ALBERT

Description: Abstract 972 Background: The IPSS published in 1997, based on cytogenetics, marrow blast % and the number of cytopenias, has played a major role in prognosis assessment in MDS. A recently presented (Greenberg, International MDS Workshop, Edinburgh 2011) IPSS provisional update (IPSS-R), using the same parameters but 5 rather than 3 cytogenetic subgroups (Schanz et al, EHA 2010), a new cut off for ANC (0.8 G/L) and different weighing of parameters, appears to refine IPSS prognostic value but, like the original IPSS, was established in pts who had received no disease modifying drugs. We assessed the prognostic value of IPSS-R in 265 higher risk MDS treated with AZA, a drug with a survival impact in those pts (Lancet Oncol, 2009). Methods: Between Sept 2004 and Jan 2009, before drug approval in EU, we enrolled 282 IPSS high and int 2 (higher) risk MDS in a compassionate patient named program of AZA and established in this cohort a prognostic scoring system (“AZA predictive score” based on Performance status (PS), cytogenetics, presence of circulating blasts, and RBC transfusion dependency) (Itzykson, Blood, 2011). We analyzed in this cohort the prognostic impact of IPSS-R in higher risk MDS treated with AZA. Results: Median age was 71 years. WHO diagnosis: 4% RA, RA RARS or RCMD, 20% RAEB-1, 54%RAEB-2, 22% RAEB-t (AML 20–30% blasts). Cytogenetics could be reclassified using new IPSS-R cytogenetic groups in 265 pts, in: 1% very good, 37% good, 18% int, 12% poor and 32% very poor. 66% pts had Hb

Description: Background We and others have shown that normal human erythroid cell maturation requires a transient activation of caspase-3 at late stages of maturation (Zermati et al, J Exp Med 2001). We further documented that, in human erythroblasts, the chaperone HSP70 is constitutively expressed and, at late stages of maturation, translocates into the nucleus and protects GATA-1, the master transcriptional factor critical for erythropoiesis, from caspase-3 cleavage (Ribeil et al, Nature 2007). During the maturation of human β-TM erythroblasts, HSP70 is sequestrated by excess of α-globin chains in the cytoplasm and as a consequence, GATA-1 is no longer protected from caspase-3 cleavage resulting in end-stage maturation arrest and apoptosis (Arlet et al, Nature 2013). Understanding the molecular mechanisms that regulate the localization of HSP70 during erythroid differentiation may help to find new therapeutic targets to reduce ineffective erythropoiesis in beta-thalassemia. Methods CD34 positive cells from normal and thalassemic peripheral blood were cultured in IMDM/BIT media in the presence of SCF, IL3, IL6 for seven days and subsequently cultured for additional 7 to 9 days in media containing SCF, IL3 and Epo. Erythroblasts differentiation, HSP70 localization were analysed by FACS, AMNIS stream, confocal microscopy and western blot analysis. RNAseq and proteomic analysis of highly purified erythroid cells at all distinct stages of differentiation were used to assess expression levels of various exportins. Duolink and Octet analyses were used to assess protein proximity and affinity of interactions, respectively. Results During erythroid differentiation, Hikeshi, the cognate nuclear importin of HSP70, is constitutively expressed and enables HSP70 nucleus entry as assessed by siRNA experiments. However, its expression was not regulated during erythroid differentiation. In contrast, exportin expression analysis showed marked differences in expression levels of XPO1 and XPO7 during erythroid differentiation. XPO1 expression being reduced at the time of c-kit down-regulation and caspase 3 activation while there was a marked increase in XPO7 expression at the late stages of terminal erythroid differentiation. XPO1 interacted in vivo (Duolink analysis) and in vitro with HSP70 (Octet analysis). Likewise, the previously described HSP70 S400A mutant (in the Leucine-rich Nuclear Export Sequence), which is constitutively located in the nucleus interacted with XPO1 with lower affinity compared to HSP70 WT. Stem Cell Factor (SCF) starvation and Pi3k inhibition led to decreased in vivo HSP70/XPO1 interactions. However, neither phosphorylation of HSP70 nor XPO1 were detected by Nanopro and proteomic analysis, and XPO1 expression was not regulated by Pi3K pathway. Expression of RanGTP Activating Protein (RanGAP), a protein critical for XPO1/cargo interaction, was down-regulated at the moment of caspase 3 activation during erythroid maturation, which may explain the decrease in HSP70/XPO-1 interactions. Inhibitors of XPO1 (leptomycin B and KPT 