Erythroid Hypoplasia Research Articles

P53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression; however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

Iron as the key modulator of hepcidin expression in erythroid antibody-mediated hypoplasia.

Erythroid hypoplasia (EH) is a rare complication associated with recombinant human erythropoietin (rHuEPO) therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU) developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy.

Megakariocytic Hypoplasia Due To TKIs In a CML Patient Can Be Overcome By TPO Receptor Agonist Eltrombopag

Background Patients who develop severe thrombocytopenia while treated with TK inhibitors may be forced to stop them, face a worse prognosis, and eventually require a treatment with a bone marrow transplant. Eltrombopag is a TPO receptor agonist that stimulates megakaryocytic proliferation. Case 62-year-old man presented in October 2008 with splenomegaly and leukocytosis. The bone marrow biopsy was diagnostic of CML. Imatinib was started at 400 mg daily resulting in hematological response and resolution of splenomegaly, but severe thrombocytopenia necessitated 50 % dose reduction. Thrombocytopenia was not responsive to Prednisone, and a trial of platelets transfusions with the standard dose of Imatinib had to be stopped because of thrombocytopenia below 10 K and the risk of bleeding – reducing the dose of the TKI was improving the platelet count. Treatment with Dasatinib and with Nilotinib also necessitated 50 % dose reductions because of thrombocytopenia. These reduced TKI dosing resulted in the best Quantitative RT-PCR for bcr/abl product reduction level at – 1.01. Bone marrow biopsy in December 2010 showed no morphologic evidence of CML, and no dysplasia; it was normocellular marrow with erythroid hyperplasia and megakaryocytic hypoplasia. Cytogenetic analysis and FISH showed presence of the Philadelphia chromosome rearrangement. The patient declined the treatment with a Bone marrow transplant. In April 2011 a TPO agonist Eltrombopag 50 mg daily was started, given on two weeks on two weeks off schedule. As platelets remained adequate since then, the Nilotinib was escalated to the full dose of 400 mg twice daily. The patient tolerated the treatment well. Results The patient required about half-a-year support with Eltrombopag; after that it was discontinued as the platelet count remained above 40 K. No dose reduction of Nilotinib was necessary. In January 2012 the patient reached the milestone of Complete Cytogentic Response with the level of bcr/abl transcript minus 2 or less, which continued to decrease and now the patient is achieving a Major Molecular Response. Conclusion Treatment of CML with Tyrosine Kinase Inhibitors – Imatinib, Dasatinib, or Nilotinib in our patient – can be complicated by severe thrombocytopenia. These patients’ next treatment option may be a Bone marrow transplant. Limited use of Eltrombopag can improve thrombocytopenia, and allow these patients to continue TKIs with attainment of the treatment milestons. Disclosures: Off Label Use: Eltrombopag. It is used in thrombocytopenia, in ITP. In this presentation it was used to treat thrombocytopenia secondary to Thyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia.

Steroid Responsive T-Cell Mediated Pure Red Cell Aplasia Following Allogeneic Hematopoietic Stem Cell Transplantation

