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Background RBC transfusion can lead to the production of alloantibodies against RBC antigens and is a clinically significant issue in transfusion medicine. Patients with sickle cell disease have an increased risk for alloimmunization; 30-50% of SS patients have alloantibodies compared to 3-10% of other hospitalized patients. These alloantibodies can cause dangerous hemolytic transfusion reactions and limit the availability of compatible antigen-negative RBC products. This is of particular importance in SS patients, who commonly make alloantibodies against multiple RBC antigens and need regular transfusions to treat their disease. However, mechanisms underlying the increased frequency of alloimmunization in sickle cell patients are poorly understood. In previous studies, inflammation in the recipient has been shown to promote alloimmunization in both mice and humans. In mouse transfusion models, type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) have been shown to promote alloimmunization. Other studies have shown that patients with inflammatory autoimmune diseases express an IFNα/β signature, which may contribute to the increased frequency of alloimmunization in these populations. Given the chronic inflammatory state in patients with sickle cell disease, we hypothesize that: SS patients may also have an IFN gene signature that may contribute to the increased frequency of alloimmunization. Methods To test this hypothesis, we initially measured the expression of the ISG, myxovirus resistance protein 1 (MxA), in the blood of previously-transfused patients (n=50) with SS disease (SS, n=13) and without SS disease (ββ, n=37) by whole blood immunoassay (ELISA). We then measured expression of another ISG, Siglec-1 (SS n=5, ββ=24), expressed on monocytes by flow cytometric analysis. To determine the degree to which ISG expression correlated with alloimmunization frequency, expression of MxA in non-alloimmunized patients was compared to the expression in patients with 1 or more alloantibodies. Statistical analysis of 2 groups was completed with a Mann-Whitney U test. Significance between 3 groups was determined using a Kruskal-Wallis test with a Dunn's post-test. Results SS patients had significantly elevated levels of MxA (mean ± standard error of the mean, SS MxA = 8.98 ng/mL ± 2.46) compared to control patients without SS (MxA = 1.25 ± 0.54, p Discussion Factors that contribute to RBC alloimmunization in sickle cell disease are poorly understood. In this study, we found that sickle cell patients had an increase in the expression of ISGs compared to other transfused patients. We also found that MxA levels are increased in patients that have 2 or more alloantibodies compared to patients without alloantibodies. These findings suggest the presence of an IFNα/β gene signature in patients with sickle cell disease. Further studies are needed to determine the relationship between interferon-stimulated responses in sickle cell patients and the increased frequency of alloantibody production. Disclosures No relevant conflicts of interest to declare.

Background: Red blood cell (RBC) alloimmunization can follow exposure to foreign RBC antigens during transfusion therapy and during pregnancy/delivery. Alloantibodies against RBC antigens can cause potentially fatal hemolytic transfusion reactions and hemolytic disease of the newborn. Recent studies indicate that inflammatory states, including autoimmunity, promote RBC alloimmunization. For example, patients with systemic lupus erythematosus (SLE) have an elevated risk of RBC alloimmunization. Approximately 50% of SLE patients have a pro-inflammatory type 1 interferon (IFNα/β) gene signature, defined as the expression of interferon stimulated genes (ISGs), that is associated with disease severity. Recent studies in mouse transfusion models indicate that IFNα/β significantly promotes RBC alloimmunization. Thus, we hypothesized that IFNα/β produced in a lupus mouse model would promote RBC alloimmunization following transfusion. Methods: To test this hypothesis, we utilized the pristane-induced lupus mouse model, known to manifest lupus autoantibodies and a lupus-like phenotype. Mice lacking the IFNα/β receptor (IFNAR1-/-), mice lacking transcription factors required for IFNα/β production (IRF3/7-/-) and wildtype (WT) mice were administered an intraperitoneal injection of pristane 14 days prior to transfusion with transgenic RBCs expressing the KEL antigen. At the time of transfusion, serum IFNα levels were measured by ELISA, and expression of interferon stimulated gene 15 (ISG15) was measured by quantitative PCR. The mean fluorescence intensity (MFI) of the anti-KEL IgM and IgG responses were measured by flow cytometry 5 days and 7-28 days, respectively, following transfusion. A Mann-Whitney U test and a Kruskall-Wallis test with a Dunn's post-test was used to compare 2 or more than 2 groups, respectively. Results: Pristane induced serum IFNα in WT mice (Day 14 post-pristane, mean IFNα=127.3 Units/ml ± 28.21 standard error of the mean, SEM, p Conclusion: Pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens, and the investigation of the contribution of IFNα/β responses to increased frequency of alloimmunization in patients with SLE. Disclosures No relevant conflicts of interest to declare.

