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Background Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. Procedure Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). Results This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. Conclusions L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

Fanconi anemia (FA), a genetic disorder affecting DNA repair, is characterized by bone marrow failure and cancer susceptibility. In FA mouse models, metformin (N,N-dimethylguanide) a biguanide metabolic agent, improves blood counts and delays tumor development. Given these findings, we conducted a single institution pilot study of metformin in non-diabetic patients with FA to assess feasibility, tolerability, and impact of metformin on hematologic response as defined by modified MDS International Working Group (IWG) criteria (Table 1). Fourteen of 15 patients with at least 1 cytopenia (hemoglobin Figure 1 Figure 1. Disclosures Pollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees. Furutani: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Esrick: bluebird bio: Consultancy. Nakano: Novartis: Consultancy. Thompson: Celgene: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Biomarin: Research Funding; Vertex: Research Funding; Graphite Bio: Research Funding; Baxalta: Research Funding; CRISPR Therapeutics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Consultancy; Beam Therapeutics: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company. OffLabel Disclosure: metformin- use in Fanconi anemia in an effort to improve hematopoiesis

BACKGROUND Red blood cell (RBC) alloimmunization is a concern for patients who receive multiple or chronic transfusions. Alloimmunization prevalence in transfused patients with bone marrow failure syndrome (BMFS) is unknown. This study aimed to determine physician practice for RBC antigen matching, immunization rates, and antibody specificities in patients with BMFS. STUDY DESIGN AND METHODS The clinical records of all patients with BMFS seen at the Children's Hospital of Philadelphia between 2001 and 2015 were reviewed. Immunization rate was determined per 100 units transfused. RESULTS ABO/D, C, E, and K (CEK) RBC matching was requested for 21.8% of patients. A total of 3782 RBC units were transfused to 87 patients, of which 2551 (67.5%) were CEK matched and 1231 (32.5%) were ABO/D only matched. The majority of units transfused to patients on a chronic transfusion regimen were CEK matched (89.6% of 2728 units). No anti-C, -E, or -K antibodies formed in any patient during the 14-year study period. Two alloantibodies and two autoantibodies formed, resulting in a rate of 0.05 alloantibodies and 0.05 autoantibodies per 100 units transfused. The prevalence of alloimmunization was 2.3%. CONCLUSION The rate and prevalence of RBC alloimmunization were low in patients with BMFS. CEK matching avoided alloimmunization to these antigens in chronically transfused patients.

Hepatitis‐associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which immune‐mediated bone marrow failure (BMF) develops following an acute episode of seronegative hepatitis. Dyskeratosis congenita (DC) is an inherited BMF syndrome characterized by the presence of short telomeres, mucocutaneous abnormalities, and cancer predisposition. While both conditions may cause BMF and hepatic impairment, therapeutic approaches are distinct, making it imperative to establish the correct diagnosis. In clinical practice, lymphocyte telomere lengths (TL) are used as a first‐line screen to rule out inherited telomeropathies before initiating treatment for AA. To evaluate the reliability of TL in the HAA population, we performed a retrospective analysis of TL in 10 consecutively enrolled HAA patients compared to 19 patients with idiopathic AA (IAA). HAA patients had significantly shorter telomeres than IAA patients (P = 0.009), including four patients with TL at or below the 1st percentile for age‐matched controls. HAA patients had no clinical features of DC and did not carry disease‐causing mutations in known genes associated with inherited telomere disorders. Instead, short TLs were significantly correlated with severe lymphopenia and skewed lymphocyte subsets, features characteristic of HAA. Our results indicate the importance of caution in the interpretation of TL measurements in HAA, because, in this patient population, short telomeres have limited specificity. Am. J. Hematol. 91:243–247, 2016. © 2015 The Authors. American Journal of Hematology Published by Wiley Periodicals, Inc.

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20–25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA , with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging—immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA. Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age > 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb >9; Partial Response (PR): Hb < 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated. Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb >9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p Conclusions: The administration of L-leucine is safe. L-leucine administration resulted in an erythroid response in 7% of subjects and an increased growth velocity in 33% of growing subjects. Based upon extrapolation from animal models, it is likely that this dose was suboptimal. We hypothesize that higher doses of L-leucine will lead to hematologic responses in more subjects who are transfusion dependent. The potential benefit of added growth in children with DBA may improve final adult height. Disclosures Glader: Agios: Consultancy, Research Funding.

