Canadian Inherited Marrow Failure Registry Research Articles

Predictors of Disease Progression and Survival in Patients with Myelodysplastic Syndrome Secondary to Inherited Bone Marrow Failure Syndromes

Introduction Inherited bone marrow failure syndromes (IBMFSs) are rare genetic disorders characterized by abnormal hematopoiesis resulting in cytopenias, risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Once patients develop MDS, the only cure is hematopoietic stem cell transplant (HSCT). Proceeding to HSCT before transformation to advanced MDS or AML leads to better outcomes (Smith et al., PBC 2013). The rate of disease progression from early MDS to advanced MDS or AML is variable and risk factors for progression in IBMFS patients are unknown. We hypothesized that certain variables could predict the likelihood of progression, 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 Canadian collaboration including >95% of pediatric IBMFS patients. Patients were diagnosed with a specific syndrome or unclassified IBMFS (ucIBMFS) based on published criteria. Diagnostic criteria for pediatric MDS defined by Cada et al. (Haematologica 2015) were used, patients with refractory cytopenia (RC) with or without dysplasia or ringed sideroblasts were included. Patients with refractory cytopenia with excess blasts (RCEB) were excluded. MDS progression was defined as 1 or more of: a new cytogenetic abnormality, more advanced cytopathology, worse cytopenias requiring HSCT. Variables evaluated included: involvement of specific organ systems, hematologic parameters within 3 months of cytogenetic clones/MDS diagnosis, type of mutation (as per Waespe et al., Genomic Medicine 2017). Univariate Cox Proportional Hazard modelling was used to estimate association with specific variables and disease progression, Kaplan-Meier analysis was used for survival evaluation. Results Between January 1, 2001 and November 30, 2018, 601 patients were enrolled in the CIMFR, and during this time 59 (9.8%) developed cytogenetic clones/MDS. Four did not meet the pre-defined criteria for IBMFS, 9 had RCEB at diagnosis with MDS and 1 did not have data available; these were excluded from our cohort of 45 patients. Twelve (26%) patients with clones/MDS had Fanconi Anemia (FA), 13 (28.3%) had Shwachman-Diamond Syndrome (SDS), 5 (10.8%) had ucIBMFS, 15 (33.3%) had other IBMFSs. The most common cytogenetic finding at diagnosis was -7/-7q (n=10, 22%). Patients who developed clones/MDS had significantly worse OS compared to those without clones/MDS (HR 3, 95% CI 2-6, p<0.0001). Among patients who developed clones/MDS, 22 (48.9%) underwent HSCT. Twenty one clones/MDS patients (46.7%) had disease progression, with a median time to progression of 7.9 months (1.2-131.4). The majority (68.4%) developed progression within one year of clones/MDS diagnosis. Nine (42.8%) developed more advanced cytopathology, 9 (42.8%) a new cytogenetic abnormality and 5 (23.8%) worsening cytopenias. Most patients with FA (67%) had disease progression, compared to 38.5% of patients with SDS. Mutation types (classified as more vs less deleterious) were similar between the groups with and without progression. The evaluated hematologic parameters (MCV, hemoglobin, platelets and neutrophils within 3 months of diagnosis) were not predictive of disease progression. Patients with disease progression, when compared to those without, were more likely to have congenital malformations of the skin (14.3% vs 0%), cardiovascular system (28.6% vs 4.2%) and kidneys (14.3% vs 4.2%). No specific system involvement predicted disease progression, although congenital malformations of the cardiovascular system had a HR of 2.63 (95% CI 0.93-7.42). Conclusion Development of MDS has a significant adverse impact on the OS of IBMFS patients, with disease progression typically occurring within the first year after MDS diagnosis. Patients with FA have a high risk of disease progression. Hematologic parameters were not predictive of the risk of disease progression, however, the presence of congenital cardiovascular anomalies is suggestive of increased risk of disease progression and warrants further investigation. Our study is limited by the rare nature of IBMFSs and MDS and the collection of additional data will help to guide treatment decisions for this high-risk patient population. Acknowledgment The authors thank Mr. Bryan Maguire for his statistical analysis of data related to this work.