[Figure][1] A 64-year-old female with neck/back pain and vaginal bleeding was found to have anemia (hemoglobin 7.4 g/dL), leukocytosis (white blood cell count 22 800/µL), thrombocytosis (platelets 549 000/µL), and elevated creatinine at 2.7 mg/dL and calcium at 17 mg/dL. Image studies

Since the IRIS Study in 2000, tyrosine kinase inhibitors (TKI) have become a standard therapy for CML patients in lieu of interferon based therapy. Nevertheless, resistance occurs in roughly 30% of imatinib-treated patients. This resistance is attributable to ABL kinase domain mutations in 50% of cases, in which T315I mutation accounts for 10 to 15%. This latter is resistant to all TKIs available except for ponatinib which enables 75% of complete cytogenetic response (CCyR) in this setting (NEJM November 29, 2012). Before Ponatinib, various strategies were proposed to overcome the T315I mutation including more importantly ‘non-targeted' therapy such as Omacetaxine. IFN is also a ‘non-targeted therapy' known to have a well-established immunological effect. There are also clues about its cellular effects: microarray analyses have shown that IFN alfa can upregulate genes that encode for apoptotic proteins (i.e TRAIL, Fas, caspases 4 and 8 and XAF-1…) conferring the anti-growth effect different from ATP competition specific to TKIs. We report here an 80 years-old imatinib-resistant CML patient harboring the T315I mutation who has been successfully treated with Peg-IFN (Pegasys). His past medical history involved hypertension, asthma and chronic obstructive pulmonary disease. He was diagnosed in July 2008 with a chronic phase CML when he presented with weight loss, splenomegaly, leukocytosis (WBC 80G/l), slight anemia (Hemoglobin 112G/l) and normal Platelet count 240G/L. Karyotyping of 26 metaphase cells in the bone marrow showed Philadelphia (Ph1) chromosome without any additional abnormality. The molecular analysis by real time PCR (Light Cycler 480) detected a BCR-ABL p210 transcript. The Sokal score was high (at 1.24) while the Eutos score was low (31). He started Imatinib 400mg daily in July 2008 and achieved 5% IS BCR-ABL ratio at 3 months and complete cytogenetic response (CCyR) at 12 months. But the patient had never reached the major molecular response (MMR) and ended up losing the cytogenetic response in February 2012 (at 40 months) with an increase of 1.5log in the BCR-ABL ratio. Sequencing analysis for ABL mutation was carried out using the applied biosystems 3500 gentic analyzer. It revealed the T315I mutation at 20%. After a request for Ponatinib was dismissed by our health authorities, we decided to treat the patient with Peg-IFN in June 2012 at the dose of 90 µg per week. At this point, the imatinib had been stopped for 3 months, 8 metaphase cells out of 20 showed Ph1 chromosome without additional abnormality, BCR-ABL ratio was 3% IS, the mutational analysis showed 50% of T315I and 20% of another acquired E255K mutation. But the patient was still in complete haematologic response. figure 1 highlights the decline in the BCR-ABL ratio with MMR reached for the first time at 9 months (from Peg-IFN treatment). Of note, the CCyR was obtained again at this landmark. At 12 months from the Peg-IFN the CCyR was confirmed, the BCR-ABL ratio came very close to MMR4 (0.02% IS) while both T315I and E255K mutations disappeared on mutational analysis. Figure 1 Figure 1. No side effect was reported except for weight loss when the dose of Peg-IFN was increased to 180 µg per week which was the dose suggested by J.H. Lipton (Leukemia and Lymphoma, March 2007). The average dose in the past 12 months was 90 µg per week. The latter dose is well tolerated and is still ongoing. This observation shows that, though we are in the ponatinib era, there still is a room for interferon, in the management of CP CML patients even in BCR-ABL domain mutated patients because of its different mechanism of action. In this case the Pegylated Interferon a-2-a at the dose of 90µg per week has allowed clearance of both T315I and E255K mutations with a response close to MMR4. Taking into account the slope of the BCR-ABL decline, deeper molecular response is expected before the ASH meeting in December 2013. Disclosures: No relevant conflicts of interest to declare.