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms. [Cancer Res 2007;67(11):5148–55]

[Figure][1] A 4-year-old girl with a known history of methylmalonic acidemia (MMA) complicated by acquired hypothyroidism, failure to thrive, and developmental delay developed pancytopenia during an admission for emesis and metabolic acidosis. She had no symptoms of a viral infection, and

[Figure][1] A 23-year-old man presented to our clinic for a second opinion. He was given the diagnosis of myelodysplastic syndrome (MDS) years prior, based on a history of iron overload and bone marrow biopsy findings of a hypercellular marrow with erythroid hyperplasia and

This article provides a practice-based and concise review of the etiology, diagnosis, and management of acquired aplastic anemia in children. Bone marrow transplantation, immunosuppressive therapy, and supportive care are discussed in detail. The aim is to provide the clinician with a better understanding of the disease and to offer guidelines for the management of children with this uncommon yet serious disorder.

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia and a propensity for transformation to acute myeloid leukemia (AML). A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene, most commonly leading to fusions of NUP98 with abd-b group HOX genes, including HOXD13. We used vav regulatory elements to direct expression of a NUP98-HOXD13 (NHD13) fusion gene in hematopoietic tissues. NHD13 transgenic mice faithfully recapitulate all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia and apoptosis, and transformation to acute leukemia. The MDS that develops in NHD13 transgenic mice is highly lethal; within 14 months, 90% of the mice died of either leukemic transformation or severe anemia and leukopenia due to progressive MDS. These mice provide a pre-clinical model that can be used for the evaluation of MDS therapy and biology.

Familial hemophagocytic lymphohistiocytosis is an inherited deficiency of natural killer cell function and excessive cytokine activity, which predominantly presents in early childhood. The initial symptoms of familial hemophagocytic lymphohistiocytosis are often nonspecific but may be predominantly neurologic. The case presented here describes an 18-month-old boy who initially presented with fever, encephalopathy, and hemiparesis. He had innumerable brain lesions visualized on magnetic resonance imaging scans. An infectious etiology was excluded, and brain, liver, and bone marrow biopsies were nonspecific but consistent with hemophagocytic lymphohistiocytosis. Cells were sent for flow cytometry perforin analysis, which demonstrated defective natural killer cell function. A diagnosis of familial hemophagocytic lymphohistiocytosis was confirmed by mutation analysis and decreased expression of the perforin gene, in the patient and immediate family members. These results showed the patient to be a compound heterozygote for perforin mutations. His case illustrates the potential for a fulminant neurological presentation of familial hemophagocytic lymphohistiocytosis with widespread lesions in the brain.