Pure red cell aplasia (PRCA) is an unusual complication following allogeneic hematopoietic stem cell transplantation (HSCT), the true incidence of which is not known. Almost all cases reported in the literature describe major ABO-incompatibility between the recipient and the donor as the major risk factor for this complication. Delayed recovery of reticulocyte counts and hypoplasia of erythroblasts in the marrow is attributed to incompatible hemagglutinins in recipients. There are reports of successful treatment of PRCA using different strategies include rapid tapering of calcineurin inhibitors, corticosteroids, donor lymphocyte infusion, rituximab, and/or plasma exchange. Additional causes of PRCA following allogeneic HSCT include parvovirus B19 infection and graft-versus-host disease (GVHD). The pathogenesis of PRCA in parvovirus infection is thought to be viral-mediated suppression of erythroid precursors in the bone marrow and this is due to the tropism of this virus for erythroid progenitor cells. Nonmyeloablative and reduced intensity conditioning regimens have been reported to have an increased risk of PRCA due to persistence of recipient lymphocytes. Here we report two cases of PRCA following allogeneic HSCT from major ABO mismatched donors. Both cases were correlated with the presence of a T-cell clone in the peripheral blood and bone marrow, and there was complete resolution of anemia with a short course of steroid treatment, as well as disappearance of the T-cell clone. Patient # 1 – A 24 year old female of Mediterranean descent underwent an HLA- 8/10 matched and major ABO-mismatched sibling donor bone marrow transplant for Sickle-Beta Thalassemia. She received a myeloablative but reduced toxicity conditioning regimen consisting of busulfan 16mg/kg, fludarabine 140mg/m2, cyclophosphamide 105mg/kg, and alemtuzumab 52mg/m2. Her immediate post-transplant course was relatively benign and significant for a mild CMV colitis which responded with resolution after treatment with anti-viral therapy. Her red cell transfusion needs had significantly decreased to every four weeks by day 130. However, at day 146 from HSCT, she started requiring red cell transfusions every two weeks, with a drop in the reticulocyte count to 0.6%. No antibodies were detected, and the patient was negative for parvovirus B19 and did not have any evidence of GVHD. However, her bone marrow showed an marked erythroid hypoplasia, and was positive for a T-cell clone with gamma chain gene rearrangement in the T-cell receptor. Due to a new onset of transfusion need, she was treated with a short course of steroids with an excellent response and disappearance of the T-cell clone from peripheral blood. She remained on immunosuppression during this entire period with a calcineurin inhibitor. Patient# 2 – A 10 year old Hispanic girl underwent a 10/10 HLA-matched and major ABO-mismatced sibling donor bone marrow transplant for idiopathic acquired severe aplastic anemia. Her preparative regimen consisted of 200 mg/kg of cyclophosphamide and 16mg/kg of rabbit-anti-thymocyte globulin. Her immediate post-transplant course was complicated by E.coli bacteremia. She had prompt engraftment and became transfusion independent on day 86. A cyclosporine taper was initiated at day 100 but held at day 119 when anemia was first noted. Peripheral blood showed a T-cell clone with gamma and beta chain gene rearrangement. She was started on a moderate dose of steroids with a good response, and has been tapered down to physiologic replacement dose of steroids with normal Hb and transfusion independence. With the resolution of anemia, the gamma chain gene rearrangement is not seen in the T-cell clonality assay, however, the beta chain gene rearrangement persists. To our knowledge, this is the first report of a T-cell mediated PRCA following allogeneic HSCT. Moreover, the PRCA was steroid responsive and not associated with GVHD in both the patients. Disclosures:No relevant conflicts of interest to declare.

Hematologic and Bone Marrow Changes in Children with Protein-Energy Malnutrition

Background: All systems in an organism are affected by protein-energy malnutrition (PEM), but one of the worst affected is the hematopoietic system. Today PEM remains a very serious problem in developing countries. We examined the relationships between clinical features, hematological, and bone marrow changes with severe PEM from Turkey. Method: We evaluated 34 (11 females and 23 males) consecutive cases of severe PEM, with no underlying diseases aged 3–20 months. The clinical nutritional conditions of the patients were determined using the Wellcome-Trust PEM classification. Ten of the patients were in the Marasmic-Kwashiorkor (M-K) group, 10 were in the Kwashiorkor (KW) group, and 14 were in the Marasmic (M) group. Full blood count, protein, albumin, serum iron (SI), iron-binding capacity (TIBC), ferritin, vitamin B12, folic acid, complement-3 (C3), complement-4 (C4), and bone marrow were investigated in all groups. Results: Anemia was detected in 97% of patients. We determined serum iron levels were low in 67.6% of the patients, TS levels were low in 76.4% of the patients and ferritin levels were low in 20.5%. The level of vitamin B12 was normal in all patients. Bone marrow analysis showed erythroid series hypoplasia in 28.5% of patients in the M group, 50% in the KW group, and 30% in the M-K group. Marrow iron was absent in 58.8% of patients. Conclusion: The most common hematologic change in the children with PEM was anemia and major cause of anemia was iron deficiency in this study. Patients with severe PEM have normal Vit B12 and serum folate levels. Most of the patients with severe PEM had normal cellularity with megaloblastic and dysplastic changes in bone marrow due to the inadequate and imbalanced intake of protein and energy.