Abstract 529 Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/−) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros−/−) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface. To extend the analysis of coagulation defects beyond the Pros−/− phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis. It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/−, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration. In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6−/−Pros−/− mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros−/− embryos. In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration. Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling. Disclosures: No relevant conflicts of interest to declare.

Abstract 4593 Introduction: As deletion of 17p13 (del 17p13) in Chronic lymphocytic leukemia (CLL) patients is associated with poor outcome and chemorefractoriness to alkylating agents and purine analogues, current diagnostic guidelines recommend its screening at diagnosis and prior to treatment initiation. TP53 is a tumor suppressor gene, maps to 17p13 and is inactivated through somatic mutations or by 17p13 deletion. Recent studies show that TP53 mutation is of clinical relevance in CLL patients and has practical implication for therapeutic stratification. The purpose of this study is to characterize the TP53 mutation pattern and its incorporation into treatment algorithms in CLL patients treated in our center. Materials and methods: A retrospective study was performed on a cohort of 42 patients with CLL for whom clinical, biological and fluorescent in situ hybridization (FISH) data were available. Ages ranged from 31 to 88 (median, 63 years), 17 patients were in Binet stage A, 12 in B and 13 in stage C. Treatment was not in controlled trials, and standard clinical criteria were used for the initiation of therapy. DNA was extracted from 52 available samples and TP53 exons 2 to 11 were screened for mutations by high resolution melting (HRM) (Roche LC480). Mutations were confirmed by sequencing both strands using different primer sets (Beckman, Ceq 8000) then validated by the IARC and UMD TP53 Mutation Databases. Results: Out of 42 patients, del 17p13 was observed in 11 (26%), del 11q22 in 8 (19%), del 13q14 in 13 (31%), trisomy 12 in 2 (5%) and normal FISH in 20 (47.6%). We found an overall incidence of TP53 mutations of 19% (8 of 42 patients), 1 new mutation was identified and 5 mutations were not previously reported in CLL but in other cancers according to the IARC and UMD TP53 mutation Databases (table 1). A total of 65 polymorphisms were detected with a prevalence for the g.11446C>G polymorphism of 0.9 compared to 0.49 in published data. Amongst the 8 patients with TP53 mutations, 7 (87%) presented a 17p deletion and mutations were mainly located in the DNA binding domain (62%). 7 of 11 patients (63%) with 17p13 deletion harbored a TP53 mutation. In the absence of del 17p13 only 3% of patients (1 of 31) had a TP53 mutation (table 2). 5 of 8 patients with mutations (62%) presented with an advanced clinical stage and a median age of 56 years. Clonal evolution leading to acquisition of the TP53 mutation was not necessary related to therapy; 3 sequential samples were available for a patient with no abnormalities at diagnosis and who developed a TP53 mutation after 2 years and before therapy initiation. Another patient who was initially devoid of TP53 mutations and responded to treatment with standard chemotherapy, developed chemorefractoriness concomitant with a TP53 mutation acquisition. An interesting case was that of a patient who had been in complete remission after treatment with rituximab, fludarabin and cyclophosphamid (RFC) despite the presence of a TP53 mutation without del 17p13 at diagnosis. Sequencing demonstrated that this patient harbored a silent mutation in exon 4 g.12441C>G. In Conclusion: This study shows similar findings to published data except for a higher incidence of patients with TP53 mutations probably due to bias selection and a low cohort of patients. We identified a new mutation and 5 other mutations not previously detected in CLL patients. As TP53 is associated with poor outcome and chemorefractoriness, and its acquisition might be a result of positive pressure selection following treatment, we think it is of utmost importance to look for TP53 mutations in parallel to FISH at diagnosis and in case of chemorefractoriness using the HRM a cheap and sensitive method followed by sequencing positive cases. Disclosures: No relevant conflicts of interest to declare.

