ALBERT

Description: Key Points Small deletions in the RPS14 region of 5q must be considered in atypical 5q− syndrome and nonclassical Diamond Blackfan anemia.

Description: Abstract 4430 Background: Diamond Blackfan anemia is a rare heritable red cell aplasia which usually presents in infancy but can also be diagnosed in childhood and even adulthood. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haplo-insufficiency have been reported in about 54% of the patients. The 5q- syndrome is an acquired myelodysplastic syndrome (MDS) characterized by a similar erythroid failure. Another RP gene included in the 5q deleted region, RPS14, has been identified as a causal gene in 5q- MDS but has not been reported in DBA. Purpose: Array Comparative Genomic Hybridization has been used to identify large deletions in patients with DBA. This report demonstrates the use of Single Nucleotide Polymorphism (SNP) genotyping array hybridization to identify a patient, previously thought to have DBA, as having a 5q- deletion consistent with 5q- syndrome. Method: Seventy-five patient samples from the Diamond Blackfan Anemia Registry of North America, a patient database of now 608 patients designed to better understand the biology and epidemiology of DBA, underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands from this cohort who did not have a mutation detected in either the resequencing project and/or the targeted sequencing efforts lead by Gazda and colleagues. Result: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one patient with presumed DBA. The patient presented at 5 years 10 months of age with anemia noted on a routine blood count. The hemoglobin was 8.4 grams/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The eADA was normal. The bone marrow showed decreased cellularity and megaloblastic changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. The cytogenetics performed at diagnosis in 1991 were reported as normal. The patient had no significant family history of anemia and was found to have no congenital physical anomalies. A diagnosis of non-classical DBA was presumed and the patient failed a trial of corticosteroids. At present the patient has marrow red cell aplasia and is on a chronic transfusion schedule. SNP array genotyping analysis identified mosaicism in two discrete regions covering ~17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33 though it excludes the 5q31 CDR associated with AML and more aggressive MDS as well as miR146a, a factor recently postulated to play a role in 5q- MDS. SNP array genotyping from purified peripheral blood populations indicated that lymphocytes were greater than 95% normal, while the myeloid cells were greater than 95% 5q-. CD34+ cells obtained from this patient showed a marked decrease in both myeloid and erythroid colony formation when compared with normal cells. Patient fibroblasts were normal and neither of the parents have any 5q anomalies by SNP array genotyping. Although the deletion was not identified in 1991 at the time of the diagnosis, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was able to be detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. Conclusion: Patients with non-classical DBA may have unique acquired 5q deletions with RPS14 haploinsufficiency. A search for other acquired somatic mutations or deletions in patients with DBA, in particular non-classical cases, is underway. SNP array genotyping is an essential diagnostic tool in this search. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 942 Background: Emerging evidence suggests that microRNAs (miRNAs) are critical in cancer and adult leukemia by functioning as tumor suppressors and/or oncogenes. Zhang et al identified 32 pediatric acute myeloid leukemia (AML)-specific miRNA patterns by analysis of bone marrow (BM) samples (1). They also established potential miRNAs as biomarkers for predicting CNS-relapse in pediatric acute lymphocytic leukemia (ALL). Altered miRNA expression disrupts normal hematopoiesis and might play a role in niche-induced oncogenesis. Dysfunction of mesenchymal stromal cells induces formation of myeloid sarcomas that infiltrate in the surrounding tissues (2). Previously, we described that mesenchymal stem cells (MSC) of the BM microenvironment participate in leukemic stem cell regulation in an in vivo model of the childhood AML stem cell niche(3). These human MSC niches, created in ectopic bioengineered 3D scaffolds, supported leukemogenesis in NOD/SCID mice. Pediatric AML engrafted at 1 month in the MSC-coated scaffolds in the mice and was retained in the niche up to 4 months, after which distant seeding to murine BM, liver and spleen occurred. The bioengineered niche created a sanctuary for quiescent leukemia cells and at 4 months the AML cells exited the niche and spread hematogenously, mimicking leukemia relapse. Analysis of miRNA patterns in our leukemia niche model could provide novel directions for individual risk-adapted therapy in childhood leukemia. Objective: To analyze miRNA expression patterns of pediatric AML after exposure to the niche microenvironment at different time points. Design and Method: miRNAs were obtained from primary CD34+ selected AML cells at (d0) Day0= no niche exposure, (1Mo) 1 month =niche engraftment, (4Mo) 4 months=hematogenous spread with leukemic exit from the niche. miRNAs were isolated from single cell suspensions with the mirVana miRNA isolation kit (Ambion) and analyzed on an Ilumina MicroRNA Expression Profiling single Beadchip (#RNA probes = 1145). Results: 498/1145 miRNAs expression profiles were selected with a detection p value 〈 0.00001. Out of 498 miRNAs expressed in the leukemic niche model, 23 were previously described as AML-specific miRNAs (2). 10/23 miRNAs were significantly upregulated and 13/23 were downregulated. Pediatric AML-specific miRNAs – miR100 and miR125b had high expression profile at baseline, but were down regulated upon contact with the niche. AML miR195 and miR193a had low expression at baseline, but miR195 was upregulated on engraftment while miR193a only upregulated at the time of hematogenous spread (niche exit). CNS-relapse in ALL might represent a physiological mechanism of leukemic exit of dormant cells from the niche sanctuary. Consistent with this notion, the same expression profile that was found in CNS-relapse in ALL patients (miR198 up – miR551a downregulated) was seen in our model when AML cells became invasive and exited the niche at 4 months. Conclusion: (1) Zhang et al. 2009, PloSOne, 4, p1 (2) Raaijmakers et al. 2010, Nature, 464, p852 (3) Vaiselbuh et al, 2010, Tissue Eng Part C Methods, June 29 (ePub) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1343 Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.