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

AbstractDiamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the “safe harbor” AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Pure red cell aplasia following autoimmune hemolytic anemia

A 26-year-old previously healthy female presented with a 6-month history of anemia. The laboratory findings revealed hemolytic anemia and direct antiglobulin test was positive. With a diagnosis of autoimmune hemolytic anemia (AIHA), prednisolone was started but was ineffective after 1 month of therapy. A bone marrow trephine biopsy revealed pure red cell aplasia (PRCA) showing severe erythroid hypoplasia. The case was considered PRCA following AIHA. This combination without clear underlying disease is rare. Human parvovirus B19 infection was not detected in the marrow aspirate during reticulocytopenia. The patient received azathioprine, and PRCA improved but significant hemolysis was once again documented with a high reticulocyte count. The short time interval between AIHA and PRCA phase suggested an increased possibility of the evolution of a single disease.

Mycophenolate mofetil-induced erythroid hypoplasia in a patient with systemic lupus erythematosus

Anaemia is prevalent in patients with systemic lupus erythematosus (SLE). The anaemia is often a consequence of the disease itself but may also be secondary to drug treatments. Mycophenolate mofetil (MMF) is increasingly used in the management of patients with SLE and its associated anaemia. We describe the case of a 19-year-old girl, who presented acutely with SLE and renal involvement. Her disease was controlled with immunosuppression but she later developed severe transfusion-dependent anaemia. Several causes were considered before a bone marrow biopsy led to the diagnosis of erythroid hypoplasia. In the absence of clinical or laboratory markers of active lupus, MMF was implicated as the cause. Its discontinuation led to a rapid and sustained correction of the anaemia. Red cell aplasia linked to the use of MMF is uncommon and the manufacturers are aware of fewer than 50 cases. This is the first case report of evolving red cell aplasia induced by MMF in SLE.

Reduction of Erythropoiesis in MDS with 5q Deletion Is Associated with Aberrant SPARC and TNF-Alpha Gene Expression and Predicts the Efficacy of Lenalidomide Therapy.

AbstractAbstract 2826Background of anemia in myelodysplastic syndromes (MDS) with 5q deletion (del(5q)) is a haplo-insufficiency of the RPS14 gene. However, degree of anemia and quantity of erythropoiesis vary markedly between MDS patients with del(5q), even with a similar degree of RPS14 insufficiency, so that the question arises whether other influences may play a further relevant role in erythroid failure in this disease.From 35 patients with low or intermediate-1 risk MDS with del(5q) who developed a transfusion-dependent anemia and were recruited and treated with lenalidomide (10 mg / d) within the MDS-003 study 38 +/− 33 months after diagnosis of MDS, bone marrow biopsies taken at 6 to 12-month time intervals after diagnosis of disease were evaluated for changes of erythropoiesis having occurred prior to lenalidomide therapy by morphometric, immunohistochemical, and molecular methods. A second group of patients with MDS treated with best supportive care (n = 307) served as a control. From each bone marrow biopsy, 20 000 nucleated cells were evaluated marking (1) nucleated erythroid cells (NEC) immunohistochemically using anti-HbA, glycophorin-C (GPC), CD71, and CD34 antibodies and (2) marrow macrophages by anti-CD68 antibody. By analogy to burst and colony forming units from in-vitro models, the numbers of erythroid precursor cells (EPC) were estimated by the numbers of erythroblastic islands (IslE) within marrow. Morphometrically assessed erythropoiesis was related to the mRNA expression of RPS14, SPARC, and TNF-alpha genes, nuclear p53 protein expression of NEC and their relevance for transfusion dependence and AML-free survival time of patients.5q deletion predicted erythroid hypoplasia independently of the type of MDS, evidence of marrow fibrosis and AML transformation (P < 0.0005). During early-phase MDS with del(5q), anemia correlated with p53 overexpression of NEC and decreased numbers of HbA+ NEC (P < 0.00005). After the first year of disease, two alterations of erythropoiesis became more relevant which did not correlate to p53 overexpression of NEC: (1) a decline in the numbers of GPC+HbA- NEC indicating impaired differentiation of EPC to proerythroblasts which correlated with RPS14 insufficiency, and (2) a progressive decrease in the number of IslE indicating a block of differentiation at an earlier stage, the IslE forming EPC, which did not correlate to RPS14 gene expression, but to two other independent factors, a reduced SPARC and an increased TNF-alpha gene expression (P < 0.00005). The degree of TNF-alpha expression correlated to the number of a central component of the IslE, the (iron-presenting) macrophages, the potential source of TNF-alpha overexpression. Both alterations contributed significantly to erythroid hypoplasia and progressive transfusion dependence (P < 0.00005). Response to lenalidomide therapy continued for > 4 years (100% AML-free survival) when started during a phase with high numbers of p53 overexpressing NEC, but was short or poor with < 20 % AML-free survival when started during a phase when differentiation of erythropoiesis was impaired at a less mature stage (P = 0.0001). On the basis of these observations, a score predicting the efficacy of lenalidomide in individual patients could be constructed. Conclusions:In MDS with del(5q), erythropoiesis significantly changes over time. In the early stage of disease, maturation defect of erythropoiesis mainly affects HbA+ NEC and correlates to p53 overexpression of NEC. In the course of disease, erythropoiesis becomes hypoplastic due to differentiation defects at the level of more immature erythroid (precursor) cells relating to a p53-independent mechanism of RPS14 insufficiency and altered SPARC as well as TNF-alpha gene expression. Efficacy of lenalidomide therapy is significantly impaired in the case of vanishing IslE, reduced SPARC and increased TNF-alpha gene expression. Disclosures:Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Knight:Celgene Corp.: Employment.

