In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)

Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders. IntroductionThe myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis, various degrees of pancytopenia, and a risk of progression to acute myeloid leukemia. 1 Low-risk myelodysplastic syndromes, including refractory anemia (RA) and RA with ringed sideroblasts (RARS), are defined as MDS with a low probability of progression to leukemia and with a relatively favorable outcome. 2 Anemia and transfusion dependency are the main clinical problems for these patients.Increased apoptosis of bone marrow precursors is a hallmark of MDS, [3][4][5] and is thought to underlie the ineffective hematopoiesis evidenced in individuals with MDS. Indeed, apoptosis mediated via the death receptor, Fas, and its ligand has been suggested to serve as an important pathogenic mechanism in MDS. [6][7][8][9] However, a clear-cut correlation between the level of expression of these apoptosis regulators and the degree of bone marrow apoptosis, or cytopenia, has not been demonstrated. In addition to extrinsic, death receptor-mediated induction of apoptosis, intrinsic signaling pathways that depend on mitochondrial events, including the release of apoptogenic factors such as cytochrome c, also exist. 10,11 Interestingly, signs of mitochondrial pathology are commonly seen in MDS, including the characteristic accumulation of iron in...

20 Congenital Factor XIII (FXIII) deficiency is a rare autosomal recessive disorder affecting approximately 1 in 2–5 million individuals without gender or ethnic predilection. The plasma form of FXIII is a heterotetramer [A2B2] comprising two FXIII-A subunits and two FXIII-B subunits. A-subunit deficiency predominates (95%). FXIII deficient patients have a high risk of life-threatening bleeds, notably spontaneous intracranial haemorrhage. They experience impaired wound healing, and recurrent first trimester spontaneous abortions. Given the severity of the disorder there is a clinical need for providing effective haemostatic replacement therapy. Currently, only plasma derived products are approved for use in the United States. These may carry a risk of blood borne infection, sensitization and allergic reactions. A multi-center, multi-national, open-label, single-arm, multiple dosing phase 3 (prophylaxis) trial was undertaken to evaluate the efficacy and safety of a novel recombinant FXIII (rFXIII) in the prevention of bleeds in congenital FXIII-A subunit deficiency. This rFXIII-A2 dimer is expressed in Saccharomyces Cerevisiae and is an exact copy of the human A-subunit, which is the active cross-linking enzyme. When rFXIII is administered, it immediately binds to the B subunit that is present in the bloodstream. Forty-one patients aged ≥7 years [mean: 26.4 (range: 7–60); 23 males; 18 females] with a diagnosis of severe congenital FXIII-A subunit deficiency were enrolled. Patients entered a 4-week run-in period followed by monthly treatment with 35 IU/kg of rFXIII for 52 weeks. Informed consent was obtained prior to any trial-related activities. Bleeding episodes and adverse events were recorded. Testing was conducted monthly to measure pre- and post-dose FXIII activity and concentration, 5M urea clot solubility and anti-FXIII antibodies. Where antibodies were found, detailed testing for neutralizing potential was performed. Data were compared with bleeding rates based on historical data. During the treatment period with rFXIII (466 patient months) five bleeding episodes treated with FXIII-containing products were observed in four patients. All five were associated with trauma. No spontaneous bleeds that require treatment or intracranial haemorrhage occurred during rFXIII treatment period in any of the patients. The annual rate of bleeds requiring treatment was estimated via a Poisson model to be 0.048 bleeds/patient/year (95% CI: [0.0094; 0.2501] - significantly lower than the historic bleeding rate of 2.91 (p<0.0001). The crude mean rate of bleeding was 0.138 bleeds/patient/year. The occurrence of bleeds requiring treatment was not associated with low FXIII activity levels in study patients. The average FXIII activity at 1 hr post-dose was 77.0 ± 20.3% (mean±SD). The mean incremental increase in activity from pre-dose to 1 hr post-dose was 1.68 ± 0.51% /IU/ kg. Four patients developed transient, non-neutralizing, low-titer anti-rFXIII antibodies. None of these patients developed anaphylactic or allergic reactions, bleeding episodes or changes in FXIII pharmacokinetics at any time. Furthermore, the non-neutralizing antibodies declined below the limit of detection in all patients despite repeated exposure to rFXIII or other FXIII containing products. Consequently, these low titer, non-neutralizing antibodies appear to be clinically insignificant. No clinical safety issues, thromboembolic events, or fatal adverse events were recorded. In conclusion, rFXIII-A2 is safe and effective prophylaxis for preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. Disclosures: Inbal: NovoNordisk: Honoraria, Research Funding. Oldenburg:novonordisk: Research Funding. Carcao:NovoNordisk: Research Funding. Rosholm:NovoNordisk: Employment. Tehranchi:NovoNordisk: Employment. Nugent:NovoNordisk: Consultancy, Research Funding.

IntroductionThe low-risk myelodysplastic syndromes (MDSs) refractory anemia (RA) and RA with ringed sideroblasts (RARS) are characterized by profound anemia and transfusion dependency, and a relatively low risk of progression to acute myeloid leukemia. 1,2 In RARS, the anemia is mirrored by hyperplastic but severely ineffective erythropoiesis due to increased apoptosis of erythroid progenitors. 3 The erythropoiesis of RA patients is also inadequate with apoptotic features, but may range from hypo-to hyperplastic, and shows no or few ringed sideroblasts. [4][5][6] The pathogenesis of RA anemia seems to be more heterogeneous, including T-cellmediated bone marrow failure in a subset of patients. 7,8 We have recently demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells and is associated with mitochondrial release of cytochrome c with subsequent activation of caspase-9 and effector caspase-3. Importantly, in RARS, granulocyte colony-stimulating factor (G-CSF) inhibits spontaneous release of cytochrome c, loss of mitochondrial membrane potential, and caspase activation, and restores erythroid proliferation. 9,10 Iron is predominantly stored in ferritin within cells. The multiple forms, or isoferritins, that can be found in human tissues are composed of variable proportions of 2 subunits: L-ferritin (light) and H-ferritin (heavy), encoded by genes located on chromosomes 11 and 19, respectively. Since free iron is potentially harmful to the cell, it is sequestered and detoxified to the less soluble ferric form by ferroxidase activity. H-ferritin (HF) exerts most of its ferroxidase activity in the cytosol. 11,12 Recently, a novel mitochondrial ferritin gene (MtF) was reported. This intronless gene contains a mitochondrial localization signal and is expressed in the mitochondrial matrix. It exhibits more than 75% sequence identity to the HF gene. 13,14 Mature erythroblasts from patients with X-linked sideroblastic anemia and RARS, in contrast to normal erythroblasts, express MtF 14 ; however, it is still unknown at which stage of erythroid differentiation this abnormal expression appears.The main task for the mitochondrion is to produce energy (adenosine triphosphate [ATP]). This occurs in the respiratory chain, consisting of 5 multiprotein enzyme complexes (I-V) and 2 15 Cytochrome c is closely associated with complex IV (cytochrome c oxidase) and has a major function to mediate the electron transport between complex III and IV. However, cytochrome c is also a key player in the regulation of apoptosis. 16,17 Erythroid differentiation requires activation of the erythropoietin (Epo) receptor followed by activation of the Jak-Stat pathway. This process is modulated by a complex network of transcription factors via activation of a set of target genes. GATA-1 plays a crucial role in erythroid development, and can switch the common lymphoid progenitors and granulocyte/monocyte progenitors toward megakaryocyte/erythrocyte lineage. 18,19 Epo induces globin gene expression an...