Poor outcome after hematopoietic stem cell transplantation of patients with unclassified inherited bone marrow failure syndromes.

Classification of inherited bone marrow failure syndromes (IBMFSs) according to clinical and genetic diagnoses enables proper adjustment of treatment. Unfortunately, 30% of patients enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR) with features suggesting hereditability could not be classified with a specific syndromic diagnosis. We analyzed the outcome of hematopoietic stem cell transplantation (HSCT) in unclassified IBMFSs (uIBMFSs) and the factors associated with outcome. Twenty-two patients with uIBMFSs and 70 patients with classified IBMFSs underwent HSCT. Five-year overall survival of uIBMFS patients after HSCT was inferior to that of patients with classified IBMFSs (56% vs 76.5%). The outcome of patients with uIBMFS who received cord blood was significantly lower than that of patients who received other stem cell sources (14.8% vs 90.9%). Engraftment failure was higher among patients with uIBMFS who received cord blood than those who received bone marrow. None of the following factors were significantly associated with poor survival: transfusion load, transplant indication, the intensity of conditioning regimen, human leukocyte antigen-identical sibling/alternative donor. We suggest that identifying the genetic diagnosis is essential to modulate the transplant procedure including conditioning agents and stem cell sources for better outcome and the standard cord blood transplantation (CBT) should be avoided in uIBMFS.

Re-Analysis of Next Generation Sequencing Gene Panel Data By Normalized Coverage Values Reveals Previously Undetected Copy Number Variations in Inherited Bone Marrow Failure Syndromes

Background: Inherited bone marrow failure syndromes (IBMFSs) comprise a group of genetic disorders affecting blood cell production in bone marrow, resulting in single or multilineage cytopenias. Patients with IBMFSs often have physical malformations and are at risk of developing leukemia and solid tumors. With advances in sequencing technologies, causal nucleotide-level mutations can readily be identified in many IBMFSs. In about half of IBMFS cases, however, a molecular mutation is not detected. Copy number variation (CNVs) have been reported to cause IBMFSs. Unfortunately, genome-wide methods to identify CNVs have major limitations as they may miss small lesions (e.g. by oligonucleotide or CGH arrays) or may have low sensitivity due to low read depths (e.g. by whole genome sequencing).Purpose: This study's purpose was to determine, after standard analysis of a next generation sequencing gene panel for causal mutations, whether re-analysis by normalized coverage value could identified CNVs and to characterize the genetic lesions.Methods & Materials: Patients with IBMFSs were enrolled in The Canadian Inherited Marrow Failure Registry (CIMFR). Genomic DNA was analyzed by next-generation panel gene panel of over 72 IBMFS-associated genes. After analysis for nucleotide level mutations, the NextGene software was used to detect copy number variation using nucleotide read depth in test samples versus normalized control coverage values. Monoalleic copy number deletions were indicated by ratio of <0.33;biallelic deletion was considered when the value was close to zero. PCR or Affymetrix SNP6.0 array were used to validate deletions.Results: Of the 258 patients who tested by the IBMFS next-generation gene panel, 93 (36%) were found to have causal nucleotide level mutations. 168 patients without identified mutations underwent analysis for copy number variation, of which 10 (6%) were found to have deletions. Genes most commonly exhibiting monoallelic deletions included RPS19, RPL11 and RPL5 (5 Diamond-Blackfan anemia patients). One patients with MDS was found to have monoalleic deletion of GATA2 . Moreover, a bialleic deletion of exon 1-5 in FANCA gene was detected in one Fanconi anemia patient. Lastly, we also identified monoalleic deletions of RBM8A in two thrombocytopenia with absent radii patients and of PARN in one unclassified pancytopenia patient. The last three patients were found to also have a nucleotide level mutation on the other allele by the NGS panel (compound heterozygousity). All the above deletions were validated by another method (PCR or Affymetrix SNP6.0 array).Conclusions: CNVs can be detected in some IBMFSs by next-generation sequencing (NGS) panels by careful analysis of normalized coverage values. Such analysis should become standard practice before usage of other mutation detection strategies such as CNV microarray or whole exome/genome sequencing, since the later techniques may not detect certain CNVs that can be detected by gene panels. DisclosuresKlaassen:Amgen: Consultancy; Baxalta: Honoraria; Biogen Canada LTD: Consultancy; Hoffman-La Roche LTD: Consultancy; Agios Pharmaceuticals: Consultancy; Octapharma: Honoraria. Dror:Alexion Canada: Research Funding.