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Abstract 513Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA.In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP).To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients.Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures:No relevant conflicts of interest to declare.

Autoantibodies to erythropoietin receptor in patients with immune‐mediated diseases: relationship to anaemia with erythroid hypoplasia

The prevalence, clinical associations and pathogenic role of newly identified autoantibodies to the erythropoietin receptor (EPOR) in patients with anaemia were investigated. Sera from 203 patients with immune-related or chronic kidney diseases were screened for anti-EPOR antibodies by enzyme-linked immunosorbent assay, and antibody specificity was evaluated by immunoprecipitating EPOR from AS-E2 cells using purified immunoglobulin (Ig) fractions. In addition, the pathogenic role of anti-EPOR antibodies was determined by examining their inhibitory effects on AS-E2 cell proliferation. Clinical findings were compared between patients with and without anti-EPOR antibodies, in all patients and those with systemic lupus erythematosus (SLE). Serum anti-EPOR antibodies were detected in 52 patients. Purified IgG or IgM fractions from anti-EPOR antibody-positive sera immunoprecipitated EPOR and inhibited the EPO-dependent proliferation of AS-E2 cells in a dose-dependent manner. Anti-EPOR antibodies were associated with low haemoglobin concentrations and reticulocytopenia in all patients enrolled and those with SLE. Further, there was a negative correlation between the levels of anti-EPOR antibodies and the number of bone marrow erythroblasts in patients who underwent bone marrow examinations. These findings suggest that EPOR autoantibodies are present in a subset of patients with anaemia and that impaired erythropoiesis can be mediated by anti-EPOR antibodies, which functionally neutralize EPO activity.

Dietary L-leucine improves the anemia in a mouse model for Diamond-Blackfan anemia

AbstractDiamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.

Fell Pony Syndrome: Characterization of Developmental Hematopoiesis Failure and Associated Gene Expression Profiles

