeloid transformation was 15/45 (33.3%) at a median follow-up of 10 months from diagnosis with clonal and malignant myeloid transformation. Overall mortality in those 282 patients on CIMFR without clonal and malignant myeloid transformation is 6.4%. Conclusion: Despite short-term follow-up of patients on the CIMFR, a relatively high prevalence of clonal and malignant myeloid transformation was found. Clonal marrow cytogenetic abnormalities are associated with a high risk of progression into advanced MDS or AML and death. Disclosures: No relevant conflicts of interest to declare.

Description: Background: DDAVP (desmopressin) is commonly used for the prophylaxis and treatment of bleeding in patients with mild forms of von Willebrand disease (VWD) and Hemophilia A (HemA). The standard dose is 0.3 ug/kg IV, to a maximum of 20 ug. In 1995, our Bleeding Disorders Program began to use a maximum dose of 15 ug both for DDAVP challenges and for therapeutic purposes. This dosing change was triggered by the new availability of 15 ug (1 ml) vials of DDAVP. We also switched to subcutaneous administration rather than intravenous, given the smaller volume (1 ml), and the body of evidence supporting this route of administration (De Sio et al, Thrombosis and Hemostasis1985; 54:387–9). With this strategy, patients weighing 50 kg with VWD or mild hemophilia A.

Description: Background. Inherited bone marrow failure syndromes (IBMFSs) comprise a genetically heterogeneous group of diseases with hematopoietic failure and varying degrees of physical malformations. The diagnosis of an IBMFS and categorizing the specific syndrome critically impact on clinical care; however, these are commonly challenging and rely on genetic testing. Since over 80 genes have been associated with IBMFSs and might be affected by different types of DNA aberrations, the best strategy to establish a diagnosis in a timely and cost effective manner is unknown. The aims of this study were to evaluate the role of genome-wide copy number variant (CNV) analysis in unraveling causal genetic alterations in IBMFS patients with unknown genotype and determine whether correlation exists between large CNVs and more severe phenotype. Methods. Patients from the Canadian Inherited Marrow Failure Registry (CIMFR) who were genetically investigated were included in this analysis. Genetic and clinical data were extracted and analyzed. Mann-Whitney test and Fisher's exact test were used to assess statistical significance. Results. Among 328 patients from the CIMFR who underwent molecular investigation, a causal genotype was identified in 185 cases (56.4%). 69 patients had genome-wide CNV analysis by SNP/CGH arrays, among which ten (14.5%) had positive results. In four out of ten cases who were genotyped by SNP/CGH array, genome-wide CNV analysis was critical for establishing the diagnosis. Among 308 patients who were tested for nucleotide-level mutations by either targeted gene analysis or next generation sequencing panels, casual mutations were found in 169 (54.9%). Three patients had compound heterozygosity for a CNV and nucleotide-level mutation. To determine whether large deletions are correlated with more severe phenotype we included nine additional patients with causal CNVs whose genotype was identified by MLPA (n=1), targeted FISH (n=1), DNA-qPCR analysis (n=1), Southern blotting (n=1) or metaphase cytogenetics (n=5). The causal CNVs among patients in our cohort ranged from 0.02 to 145.5 Mb in size. The most common disease associated with causal CNVs was Diamond-Blackfan anemia (four patients). Patients with CNVs tended to have significantly more non-hematological organ system involvement (p=0.03), developmental delay (mean=56% vs. 28%, p=0.03) and short stature (mean=67% vs. 40%, p=0.04) than patients with nucleotide-level mutations. The difference remained significant when we compared all patients with mutations that are predicted to result in truncation or lack of protein from the respective allele (large CNV, nonsense, and indel/ frameshift) to patients with mutations that are predicted to be hypomorphic or affect function (splicing, indel/ inframe and missense). There was no correlation between CNVs and the severity of the hematological disease. Conclusions. Most patients with IBMFSs have nucleotide-level mutations. However, a significant proportion of patients without such mutations have large CNVs that are not efficiently detected by current nucleotide-level testing methods. Therefore, genome-wide CNV analysis should be considered in IBMFS cases, where nucleotide-level sequencing does not reveal the causal mutation. Patients with IBMFSs and large CNVs had more non-hematological organ system involvement, a higher prevalence of developmental delay and short stature. This might be related to an additional impact of the CNVs on other genes close to the affected IBMFS gene or the severe damaging effect of the CNVs. Disclosures Lipton: Teva: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Description: Introduction: Inherited bone marrow failure syndromes (IBMFS) are characterized by single or multi-lineage cytopenias as well as non-hematologic manifestations. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the hematological abnormalities. However, high rates of mortality and morbidity from this procedure have been reported in patients with IBMFSs. Objective: The study aim was to investigate the impact of patient, donor and treatment-related variables on the outcome of HSCT in IBMFS patients. Methods: Data of patients who were prospectively enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR) from 2001 to 2012 and underwent HSCT were analyzed. The CIMFR is a population-based multicenter study, which includes all 16 pediatric tertiary care centers across all provinces in Canada. These centers care for 〉95% of the eligible pediatric IBMFS population in Canada. Descriptive analyses, as well as univariate and multivariate analyses (using a Cox proportional hazards model) were performed to assess the impact of multiple factors on probability of survival following transplant. Results: Among 363 patients enrolled in CIMFR, 65 underwent allogeneic HSCT. Thiry-four patients were male and 30 were female (gender unknown for 1 patient). Median age at diagnosis with IBMFS was 3.0 years (range: prenatal diagnosis to 32.0 years) and median age at HSCT was 6.5 years (range: 0.25-20.1 years). Median follow-up time post-HSCT (time to death or last follow-up) was 2.8 years (range 0.01–15.9 years). Indications for transplant included severe cytopenia (n=44), myelodysplastic syndrome (n=18) acute myeloid leukemia (n=2) and unavailable cause (n=1). Cell type were bone marrow (n=40), cord blood (n=17), peripheral blood (n=5), unknown (n=3). Sixty-two percent of patients (n=40) received cells from an unrelated donor. Seventy-four percent (n=47) of patients had a full HLA-matched donor; 19 of those were related and 28 were unrelated donors. The most common conditioning regimen combined high dose cyclophosphamide, fludarabine and anti-thymocyte globulin (n=17). Incidence of graft failure, acute (grade II-IV) graft versus host disease (GVHD) and chronic GVHD was 14%, 30% and 22%, respectively. Five-year probability of survival for HSCT recipients was 73.4% ± 6.1% (SE). Causes of death included infections (CMV, fungal infections and bacterial), GVHD, lymphoproliferative disorder and congestive heart failure, pulmonary fibrosis, bronchiolitis obliterans. In the univariate analysis, factors significantly associated with increased mortality post-HSCT included ≥20 pre-HSCT platelet transfusions, pre-transplant administration of granulocyte colony-stimulating factor, 1 or more HLA mismatches, donor type (divided into four categories: matched related, partially matched related, matched unrelated, partially matched unrelated) and conditioning regimens combining high doses of cyclophosphamide with fludarabine and anti-thymocyte globulin (while comparing this combination against all others). The former 3 variables remained significant in the multivariate analysis. Conclusion: Number of pre-transplant platelet transfusions, use of granulocyte colony-stimulating factor and one or more donor-recipient HLA mismatches were shown to increase the risk of mortality in patients with IBMFSs undergoing HSCT. Novel strategies are needed to improve outcome in patients with a high risk of HSCT-related complications. Disclosures No relevant conflicts of interest to declare.

Description: There is an Inside Blood Commentary on this article in this issue.