Genotypic and Phenotypic Spectrum of Dyskeratosis Congenita: Results from the Canadian Inherited Marrow Failure Registry

Introduction: Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome caused by mutations in one of 13 telomere-related genes, resulting in disruption of normal telomere maintenance; however, about 30% of patients do not have a molecular diagnosis. DC patients are at increased risk for severe bone marrow failure (SBMF), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumours. Life expectancy is compromised by SBMF, malignancy, pulmonary and liver fibrosis, and GI bleeding. Objectives: Among patients with DC in Canada, aims were to: (1) characterize the genetic profile of DC in Canada, (2) define the spectrum of clinical features of DC, (3) determine the incidence and age when SBMF, MDS, AML or solid tumours develop, (4) identify factors that are associated with higher mortality risk, and (5) describe the causes of death. Methods: Data of patients enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR) and meeting diagnostic criteria for DC between January 1, 2001 and March 1, 2018 were included. The CIMFR is a multicentre registry that captures data on patients with inherited marrow failure syndromes from pediatric tertiary referral centres across all Canadian provinces. We investigated several continuous (e.g. age at diagnosis of SBMF/MDS/AML) and categorical (e.g. mutated gene) variables that are associated with specific outcomes, namely overall survival and development of SBMF. Cox proportional hazard models were used to assess risk of death based on age at diagnosis and presence of SBMF. Kaplan-Meier curves were used to assess overall survival. Results: As of March 1st, 2018, 35 patients with DC were enrolled. The mean age of diagnosis was 10.94 years (0-39.9). The underlying genotypes were: DKC1 (7), TERT (6), TINF2 (5), RTEL1 (3), PARN (2), TERC (2) but remained undetermined in the others (10). Twenty-seven patients were classified as classical DC, 7 had Hoyeraal-Hreidarsson syndrome and 1 patient had Coats plus syndrome. Eight patients (23%) developed SBMF. The mean age of SBMF was 4.22 years (1-8.66). No statistical difference was found between genotypes and progression to SBMF (P=0.1). Modelling death as a function of time varying SBMF status using a cox proportional hazard regression model showed that the presence of SBMF in DC patients was predictive of higher mortality rate (P= 0.009, hazard ratio 5.7, CI 1.54-21.5). None of the patients developed malignancy during childhood (0-18 years). One adult patient developed skin cancer. Eleven patients (31%) received a hematopoietic stem cell transplant (HSCT). The mean age of HSCT was 9.5 years (0.5-37). Ten (29%) patients died, five of whom were recipients of HSCT. Mean age of death was 12.98 years (2-24.6). Extra-hematological complications included gastrointestinal bleeding (50%), pulmonary fibrosis (40%), overwhelming infection (40%), liver fibrosis (20%), cardiomyopathy (10%), hemolytic uremic syndrome (HUS) (10%) and thrombotic microangiopathy (TMA) (10%). Most patients had more than one organ dysfunction. Analysis of survival showed that all patients with TINF2 mutations have died (at median age of 10.8 years, range 2.5-23.25) whereas none died in the TERT group. Patients diagnosed at younger age had lower overall survival compared to patients diagnosed at older ages (P= 0.03, HR: 0.72, CI: 0.57-0.90). All deaths were due to organ dysfunction related to DC. Fifty percent of the patients had concurrent SBMF at the time of death. Conclusion: In this analysis, we characterised the genetic and phenotypic spectrum of DC patients registered in the CIMFR. We found a high mortality rate mainly related to organ dysfunction and SBMF, and described the impact of genotype, earlier age at diagnosis and presence of SBMF in predicting survival. We found that malignancy is an uncommon complication in the pediatric age group. Figure Disclosures Klaassen: Amgen Inc: Consultancy; TranQoL and KIT: Other: creater and owner of Kids ITP tool and TranQoL; Octapharma AG: Speakers Bureau; Baxalta: Speakers Bureau; Biogen Canada Limited: Speakers Bureau; Novo Nordisk Canada Inc: Consultancy; Hoffman-LaRoche Ltd: Consultancy; Agios Pharmaceuticals Inc: Consultancy; Shire Pharma Canada Inc: Consultancy. Pastore:Pfizer: Honoraria. Lipton:BMS: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