Fell Pony syndrome (FPS) is a fatal immunodeficiency that occurs in foals of the Fell Pony breed. Affected foals present with severe anemia, B cell lymphopenia, and opportunistic infections. Our objective was to conduct a prospective study of potential FPS-affected Fell Pony foals to establish clinical, immunological, and molecular parameters at birth and in the first few weeks of life. Complete blood counts, peripheral blood lymphocyte phenotyping, and serum immunoglobulin concentrations were determined for 3 FPS-affected foals, 49 unaffected foals, and 6 adult horses. In addition, cytology of bone marrow aspirates was performed sequentially in a subset of foals. At birth, the FPS-affected foals were not noticeably ill and had hematocrit and circulating B cell counts comparable to those of unaffected foals; however, over 6 weeks, values for both parameters steadily declined. A bone marrow aspirate from a 3-week-old FPS-affected foal revealed erythroid hyperplasia and concurrent erythroid and myeloid dysplasia, which progressed to a severe erythroid hypoplasia at 5 weeks of life. Immunohistochemical staining confirmed the paucity of B cells in primary and secondary lymphoid tissues. The mRNA expression of genes involved in B cell development, signaling, and maturation was investigated using qualitative and quantitative reverse transcriptase PCR (RT-PCR). Several genes, including CREB1, EP300, MYB, PAX5, and SPI1/PU.1, were sequenced from FPS-affected and unaffected foals. Our study presents evidence of fetal erythrocyte and B cell hematopoiesis with rapid postnatal development of anemia and B lymphopenia in FPS-affected foals. The transition between fetal/neonatal and adult-like hematopoiesis may be an important aspect of the pathogenesis of FPS.

Different boundaries characterize isolated and non-isolated 5q deletions in myelodysplastic syndromes and acute myeloid leukemias

Chromosome 5 long arm deletions, del(5q), are heterogeneous cytogenetic changes. An isolated 5q interstitial deletion is typically associated with erythroid hypoplasia, hypolobated micromegakaryocytes, less than 5% bone marrow blasts, macrocytic anemia, mild leukopenia, normal or increased

Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

AbstractDiamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

Pure red cell aplasia with follicular lymphoma showing regression and progression parallel to lymphoma

Malignant lymphomas occasionally accompany autoimmune diseases. Pure red cell aplasia (PRCA) is thought to arise via an autoimmune mechanism and rarely occurs in lymphoma. Here, we report a case of follicular lymphoma accompanying PRCA, in which lymphoma and PRCA showed parallel regression and progression in response to chemotherapy for lymphoma. An 83-year-old Japanese man presented with a chief complaint of dizziness. He was found to be anemic, and had a gastric ulcer and retroperitoneal mass. Biopsy of the gastric lesion suggested malignant lymphoma, and he was referred to our hospital. The initial laboratory results were as follows: hemoglobin, 73 g/L; mean corpuscular volume, 92.6 fL; and reticulocyte count, 2 9 10/L. Computed tomography (CT) imaging showed general lymphadenopathy, and excision biopsy of the axillary lymph node confirmed grade 1 follicular lymphoma. Fluorescence in situ hybridization (FISH) analysis of the lymph node was positive for fusion of the Bcl-2 and IgH genes. Bone marrow examination showed no lymphoma infiltration. He received six courses of R-CVP (rituximab, cyclophosphamide, vincristine, and prednisolone) chemotherapy and complete response was achieved. Even after completion of chemotherapy, his anemia persisted and he required regular transfusion of red blood cells. White blood cell and platelet counts were normal. Bone marrow examination showed remarkable erythroid hypoplasia without dysplasia, and lymphoma infiltration was not detected by FISH analysis. CT imaging indicated that thymoma was absent. Hemolytic anemia was unlikely, as lactic dehydrogenase, bilirubin, and haptoglobin were normal. The patient further did not show indications of thymoma or hemolytic anemia in his clinical course. Nine months after completion of chemotherapy, his anemia spontaneously resolved and he no longer required transfusions. Approximately 2 years after the initial therapy, CT imaging showed enlarged abdominal lymph nodes and the patient was diagnosed with recurrent lymphoma. He was observed without therapy, and 1 year after the recurrence, the disease gradually progressed accompanied by concomitant progression of anemia. At initiation of salvage therapy, hemoglobin was 82 g/L and reticulocyte count was 5 9 10/L. Bone marrow examination again showed erythroid hypoplasia without dysplasia or infiltration of lymphoma. Immunoglobulin M (IgM) antibody to parvovirus B19 was negative. With modified R-ESHAP chemotherapy (rituximab, etoposide, methylprednisolone, cytarabine, and carboplatin), his lymphoma achieved partial response. His anemia also improved; hemoglobin was 100 g/L and reticulocyte count was 85 9 10/L. However, as he suffered severe discitis, he could not continue chemotherapy. His lymphoma grew slowly, and his anemia progressed again. Bone marrow examination showed neither dysplasia nor infiltration of lymphoma. Rituximab mono-therapy was ineffective for lymphoma and anemia. Seven months after salvage chemotherapy, at the time of this report, the patient is alive with slowly progressive lymphoma and red blood cell transfusion dependence. His clinical course is shown in Fig. 1. PRCA has been reported to occur rarely in lymphoma [1–4]. In most reported cases, PRCA improved after the chemotherapy for lymphoma. However, steroids and other immunosuppressive agents, such as cyclophosphamide [5] and rituximab [6], used for lymphoma therapy are also G. Yamamoto H. Maki M. Ichikawa M. Kurokawa (&) Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Morphologic and Phenotypic Features of Concurrent Clonal T-Cell Large Granular Lymphocytic Expansion and Myelodysplastic Syndrome