251) were able to induce HSP70 nuclear localization at early stages of differentiation (proE). In erythroid progenitors from β-TM patients, treatment with the Selective Inhibitor of Nuclear Export compound KPT-251 rescued nuclear HSP70 localization and GATA1 expression, and resulted in improved of erythroid terminal differentiation, without cytotoxicity, of thalassemic erythroid progenitors. Conclusion XPO1 is a major regulator of erythropoiesis through the regulation of HSP70 nuclear localization and is a potential new target to decrease ineffective erythropoiesis of thalassemia. Specific XPO-1 inhibitors currently in clinical development are being tested for potential therapy in thalassemic erythroid progenitors. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 422 Background: The IPSS published in 1997, based on cytogenetics, marrow blast % and the number of cytopenias, has played a major role in prognosis assessment in MDS. A provisional revised IPSS had been presented in 2011, which in our experience brought limited additional prognostic value for outcome of AZA treatment (Lamarque, ASH 2011). A final IPSS-R has now been published (Greenberg, Blood 2012), using the same parameters but 5 rather than 3 cytogenetic subgroups (Schanz et al, JCO, 2011), new cut off values for cytopenias and bone marrow blast % and different weighing of parameters. It appears to refine IPSS prognostic value but, like the original IPSS, was established in pts who had received no disease modifying drugs. We assessed the prognostic value of IPSS-R in 264 higher risk MDS treated with AZA, a drug with a survival impact in those pts. Methods: Between Sept 2004 and Jan 2009, before drug approval in EU, we enrolled 282 IPSS high and int 2 (higher) risk MDS in a compassionate patient named program of AZA and established in this cohort a prognostic scoring system (“AZA predictive score” based on Performance status (PS), cytogenetics, presence of circulating blasts, and RBC transfusion dependency) (Itzykson, Blood, 2011). We took advantage of this cohort to evaluate the prognostic impact of IPSS-R in higher risk MDS treated with AZA. Results: Median age was 71 years. WHO diagnosis: 4% RA, RARS or RCMD, 20% RAEB-1, 54%RAEB-2, 22% RAEB-t/AML. Cytogenetics could be reclassified using IPSS-R cytogenetic groups (Shanz, JCO 2011) in 265 pts, in: 1% very good, 37% good, 18% int, 12% poor and 32% very poor. 18%, 48% and 34% pts had Hb10 g/dl, respectively. 43%, 32% and 25% had baseline platelet count 100 G/L, respectively. ANC was

Description: Late stage of erythroidmaturation requires transient mitochondria depolarization (DYM) leading to caspasesactivation without apoptosis (Zermati, J Exp Med 2001). Thus, the control ofDY M (transient vs irreversible) determines the fate of erythroblast (Differentiaiton vs Apoptosis). Here we show thatDY Mduringdifferentiation is associated to a conformational change ofBax, which is dependent on Bid but neither onBim nor Puma. Likewise, decrease expression ofBax or Bid bylentiviral transduction oferythroid progenitors withshRNAs inhibit bothDY Mand terminalerythroid differentiation (ED) (figA). These results suggest that the extrinsic pathway of apoptosis is involved in ED.Caspase-10 but not caspase-8 is cleaved at the onset of terminal ED and its inhibition in immature erythroblasts withshRNA induced an end-stage maturation arrest and apoptosis. Both caspase-8 and caspases-10 can cleave Bid on LQTD-60 site, generating the typical proapoptoticP15 tBidfragment. This cleavage exposes a glycine residue to N-myristoylation, targeting Bid to mitochondria and promoting apoptosis. Caspase-10 can also recognize the IEAD-75 site, resulting in the generation of a P13 fragment. To determine whether Bid is cleaved during ED, we performed immunoblotanalyses on non-apoptotic sorted erythroidcells at different stages of maturation. We showed that Bid is sequentially cleaved during ED, generating two major truncated forms of 18 and 13 kDa, respectively. The truncated P18 Bid could result from a putative caspase-10 cleavage site located on RELD-38 and has never been described. To investigate the contribution of each Bid fragment on ED, we design N-terminal deletion mutants of Bid, starting on residue 39 (DEL38) or residue 76 (DEL75), generating Bid P18 and Bid P13 respectively, cloned in a lentiviralvector. While P18 overexpression induced apoptosis in 40% of erythroidcells, P13 did not and inducedouter DYMwithin a few hours. In contrast, both deletion mutants induced full ED after 3 days of culture compared to 8 days in controls, as shown by morphological and FACS analyses that show a higher percentage of Band3high/CD49dlow population (4.4% for control vs 39.35% for P18 p