TINF2 Mutations Are Associated with Poor Outcome Post Hematopoietic Stem Cell Transplantation for Dyskeratosis Congenita

Background. Dyskeratosis congenita (DC) is a telomere biology disorder with a high risk of bone marrow failure, cancer, pulmonary and liver disease. Mutation in multiple telomere related genes including DKC1, TINF2, RTEL1, TERC, TERT, WRAP53, CTC1, NOP10, NHP2 and TPP1 have been reported. Patients with mutations in several DC genes (e.g. heterozygous TINF2, hemizygous DKC1 and biallelic RTEL1 mutations) typically tend to have a particularly serious disease with severe bone marrow failure (SBMF) at a young age, non-hematological manifestations and very short telomeres. Currently, allogeneic hematopoietic stem cell transplantation (HSCT) is the only available curative treatment for bone marrow failure and leukemia in DC patients. Relatively few case series of patients with DC undergoing HSCT have been reported, which generally suggested a poor outcome. The present study aimed to characterize the outcome of HSCT in a Canadian cohort of patients with DC and determine potential relationships between outcome and genotype. Methods. The Canadian Inherited Marrow Failure Registry (CIMFR) is a multicenter registry that includes tertiary centers that care for IBMFS patients across all Canadian provinces. Patients with DC who had been enrolled in CIMFR and underwent HSCT between January 2001 and December 2019 were included. Data were extracted from the CIMFR database. Results. Among 35 patients with DC enrolled in the CIMFR, 11 underwent HSCT. Seven patients were male. Median age at presentation, diagnosis and HSCT was 2.1 years (range: 0 to 9.13s), 5.5 years (range: 1.94 to 35.25), and 7.0 years (range; 0.5-37), respectively. The diagnosis of 3 patients was made after HSCT. Median follow up time from HSCT was 5.89 years (range; 0.2-14.0 years). Among transplanted patients, five had TINF2 mutations, two had RTEL1 mutations, and one patient had DKC1 mutation. Eight patients underwent HSCT for severe bone marrow failure, and three patients for single or multilneage cytopenia. All patients had normal bone marrow karyotype before HSCT. All patients had a full matched donor; two were related and nine were unrelated. Ten patients received reduced-intensity conditioning, and one received myeloablative conditioning. Two patients experienced engraftment failure and underwent a second HSCT. Five years and ten years overall survival after HSCT were 90.9% (95% CI 73.9-100%) and 80% (95% CI 27.2- 97.5%), respectively; however, complications and deaths started appearing thereafter, mainly in patients with TINF2 mutation. All five patients with TINF2 mutation died, and other patients were alive. The causes of death were: 1) pulmonary fibrosis (N=2), 2) gastrointestinal bleeding (N=2), and 3) EBV infection (N=1). Two patients were diagnosed with pulmonary fibrosis after 8 and 11 years from HSCT and died 13.7 and 14 years post-transplant. Two patients had gastrointestinal bleeding after 3.9 years and 4.8 years from HSCT and died 6.6 and 5.7 years post-transplant. Of the patients with GI bleeding, both had hepatic fibrosis and one had pulmonary fibrosis. Summary: In this series, most patients with DC had resolution of the bone marrow failure and relatively good quality of life in the first few years post HSCT. However, longer outcome in the patients with TINF2 mutation was dismal because of DC-related complications, especially pulmonary fibrosis and gastrointestinal bleeding. Effective therapies to prevent these complications are critically needed. Additional reports about HSCT outcome of patients with DC are necessary to characterize HSCT in patients with other genetic groups and to replicate the above results in TINF2 patients. Disclosures No relevant conflicts of interest to declare.