Abstract 2803Myelodysplastic syndrome (MDS) and T-cell large granular (T-LGL) leukemia are both bone marrow failure disorders. It has been reported in a small number of cases that clonal T-LGL proliferation or leukemia can coincidentally occur with MDS. Also, clonal CD8+/CD57+ effector T cells expansion was detected in as many as 50% of MDS bone marrows [Epling-Burnette, 2007]. How clonal LGL cells that reside in the bone marrow interfere with hematopoiesis remains unclear, particularly in the setting of MDS. We analyzed the clinicopathological features of concomitant MDS and T-LGL, and evaluated bone marrow status for lineage or pan-hypoplasia in these patients. Design:Clinical and pathologic data from patients with a diagnosis of MDS and flow cytometry performed on the peripheral blood between 1/2005 and 12/2009 were reviewed. The concurrent bone marrow biopsies from each patient at the time of flow cytometric analysis were reviewed by two hematopathologists. Bone marrow cellularity, lineage hypoplasia (M:E >5: 1 or <1:2) were documented. Peripheral lymphocyte count and CD3+/CD57+ and CD8+/CD57+ populations by flow cytometry were calculated and T cell gene receptor (TCR) rearrangements were correlated. Results:We performed LGL flow cytometry panel on 76 MDS patients (high grade MDS, n=23; low grade, n=54), as well as TCR gene rearrangements, and identified clonal T-LGL cells in peripheral blood of 37 patients (48.7%), including 15 high grade MDS (40.5%, RAEB-I and RAEB-II), and 22 low grade MDS (59.4%), including RCMD(13), RA(1), RS(1), RCMD-RA(1), RCMD-RS (2), 5q- MDS(1), and MDS unclassifiable(3). The immunophenotype of the T-LGL cells was typically CD3+/CD57+/CD7 dim+/CD5 dim+/CD8+ with variable CD11b,CD11c, CD16, CD56 and HLA-DR. A frequent variant in these MDS patients was CD11b-,CD11c -, CD16+/−, CD56+/−, HLA-DR- and CD62L+.The TCRβ or/and TCRγ gene rearrangements were positive in 35 of the 38 cases (92.1%). The peripheral blood lymphocyte counts were 300–3820 cells/μL (1199±799 cells/μL); the CD3+/CD8+/CD57+ T-LGL cell counts were 30–624 cells/μL (229±154 cells/μL). In comparison, the remaining 39 patients with non-clonal T-LGL included 11 high grade MDS cases, and 28 low grade MDS cases. The peripheral blood lymphocyte counts were 308–2210 cells/μL (1030±461 cells/μL). CD3+/CD57+ cells were 1–425 cells/μL (105±98 cells/μL). There was no identifiable phenotypic features suggestive of clonal T-LGL cells such as dim CD5 and/or dim CD7 with aforementioned aberrant expressions on T-cells, although 7 of the 39 cases had TCRβ or/and TCRγ gene rearrangements. Thirty healthy donors were included for controls with absolute lymphocyte counts of 2136±661 cells/μL and baseline CD3+/CD57+ cells of 162±109 cells/μL. All showed no clonal LGL phenotype and negative TCR gene rearrangements.Since the presence of T-LGL cells may impair bone marrow hematopoiesis, we examined if there are bone marrow status differences between these two groups. All the bone marrows were obtained at diagnosis or not on chemotherapy. The bone marrow cellularity of the MDS patients with clonal T-LGL ranged from <3% to almost 100%, averaging 56%, with 8 cases with dramatic hypocellularity (<3%-20%), while the bone marrow cellularity of the MDS patients without clonal T-LGL ranged from 20% to 90%, averaging 62%, with only 2 cases with mild hypocellularity (20% in 73- and 65-year-old). In addition, among MDS patients with clonal T-LGL cells, 14 of 37 (37.8%; 5 high grade, and 9 low grade) bone marrows had certain lineage hypoplasia, including 3 cases of trilineal hypoplasia, 9 cases of erythroid hypoplasia, and 2 cases of myeloid hypoplasia. In contrast, among 39 MDS patients without T-LGL, there were only 1 bone marrow with trilineal hypoplasia and 3 others with erythroid hypoplasia (10.2%). The difference between the two groups was statistically significant (p=0.004, chi square test).In conclusion, our studies indicate that clonal T-LGL cells expansion is a fairly common finding in high grade as well as low grade MDS. The clonal T-LGL cells have more than one variant immunophenotypes and are typically positive for TCR gene rearrangements. Additionally, we observed that the clonal LGL cells present in MDS bone marrows could be associated with lineage hypoplasia, which, in this respect, might impact clinical treatment. Disclosures:No relevant conflicts of interest to declare.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Abstract SCI-2Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Failure in RPS19-Deficient Mice Is Partly Caused by p53 Activation and Responds to L-Leucine Treatment