Abstract 2320 Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related death. With a decrease in antibody-mediated, plasma-induced TRALI, packed red blood cells (PRBCs) have become implicated more often. We have developed 3 experimental filters that concomitantly remove leukocytes, platelets, antibodies (abs), and lipids from PRBCs. We hypothesize that these filters remove antibodies against HLA class I and class II antigens and lipids without affecting the quality of stored PRBCs. Methods: 31 female donors whose plasma was historically positive for HLA by both Luminex™ beads (flow cytometry) and ELISA (GTI) testing were recruited and 4 male controls per filter were also drawn. Whole blood was drawn, PRBCs separated, then filtered with one of the experimental filters (B (n=11), P (n=10), or M (n=10)) or the FDA-approved Pall BPF4. Samples were drawn prior to filtration (Pre) or immediately after (Post) and at days 21 and 42 of storage. IgG, IgM, and HLA abs (by both flow cytometry and ELISA) were measured, plus a CBC was performed, pre-& post-filtration. Adenosine triphosphate (ATP), 2,3-diphoshoglycerate (2,3-DPG), pH, and neutrophil (PMN) priming activity were performed on all samples with the latter completed as the augmentation of the maximal rate of fMLP-activated PMNs measured as the superoxide dismutase inhibitable reduction of cytochrome c (nmol O2−/min). Statistical differences were determined by independent or paired (priming) ANOVA, and the data is expressed as the mean±SEM with *=p Disclosures: Silliman: Pall Corporation: Honoraria. Mish:Pall Corporation: Employment. Ceriano:Pall Corporation: Employment. Sowemimo-Coker:Pall Medical Corporation: Employment.

Late in erythropoiesis, nuclei are expelled from erythroblasts and 2×1011 anucleated new red blood cells are daily delivered in the peripheral blood. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as an “eat me” signal for their engulfment by macrophages located in the blood island. The two PS opsonins, milk-fatglobule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors αvβ5/αvβ3 and TAM (TYRO3, AXL and MER), are involved in the phagocytosis of apoptotic cells, but their role in the phagocytosis of expelled nuclei from erythroblasts is not determined. Because fetal liver and bone marrow macrophages do not express MFG-E8, the GAS6-MER pathway might constitute a crucial pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from spleens of phlebotomized mice, and studied nuclei internalization capacity of bone marrow derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), AXL (AXL−/−) or TYRO3 (TYRO3−/−), or lacking MER kinase domain (MERkd). Released nuclei were identified by flow cytometry according to their size and their double positive staining for the erythroid lineage marker Ter119 and Annexin V for PS. Purity of the preparation was checked by morphological examination of cytospin preparations. In vitro phagocytosis assays show that GAS6−/− BMDM cleared 30% less nuclei than wild-type (WT) BMDM. We observed a slight decrease of internalization capacity for AXL−/− BMDM, whereas TYRO3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, MER deficiency nearly abolished nuclei phagocytosis. AXL−/−/TYRO3−/− and AXL−/−/MERkd BMDM were tested and did not show any cumulative effects when compared to WT and single knockouts. We also investigated the signalling pathway downstream of MER in BMDM. In particular, we assessed the expression of the activated form of Rac1, which is crucial for the cytoskeletal reorganization in phagocytosis. Activation of Rac1 after the initiation of the phagocytosis was delayed for 45 minutes in MERkd as compared to WT BMDM. In vivo, we found an accumulation of nuclei in MERkd mice 4 days post phlebotomy, when erythropoiesis is increased in response to anemia. Nuclei circulated in the blood of MERkd mice at a level of 0.08 ± 0.042 G/L and were identified on peripheral blood smears of these mice whereas they were undetectable in the blood of WT mice. We demonstrated an increase of a double labelled Ter119/AnnexinV population corresponding to nuclei in BM (2-fold) and spleen (1.5-fold) of MERkd mice as compared to WT mice. The augmentation of this double labelled population in the MERkd mice translated the phenotype of splenomegaly of these mice. Hematocrit and reticulocyte levels were comparable between WT and MERkd as previously reported (JCI118:583–596, 2008). Thus, MER was critical for in vitro phagocytosis of nuclei from erythroblasts whereas the role of AXL and TYRO3 appeared to be negligible. GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and MER receptor on BMDM, allowing nuclei clearance. In vivo, the absence of MER caused an accumulation of nuclei in BM and spleen and their appearance in circulating blood due to their inefficient elimination during erythropoietic response to anemia. In conclusion, we postulate that GAS6 and its receptor MER were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island, through Rac1 activation.