Androgen therapy in inherited bone marrow failure syndromes: analysis from the Canadian Inherited Marrow Failure Registry.

Progressive cytopenia is a serious complication among paediatric patients with inherited bone marrow failure syndromes (IBMFS). Androgens have been used to improve blood counts in different bone marrow failure conditions. Little is known about efficacy and toxicity with new androgens (i.e., danazol) in different types of IBMFS. We identified 29 patients from the Canadian Inherited Marrow Failure Registry, who received oxymetholone or danazol. Sixteen (55%) had haematological response including patients with unclassified IBMFS (45%). Danazol showed a better toxicity profile and similar efficacy compared to oxymetholone. Androgens are an effective and safe option to ameliorate bone marrow failure in IBMFS.

The Impact of Identifying the Syndromic and Genetic Diagnoses on Hematopoietic Stem Cell Transplantation Outcome in Patients with Inherited Bone Marrow Failure Syndromes

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.

Predictors of Disease Progression and Survival in Patients with Myelodysplastic Syndrome Secondary to Inherited Bone Marrow Failure Syndromes

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 &gt;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&lt;0.0001), which varied by IBMFS category. MDS patients with UCIBMFS had a statistically significant lower OS compared to those without MDS (HR 5.7, 95% CI 1.7 to 18.6, p=0.004). In contrast, patients with FA had poor OS regardless of whether or not they developed MDS. Twenty four MDS patients (40%) had disease progression, with a median time to progression of 4.7 months (1.14-131). Nine patients (38%) with disease progression had FA, 5 (21%) had SDS, 4 (17%) had UC, and 6 (25%) had other marrow failure syndromes. Ten patients (42%) developed more advanced cytopathology, 10 (42%) a new cytogenetic abnormality, and 5 (20%) worsening cytopenias. Eight patients progressed from RC to RCEB, with a median time to progression of 5.7 months (1.14 to 113). Progression to more advanced cytopathology was associated with lower OS (HR 2.7, 95% CI 1.0 to 7.4, p=0.046). Median time to progression of cytogenetics was 4.13 months (1.14 to 131), which was not predictive of worse OS (p=0.22). Notably, there was no difference in OS or risk of progression between the -7/-7q and isochromosome 7q groups (p=0.644). Finally, patients who progressed due to worsening cytopenias had significantly lower OS compared to those who did not (p=0.011), but numbers were small. Seventeen (71%) of the MDS patients who progressed underwent HSCT. Conclusion Development of MDS has a significant adverse impact on the OS of IBMFS patients, with disease progression occurring 4.7 months from MDS diagnosis. Progression to advanced cytopathology is associated with decreased survival, while worsening cytogenetic clones and cytopenias may not carry the same risk. Importantly, isochromosome 7q10 is also associated with a risk of progression to more severe hematological disease, and may have an impact on survival. Further analysis of additional variables (i.e. HSCT) will provide insight into the important predictors of survival and disease progression, and help to guide treatment decisions for this high-risk patient population. Disclosures No relevant conflicts of interest to declare.

The clinical impact of copy number variants in inherited bone marrow failure syndromes