Description: Introduction To better describe the baseline characteristics, the management and the outcome of patients diagnosed with cold agglutinin disease (CAD) in the “real life”, a retrospective multicentre study was performed. Methods All patients diagnosed with CAD in one of the 3 participating university centers in Paris area over a 20-year period were included. The diagnosis of CAD was based on features of active hemolysis (with or without anemia) with a positive direct antiglobulin test (C3 ± IgG pattern) and the presence of cold agglutinins in the serum at a significant titer (〉1/32), in the absence of any other cause of inherited or acquired hemolytic anemia. All patients’ characteristics were collected and retrospectively analyzed by the same investigator (ML) using a standardized study form. Results Forty eight patients (64.5 % of females, median age at diagnosis = 65.5 years [range: 30-93 years]) were included. The main symptom or biological abnormality leading to the diagnosis was: unexplained anemia (31%), acrocyanosis (25%), unusual fatigue (20%), hemoglobinuria (10%), neuropathy (8%), jaundice (8%), venous thrombosis (4%) or persistent lymphocytosis (4%). Of note, the diagnosis was incidental in 15% of the cases. The median hemoglobin (Hb) level at diagnosis was 9.3 g/dl [4-16.2 g/dl] and the titer of cold agglutinins varied from 1/32 to 1/64,000 with no obvious correlation with disease activity. Thirty-eight patients (77%) had a monoclonal IgM detected in the serum (kappa light chain in 92% of the cases). At time of diagnosis, a bone marrow aspirate (n= 19) or biopsy (n=11) was performed in 62.5% of the cases, an immunophenotyping of B-cell lymphocytes in the peripheral blood and/or in the bone marrow in 32 patients (66%), and a diagnostic imaging of chest and abdomen in 75% of the cases. Based on these tests, an underlying lymphoproliferative disorder (beyond the sole presence of monoclonal IgM) was found concomitantly in only 40% of the cases: unclassified clonal B cell- lymphoproliferative disorder (21%), chronic lymphocytic leukemia (11%), Waldenström macroglobulinemia (6%), and B-cell prolymphocytic leukemia (2%). The diagnosis of CAD was made after the diagnosis of lymphoma (1 follicular lymphoma and 1 case of marginal zone lymphoma) in 2 cases and before the onset of a diffuse large B-cell lymphoma in 1 case. After a median follow-up of 5 years [0.6-25 years], 12 patients (25%) did not require any other measures than folic acid supplementation and cold avoidance. At least one transfusion of packed-red blood cells (PRBCs) was required in 23 patients (48%), with a median of 10.5 PRBCs [2-40]. Thirty patients (62.5%) were given at least one treatment-line including rituximab (RTX, n=19), corticosteroids(n=14), alkylating agents (n=5), RTX + chemotherapy (n=7) or others (danazol: n=3, azathioprine: n=1, or intravenous immunoglobulin: n=3) The main treatment indication was an active haemolytic anemia (73%), marked cold-induced circulatory manifestations (7%) including 2 cases of cutaneous necrosis or both (13%), or tumor progression (7%). Of note, 15 patients (31%) were treated at least transiently with an erythropoietic-stimulating agent (ESA), mainly darbepoietin alpha, at various doses (ranging from 80µg to 300 µg/week) with a clear benefit observed in at least 8/15 cases (53%). In 5 patients, the use of ESA as a single agent was helpful to overcome one or several hemolytic episodes without the need of transfusion. Four patients (8%) have died during the follow-up period: CLL progression with sepsis (n = 1), myelodysplatic syndrome and sepsis (n =1), unknown cause in 2 elderly patients). Conclusion The initial workup and the management was highly heterogeneous and the observed rate of underlying lymphoproliferative disease was lower then previously reported in the literature. Whereas CAD has a relatively good long-term prognosis, a transfusion was required in almost 50% of the cases during follow-up and up to 65% of the patients received at least one treatment-line. The use of ESA off-label seems promising must needs to be better assessed prospectively. In the last decade, the use of rituximab alone or in combination with chemotherapy has emerged but its indications are still far from being consensual. There is definitely a need for international guidelines in order to harmonize the initial workup and treatment’s indications in patients with CAD. Disclosures: No relevant conflicts of interest to declare.