Late in erythropoiesis, nuclei are expelled from erythroblasts and 2×10 11 anucleated new red blood cells are daily delivered in the peripheral blood. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as an “eat me” signal for their engulfment by macrophages located in the blood island. The two PS opsonins, milk-fatglobule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors αvβ5/αvβ3 and TAM (TYRO3, AXL and MER), are involved in the phagocytosis of apoptotic cells, but their role in the phagocytosis of expelled nuclei from erythroblasts is not determined. Because fetal liver and bone marrow macrophages do not express MFG-E8, the GAS6-MER pathway might constitute a crucial pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from spleens of phlebotomized mice, and studied nuclei internalization capacity of bone marrow derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6 −/− ) , AXL (AXL −/− ) or TYRO3 (TYRO3 −/− ) , or lacking MER kinase domain (MER kd ) . Released nuclei were identified by flow cytometry according to their size and their double positive staining for the erythroid lineage marker Ter119 and Annexin V for PS. Purity of the preparation was checked by morphological examination of cytospin preparations. In vitro phagocytosis assays show that GAS6 −/− BMDM cleared 30% less nuclei than wild-type (WT) BMDM. We observed a slight decrease of internalization capacity for AXL −/− BMDM, whereas TYRO3 −/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, MER deficiency nearly abolished nuclei phagocytosis. AXL −/− /TYRO3 −/− and AXL −/− /MER kd BMDM were tested and did not show any cumulative effects when compared to WT and single knockouts. We also investigated the signalling pathway downstream of MER in BMDM. In particular, we assessed the expression of the activated form of Rac1, which is crucial for the cytoskeletal reorganization in phagocytosis. Activation of Rac1 after the initiation of the phagocytosis was delayed for 45 minutes in MER kd as compared to WT BMDM. In vivo, we found an accumulation of nuclei in MER kd mice 4 days post phlebotomy, when erythropoiesis is increased in response to anemia. Nuclei circulated in the blood of MER kd mice at a level of 0.08 ± 0.042 G/L and were identified on peripheral blood smears of these mice whereas they were undetectable in the blood of WT mice. We demonstrated an increase of a double labelled Ter119/AnnexinV population corresponding to nuclei in BM (2-fold) and spleen (1.5-fold) of MER kd mice as compared to WT mice. The augmentation of this double labelled population in the MER kd mice translated the phenotype of splenomegaly of these mice. Hematocrit and reticulocyte levels were comparable between WT and MER kd as previously reported (JCI118:583–596, 2008). Thus, MER was critical for in vitro phagocytosis of nuclei from erythroblasts whereas the role of AXL and TYRO3 appeared to be negligible. GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and MER receptor on BMDM, allowing nuclei clearance. In vivo , the absence of MER caused an accumulation of nuclei in BM and spleen and their appearance in circulating blood due to their inefficient elimination during erythropoietic response to anemia. In conclusion, we postulate that GAS6 and its receptor MER were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island, through Rac1 activation.