Inherited bone marrow failure syndromes comprise a genetically heterogeneous group of diseases with hematopoietic failure and a wide array of physical malformations. Copy number variants were reported in some inherited bone marrow failure syndromes. It is unclear what impact copy number variants play in patients evaluated for a suspected diagnosis of inherited bone marrow failure syndromes. Clinical and genetic data of 323 patients from the Canadian Inherited Marrow Failure Registry from 2001 to 2014, who had a documented genetic work-up, were analyzed. Cases with pathogenic copy number variants (at least 1 kilobasepairs) were compared to cases with other mutations. Genotype-phenotype correlations were performed to assess the impact of copy number variants. Pathogenic nucleotide-level mutations were found in 157 of 303 tested patients (51.8%). Genome-wide copy number variant analysis by single-nucleotide polymorphism arrays or comparative genomic hybridization arrays revealed pathogenic copy number variants in 11 of 67 patients tested (16.4%). In four of these patients, identification of copy number variant was crucial for establishing the correct diagnosis as their clinical presentation was ambiguous. Eight additional patients were identified to harbor pathogenic copy number variants by other methods. Of the 19 patients with pathogenic copy number variants, four had compound-heterozygosity of a copy number variant with a nucleotide-level mutation. Pathogenic copy number variants were associated with more extensive non-hematological organ system involvement (p = 0.0006), developmental delay (p = 0.006) and short stature (p = 0.04) compared to nucleotide-level mutations. In conclusion, a significant proportion of patients with inherited bone marrow failure syndromes harbor pathogenic copy number variants which were associated with a more extensive non-hematological phenotype in this cohort. Patients with a phenotype suggestive of inherited bone marrow failure syndromes but without identification of pathogenic nucleotide-level mutations should undergo specific testing for copy number variants.

Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

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. DisclosuresLipton:Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Copy Number Variants Underlying Inherited Bone Marrow Failure Syndromes

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. DisclosuresLipton:Teva: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

The impact of category, cytopathology and cytogenetics on development and progression of clonal and malignant myeloid transformation in inherited bone marrow failure syndromes.

Inherited bone marrow failure syndromes are a group of rare, heterogeneous genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome and acute myeloid leukemia. The clinical characteristics, risk classification, prognostic factors and outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes are largely unknown. The aims of this study were to determine the impact of category, cytopathology and cytogenetics, the three components of the "Category Cytology Cytogenetics" classification of pediatric myelodysplastic syndrome, on the outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure. We used data from the Canadian Inherited Marrow Failure Registry. Among 327 patients with inherited bone marrow failure syndrome enrolled in the registry, the estimated risk of clonal and malignant myeloid transformation by the age of 18 years was 37%. The risk of clonal and malignant myeloid transformation varied according to the type of inherited bone marrow failure syndrome but was highest in Fanconi anemia. The development of clonal and malignant myeloid transformation significantly affected overall survival. Mortality varied based on cytopathological group. The largest group of patients had refractory cytopenia. Clonal marrow cytogenetic abnormalities were identified in 87% of patients with clonal and malignant myeloid transformation, and different cytogenetic groups had different impacts on disease progression. We conclude that category, cytopathology and cytogenetics in cases of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes have an important impact on outcome and that the classification of such cases should incorporate these factors.

Risk Factors for Poor Survival after Hematopoietic Stem Cell Transplantation in Inherited Bone Marrow Failure Syndromes

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. DisclosuresNo relevant conflicts of interest to declare.

Application of Novel Next Generation Sequencing Gene Panel Assay to Genetic and Clinical Diagnosis of Inherited Bone Marrow Failure Syndromes

▪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. DisclosuresNo relevant conflicts of interest to declare.

Combined De-Novo Mutation and Non-Random X-Chromosome Inactivation Causing Wiskott-Aldrich Syndrome in a Female with Thrombocytopenia

Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome. Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome. A female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G > A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome. Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.

Outcome and Clinical Characteristics of Clonal and Malignant Myeloid Transformation in Inherited Bone Marrow Failure Syndromes

AbstractAbstract 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.