Acquired aplastic anemia (AA) is a hematologic disorder characterized by low blood counts and a hypocellular bone marrow, caused by autoimmune destruction of early hematopoietic cells. The diagnosis of AA is made by excluding other disorders that can present with bone marrow failure (BMF). Such disorders include dyskeratosis congenita (DC), a multisystem BMF syndrome, caused by an inherited defect in telomere maintenance. Although classical DC presents in childhood with stereotypical mucocutaneous changes, milder forms of telomere dysfunction associated with mutations in TERT and TERC genes can present with non-syndromic bone marrow failure clinically indistinguishable from AA. In clinical practice, lymphocyte telomere length measurements are used as a first-line screen for inherited telomeropathies before initiating treatment for AA. In our BMF center, we have observed that several patients with features of hepatitis-associated AA (HAA) had lymphocyte telomere lengths at diagnosis at or below the first percentile of age-matched controls, in the range similar to inherited telomere disorders. To confirm our initial observation, we performed a retrospective analysis of telomere lengths of consecutively enrolled HAA patients with non-hepatitis associated AA patients in our institution. A total of 30 patients with AA were included in this study: 10 had HAA and 20 had other AA (Table 1). The median age at telomere testing was 8.0 years (range 1-19 years). There was no significant difference in age or disease severity between the two groups (p=0.827). The patients' median lymphocyte telomere length (TL) was significantly lower in the HAA patients compared to AA (7.4kb versus 9.1kb, P= 0.021); the difference remained significant after adjusting for patient age (p Because differences in lymphocyte activation and subset composition are known to impact telomerase activity, we hypothesized that alterations in lymphocyte populations caused by the unique inflammatory state of HAA could partly account for significantly shorter TL in this population. HAA patients exhibited significantly lower absolute lymphocyte counts and lower lymphocyte subsets across the board, as well as the decreased CD4/CD8 ratio compared to non-hepatitis AA patients (Figure 2). The median telomere length in the two groups was significantly correlated with lymphocyte counts (Pearson correlation coefficient 0.52, p=0.003). An altered lymphocyte homeostasis such as the one characteristic for HAA limits the specificity of telomere measurements as a screening method to identify patients with AA due to a genetic defect in telomere maintaining genes. As such, short telomeres in HAA in the absence of other features suggestive for DC does not necessarily warrant genetic testing for telomere length. Longitudinal studies of telomeres and study of clonal hematopoiesis in this population is ongoing. Table 1. Overall (n = 30) AA (n=20) HAA (n=10) P value* Patient Characteristic Gender, female n (%) 12 (40) 9 (45) 3 (30) 0.694 Gender, male n (%) 18 (60) 11 (55) 7 (70) Age at diagnosis, y, median (range) 8.0 (1-19) 8.3 (1-19) 7.5 (3-17) 0.827 Disease Severity, n (%) 0.999 Moderate 6 (20) 4 (20) 2 (20) Severe 22 (73) 15 (75) 7 (70) Very Severe 2 (7) 1 (5) 1 (10) Median Lymphocyte Telomere Length, kb (range) 8.9 (5.9-11.3) 9.1 (7.5-11.3) 7.4 (5.9-9.8) 0.021 ≤ 1st percentile of age-matched controls 5 0 5 1-10th percentile of age-matched controls 5 5 0 > 10th percentile of age-matched controls 20 15 5 *P-values are obtained by Fisher's exact test for gender and disease severity and by Wilcoxon test for age and telomere length. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract 262 Introduction: The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI. Patients and Methods: In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated. Results: A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI. Conclusion: SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI. Disclosures: No relevant conflicts of interest to declare.

The NUP98-HOXD13 (NHD13) fusion gene is formed by the t(2;11)(q31;p15) in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). This fusion gene encodes a protein that fuses the N-terminal portion of NUP98, a nucleoporin involved in mediating RNA and protein transport in and out of the nucleus, with the C-terminal portion of HOXD13, a homeodomain protein not expressed during normal haematopoietic development. We have previously demonstrated that expression of the NHD13 fusion gene in a transgenic mouse model results in an invariably fatal MDS. These mice either die of complications of severe pancytopenia or progress to a fatal acute leukemia; greater than 90% of mice die by 14 months of age. The prolonged latency suggests that additional genetic events are required for leukemic transformation. We used a MOL4070LTR retroviral mutagenesis screen to identify candidate genes that might collaborate with the NHD13 transgene. All transgenic mice infected with the MOL4070LTR virus developed an acute leukemia, and the median survival of these mice was only four months, with none surviving beyond seven months of age. These survival figures are significantly decreased compared to either the wild type infected group or the transgenic uninfected group, suggesting a true synergistic effect between the NUP98-HOXD13 transgene and the genes affected by retroviral insertion events. Cloning of the insertion sites allowed identification of candidate collaborating genes. The most frequently identified gene was Meis1, a gene known to collaborate with HOX genes during leukemic transformation. This result supports the contention that the retroviral infection screen identifies specific collaboration between retrovirus and transgene. The second most common insertion site was near Mn1. These insertions clustered in two regions: four occurred within the final intron of Mn1, and four occurred between 80 kb and 160 kb 3′ of the 3′ end of the gene. Of note, MN1 is involved in human leukemia by fusion to the TEL oncogene via the t(12;22)(p13;q11) in myeloid leukemia. This translocation occurs within the intron of the human MN1 gene equivalent to that in which the four retroviral insertions occur in the mouse Mn1 gene. Moreover, this fusion transcript has recently been shown to collaborate with HOXA9 overexpression in leukemogenesis (Kawagoe and Grosveld, Blood 106(13):4269–77). We identified a fusion transcript formed in these four mice by RNA splicing between the 5′ part of Mn1 and the retroviral env gene. In addition, all eight of the mice with Mn1 insertions (both intronic and 3′) showed elevated wild-type Mn1 expression, suggesting that overexpression of wild-type Mn1 as well as a retroviral Mn1-env fusion can collaborate with the NHD13 transgene during leukemic transformation. Further studies are underway to investigate the oncogenic potential of an Mn1-env fusion as well as overexpression of the wild-type Mn1.