Abstract 727Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al. Blood. 2010;116:193. ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. In the current study we have analyzed the role of p53 in RPS19-deficient hematopoiesis by crossing the transgenic mice into Trp53 null background. To isolate the hematopoietic phenotype we transplanted bone marrow cells from these mice into lethally irradiated wild-type recipients. We have previously shown that the severity of the hematopoietic phenotype in transplanted recipients is highly dependent on the level of RPS19 downregulation, and the recipients with low RPS19 expression die 2–3 weeks after induction because of bone marrow failure. Remarkably, the inactivation of Trp53 rescued the early mortality in these recipients. However, although the inactivation of Trp53 completely reversed the erythrocyte and leukocyte numbers in the recipients with intermediate RPS19 downregulation, the recipients with low RPS19 expression still developed a mild anemia and macrocytosis. p53 activation is known to inhibit the AKT/mTOR pathway, a central regulator of cell growth and survival. Although the role of this pathway in DBA pathogenesis remains poorly defined, some patients positively respond to treatment with amino acid L-leucine, a nutrient signal that stimulates mTOR activity. Currently we are studying the role of L-leucine in RPS19-deficient hematopoiesis both in vitro and in vivo. Our preliminary data confirm that L-leucine modestly enhances the proliferation of RPS19-deficient c-Kit -enriched hematopoietic progenitors in vitro (1.2 fold in 8 days), while there is no effect on wild-type cultures. Interestingly, the proliferative response in RPS19-deficient cultures appears more pronounced when cells are cultured in low cytokine concentration (1.6 fold in 8 days). Since primary cells from DBA patients are highly responsive to stem cell factor (SCF), which also mediates its effect partly via PI3K/AKT/mTOR pathway, we are studying whether L-leucine has a synergistic role with SCF enhancing the proliferation of hematopoietic progenitors. Finally, a 15% L-leucine supplement in drinking water partly rescues the erythrocyte and leukocyte number in RPS19-deficient mice. In summary, our results demonstrate a key role of p53 activation in RPS19-deficient DBA, although they also suggest that p53-independent pathways may contribute towards phenotype upon severe RPS19 deficiency. Furthermore, our preliminary data supports the role of L-leucine as a therapeutic agent in the treatment of DBA. Disclosures:No relevant conflicts of interest to declare.

Del(5q) in myeloid neoplasms

Note Interstitial del(5q) was first reported as a type of refractory anemia with characteristic clinical features; female predominance (unlike other MDS), macrocytosis, erythroid hypoplasia, frequent thrombocytosis and dysmegakaryopoiesis. It is one of the most common structural rearrangements in MDS (10%), seen as an isolated abnormality or with additional karyotypic anomalies. It is also observed in AML, with important prognostic significance.