Frequency and Natural History of Inherited Bone Marrow Failure Syndromes: The Israeli Inherited Bone Marrow Failure Registry

Inherited bone marrow failures syndromes (IBMFS) are rare genetic disorders usually characterized by congenital anomalies, development of bone marrow failure and a tendency to develop malignant diseases. Although single disease registries for Fanconi anemia (FA), Diamond-Blackfan Anemia (DBA), Severe Congenital Neutropenia (SCN) and Dyskeratois Congenita (DC) have been established the true incidence of many of the IBMFS is still unknown. To investigate the prevalence of each of the IBMFS and to the types and frequencies of complications we set up a retrospective national registry of these disorders in Israel. We reviewed charts of 127 patients with IBMFS diagnosed between 1964 and 2005. This represents the majority of such patients in the country. Consanguinity was recorded in 50 (40%) patients. Median time since diagnosis was 6 years (range: 1 month-39 years). Genetic analysis was available in 60 (47%) patients. The number of patients within each disease category is presented in the Table. The majority of patients who succumbed to their disease had FA. Of the 29 patients who developed malignancies (hematological and solid) the majority 23 (79%) had FA. All of the 6 patients who developed solid tumors had FA. The solid tumors were squamous cell carcinoma of head and neck, esophagus, cervix and vulva.Table: Israeli Inherited Bone Marrow Failure Cohort - Diagnosis, Complications and SurvivalNo. of pts.FADBASCNCAMTDCSDSTARNOSAllTotal (%)66 (52)18 (14)21 (16.5)8 (6)6 (5)3 (2)3 (2)2 (1.5)127 (100)Per 106 people5.32.62.11.10.860.430.29Deceased (%)24 (36)-6 (29)-2 (33)1 (33)--33 (26)Molecular diagnosis (%)34 (51)6 (33)7 (33)8 (100)4 (67)1 (33)--60 (47)MDS/AML/ALL (%)19 (29)-4 (19)2 (25)----25 (20)Solid tumor (%)6 (9)-------6 (5)CAMT, Congenital Amegakaryocytic Thrombocytopenia; SDS-Shwachman-Diamond Syndrome; TAR, Thrombocytopenia Absent Radii; NOS-not otherwise specified. This is the largest population-based study which has examined the relative frequency of each of the IBMFS. In this cohort, FA was by far the most common form of an IBMFS (52% of pts), followed by DBA (16.5%) and SCN (14%), while CAMT, DC, SDS and TAR were far less common. These findings agree with the frequencies of FA, DBA and SCN reported by individual disease registries, and contrast with the results from the Canadian inherited marrow failure registry (Pediatr Blood Cancer 47:918, 2006), where among a smaller number (39) of patients with an IBMFS the frequencies of FA, SDS, DBA and SCN were similar (12% each). One caveat is that the Israeli cohort has a large proportion of consanguineous families, which differs from most cohorts in other countries. The number of undiagnosed patients with IBMFS in our study was very small. SDS and DC are either rare in our region or under-diagnosed. The implementation of new diagnostic tests will help to resolve this issue. These data provide a rational basis for longitudinal surveillance and prevention of complications in the severe forms of IBMFS.

Clinical and Genetic Analysis of Unclassifiable Inherited Bone Marrow Failure Syndromes

Unclassified inherited bone marrow failure syndromes are a heterogeneous group of genetic disorders that represent either new syndromes or atypical clinical courses of known inherited bone marrow failure syndromes. The relative prevalence of the unclassified inherited bone marrow failure syndromes and their characteristics and the clinical and economic challenges that they create have never been studied. We analyzed cases of inherited bone marrow failure syndrome in the Canadian Inherited Marrow Failure Registry that were deemed unclassifiable at study entry. From October 2001 to March 2006, 39 of the 162 patients enrolled in the Canadian Inherited Marrow Failure Registry were registered as having unclassified inherited bone marrow failure syndromes. These patients presented at a significantly older age (median: 9 months) than the patients with classified inherited bone marrow failure syndrome (median: 1 month) and had substantial variation in the clinical presentations. The hematologic phenotype, however, was similar to the classified inherited bone marrow failure syndromes and included single- or multiple-lineage cytopenia, severe aplastic anemia, myelodysplasia, and malignancy. Grouping patients according to the affected blood cell lineage(s) and to the presence of associated physical malformations was not always sufficient to characterize a condition, because affected members from several families fit into different phenotypic groups. Compared with the classified inherited bone marrow failure syndromes, the patients with unclassified inherited bone marrow failure syndromes had 3.2 more specific diagnostic tests at 4.5 times higher cost per evaluated patient to attempt to categorize their syndrome. At last follow-up, only 20% of the unclassified inherited bone marrow failure syndromes were ultimately diagnosed with a specific syndrome on the basis of the development of new clinical findings or positive genetic tests. Unclassified inherited bone marrow failure syndromes are relatively common among the inherited bone marrow failure syndromes and present a major diagnostic and therapeutic dilemma.

