ALBERT

Description: Abstract 2119 Homozygous patients (SS) display the most severe form of sickle cell disease (SCD) with high risk of painful complications during their lifetime including vaso-occlusions (VOC), acute chest syndromes (ACS) and strokes. Even though multiple factors seem to be involved in those sickle related events, hypoxia is the most important triggering factor. Transfusing patients with IHP-RBCs (homologous RBCs loaded with Inositol Hexaphosphate) could potentiate existing transfusion therapy. Indeed, IHP by reducing the oxygen-hemoglobin affinity improves the capacity of transfused RBCs to release more oxygen in the blood stream and to the tissues. In a previous work, we showed that IHP-RBCs were 7-fold more effective than normal RBCs to reduce in vitro sickling of homozygous patient RBCs (Bourgeaux et al, Transfusion, 2010). We hypothesized that in vivo, IHP-RBCs with increased oxygenation properties may reduce the severity of SCD pathophysiology in transgenic mice. In a first study, BERK transgenic mice which are more representative of SCD in childhood (splenomegaly and RBC sequestration crisis) were used to evaluate the effect of chronic IHP-RBCs transfusions. Mice (n=5-7/group) were subjected to 4 repeated RBC exchanges (every 2 weeks) using IHP-RBCs or control RBCs. One week after the last exchange, mice were challenged with hypoxia and sacrificed for organ and hematological analysis. It was observed an improved survival rate and a prevention of anemia under hypoxic conditions (Hb=7.0±0.6 g/dl vs 5.9±0.4 g/dl for mice receiving control RBCs). On the contrary to control RBCs, IHP-RBCs treatment also corrected impaired brain development observed in female BERK mice with higher brain weight to total body weight ratio (2.12±0.07% versus 1.89±0.08% for untreated mice, p

Description: Abstract 1625 Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin S (HbS) that polymerizes under hypoxic conditions leading to sickled-shape red blood cells (RBCs). Vaso-occlusive crisis (VOC) is one of the major clinical manifestations of the disease, very painful for patients and causing irreversible organ damages. RBC exchange is commonly used as preventive and curative treatment of the disease. However, the therapeutic action of RBC exchange only relies on the removal of HbS-containing RBCs (SS-RBCs) and their transient replacement by normal RBCs (AA-RBCs). Recent works have shown that sickled reticulocytes, activated platelets and leukocytes play a critical role in the onset of VOC. They aggregate with endothelial cells creating local hypoxia, enhancing sickling and thus capillary blockade. Oxygen deprivation that occurs in venous capillaries may widely contribute to the severity of the occlusion. Therefore, increasing the oxygenation level in capillaries could help to prevent SS-RBCs from sickling and avoid crisis. This may be possible by transfusing patients with AA-RBCs loaded with Inositol HexaPhosphate (IHP), an allosteric effector that binds tightly to hemoglobin. The resulting suspension (IHP-RBCs) has the ability to increase oxygen release by 2 to 3 fold compared to normal AA-RBCs. The objective of the present study was to evaluate in vivo the benefit of using IHP-RBCs treatment in SCD. We used BERK transgenic mouse model that fully mimics human SCD in childhood with specific features of splenomegaly, reticulocytosis and leukocytosis. IHP-RBCs were prepared by loading IHP into murine C57BL6J RBCs using reversible hypotonic lysis method. RBCs subjected to reversible hypotonic lysis but without IHP were used as control suspension. Study was designed with repeated RBC exchanges scheduled every 2 weeks. First RBC exchange using IHP-RBCs or control suspension was performed on 6–7 week-old mice followed by 2 further injections. Mice were sacrificed one week after last RBC exchange and critical hematological parameters (reticulocyte, leukocyte, platelet counts and sickled cells) as well as serum inflammation markers were used as readouts to evaluate the risk of VOC. The first study was performed in normoxic conditions. After the therapy, approximately 42% of mouse RBCs had been replaced by IHP-RBCs or control suspension. Strong reduction of spleen weight (50%) and circulating sickled RBCs was observed in both cases due to the dilution of SS-RBCs. Interestingly, IHP-RBCs treatment enabled to significantly lower reticulocytes (18% vs 31%), leukocytes (5.3 vs 8.4 103/μl) and platelet counts (1057 vs 1518 103/μl) compared to not treated mice. Additionally, Serum Amyeloid Protein (SAP), an inflammation marker analogous of human C-Reactive Protein was also significantly reduced with IHP-RBCs (450 vs 750 μg/ml) indicating lowered severity of inflammation. The analysis of VCAM and HIF-1 factors in both spleen and lungs were very low in both treated and not treated mice. Further experiments demonstrated that hypoxic stress is needed to induce significative inflammation at the organ level. The study will thus be repeated in hypoxic conditions to evaluate the effect of IHP-RBCs treatment on organ damaging. We had in a previous study demonstrated in vitro the ability of IHP-RBCs to reduce sickling of human SS-RBCs (Bourgeaux et al, Transfusion, in press). The present in vivo study brings new evidence of the therapeutic potential of IHP-RBCs with the observation of a significant reduction of VOC risk factors and SAP level in treated mice. These results strongly support the fact that loading IHP into AA-RBCs may improve the effectiveness of conventional transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4333 Introduction L-asparaginase (L-Aspa) is one of the standard components of acute lymphoid leukemia (ALL) therapy. Its efficacy as an antineoplasic agent is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. In vitro studies have already shown the sensitivity to L-Aspa of leukemic cells from patients with acute myeloid leukemia (AML), depending on the French-American-British (FAB) subtype (Zwaan et al., 2000; Okada et al., 2003). Despite promising results in clinical trials (Capizzi et al., 1988; Wells et al., 1993), L-Aspa has only been used scarcely in the treatment of AML, mainly because of the adverse effects observed commonly that impair its use. The existence of a new formulation of L-Aspa (L-Aspa encapsulated in heterologous red blood cells) with a better safety profile allows considering its use for the treatment of AML (Godfrin et al., 2012). The sensitivity to L-Aspa may be inversely correlated to their expression of asparagine synthetase (ASNS, an enzyme catalyzing the intracellular synthesis of asparagine). The aim of this study was to investigate the potential of L-Aspa for AML treatment by determining the leukemic cell expression of ASNS and their sensitivity to L-Aspa. Materials and methods Studies were performed on an AML cell line (HL-60) and on leukemic cells isolated from the bone marrow of AML patients by Ficoll density gradient. The IC50 (concentration inhibiting 50% of cell viability) was determined in vitro by incubating various concentrations of L-Aspa with the cells and measuring the cell viability with a cell counting kit (CCK-8 viability assay). ASNS expression was determined by western-blot. Results As a preliminary, determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro(0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low in comparison with other cancer cell lines. Concerning blasts isolated from AML patients, 2/3 patients displayed an IC50 〈 0.01 IU/mL whereas 1/3 displayed an IC50 as low as 0.13 IU/mL, which means a high sensitivity to L-Aspa. All these patients were negative for ASNS expression. On 6 patients tested for ASNS expression, 5 had their blasts negative whereas one was positive. Patients with blasts negative for ASNS expression were all affected by FAB M5 or M1 AML whereas patient with blasts positive was diagnosed with a M2 AML. Conclusion Both AML cell line and cells isolated from AML patients were sensitive to L-Aspa. In all clinical cases, high sensitivity and/or lack of ASNS expression were linked to a FAB M5 or M1 AML, consistent with data from literature suggesting a higher sensitivity of M1, M4 and M5 AML subtypes. The only clinical case positive for ASNS expression was a FAB M2 AML, consistent with literature that indicates an in vitro resistance of the M2 AML subtype to L-Aspa. Globally, these results suggest that L-Aspa is effective for killing AML cells with low/no ASNS expression. Thus, thanks to its low toxicity profile, L-Aspa may be considered as a potential therapy for FAB subtypes or individuals characterized by low/no ASNS expression. Based on the epidemiology of AML subtypes (Selter et al., 2011), L-Aspa therapy may be beneficial for 50% of patients with AML. These results finally highlight the necessity of the determination of the grade and/or the expression of ASNS in leukemic cells to select AML patients at potential for L-Aspa therapies. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Campion:ERYTECH Pharma: Employment. Gay:ERYTECH Pharma: Employment. Godfrin:ERYTECH Pharma: COO Other.