Disease progression in recently diagnosed patients with inherited marrow failure syndromes: A Canadian inherited marrow failure registry (CIMFR) report

Inherited bone marrow failure syndromes (IMFSs) are genetic disorders characterized by defective single-lineage or multi-lineage hematopoiesis. IMFS patients are at risk for severe cytopenias, development of marrow cytogenetic abnormalities (MCA), myelodysplasia (MDS), and malignancy. The rate of disease progression and proportion of patients at risk for these complications is currently unclear. We examined recently diagnosed IMFS patients to determine distribution of diagnoses, disease progression and development of significant outcomes. The CIMFR is a prospective multi-center study established in 2001 to register all IMFS patients in Canada. Analysis was restricted to patients diagnosed after November 30, 1997. Summary statistics were used to depict the study population while survival was described using the Kaplan-Meier method. 74 CIMFR patients were considered recently diagnosed. Median age at diagnosis was 2.7 years (range, birth to 40.6). Annual follow-up data were available for 53 (72%) patients. The five most prevalent diagnoses were Fanconi anemia (FA), Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), dyskeratosis congenita (DKC), and Kostmann's neutropenia (KS). Eighteen (24%) patients were unclassifiable. Twenty-eight (53%) follow-up patients had disease progression as indicated by new or worsening cytopenias, new marrow changes, or initiation of transfusion support and/or medical therapy. Fourteen (19%) fulfilled minimal diagnostic criteria for myelodysplasia. Eleven patients had hematopoietic stem cell transplantation (HSCT) by first follow-up. Five patients have died. Survival at 36 months is 89.8 +/- 5.7%. IMFS patients are often diagnosed at a young age. The relative distribution of diagnoses is similar to previous reviews of published cases; however, 25% of patients are currently unclassifiable. Disease progression has occurred in approximately 50% of follow-up patients. Early mortality is noted. Continued prospective observation of these patients is warranted.

Mutation analysis of SBDS in pediatric acute myeloblastic leukemia

Shwachman-Diamond syndrome (SDS) is associated with a high risk of myelodysplasia, acute myeloid leukemia (AML), and chromosome 7 abnormalities. Ninety percent of SDS patients have mutations in SBDS on 7q11. Herein, we studied the role of genetic alterations in SBDS in AML. DNA was extracted from marrows of SDS patients with AML, as well as from children with de novo AML. Direct sequencing of PCR amplified genomic DNA was performed using specific primers flanking each exon. To study whether SBDS heterozygosity confers a risk for MDS/AML, data on family members of SDS patients on the Canadian Inherited Marrow Failure Registry (CIMFR) was analyzed. Of two SDS patients with SDS/AML one was homozygous 258 + 2T > C, and one was compound heterozygous 183-184TA > CT/258 + 2T > C. To determine whether a subset of patients with SDS can present with AML, we analyzed 48 AML samples at remission, but no mutations were identified. To address whether acquired mutated SBDS gene is associated with leukemic transformation in de novo AML, we analyzed 77 AML samples at diagnosis or relapse (4 with -7 and 7q-) for SBDS mutations; no alterations were detected. Also, among the relatives of an SDS patient cohort on the registry no cases of MDS/AML were reported. Common mutations occurred in our SDS patients who develop AML, and thus, AML is not confined to a rare genetic subgroup of SDS. Newly diagnosed patients with AML are unlikely to have an underlying undiagnosed SDS. Acquired SBDS gene mutations also would appear unlikely to play a mechanistic role in de novo AML, and might not be involved in the pathogenesis of chromosome 7 abnormalities as well.