ALBERT

Description: Key Points FLT3 activation cooperates with the MLL-AF4 fusion gene to fully abolish blood formation from hESCs. FLT3 activation does not cooperate with the MLL-AF4 fusion oncogene to transform hESCs or hESC-derived hematopoietic progeny.

Description: Abstract 1947 Background: Thrombocytopenia requiring platelet transfusions is a constant in the hematopoietic transplantation (HT). In some situations, like the adult non-related donor and cord blood HT, the platelet engraftment is delayed for a long time. Hemorrhagic cystitis, venooclusive disease, graft-vs-host disease could to worse these procedures with a very high risk of bleeding and to increase the transplantation morbi-mortality. The agonists of thrombopoietin receptor (TRAs) have demonstrated to increase the platelet production in different pathological situations, like in the ITP and MDS patients. Thus, these new drugs could have a potential benefit in other clinical situations with low platelet production. Methods: We describe our experience in seven patients with Allogeneic HT using Romiplostim (NPlate®, Amgen Inc.), a parenteral TRA peptide, to accelerate the platelet engraftment or to increase the platelet level in the thrombocytopenia induced by HT conditioning or HT related complications. We have administrated Romiplostim in a compassionate basis (off-label). In all cases the drug was administered subcutaneously at a dose of 250 mcg. Most of the patients received only one dose, with the exception of patients #1 and 7, whom received two doses separated by seven days. The first case, a woman diagnosed as Acute Lymphoblastic Leukemia (ALL) with severe HLA platelet refractoriness acquired in the induction and consolidation chemotherapy treatments previous to HT, received two doses of 250 mcg of Romiplostim on days +4 and +12 after peripheral blood progenitor cells infusion from an HLA matched brother. Results: In the first case, a rapid and sustained platelet level increase was obtained, without platelet transfusional support. Encouraged by this successful result, we have used Romiplostim in six more patients with platelet refractoriness to platelet transfusions with or without bleeding. In all the patients the spleen was present. The patient #6, obtained a previous platelet engraftment that was loosed with the beginning of severe cGVHD. (see table) Conclusion: The use of Romiplostim could be very useful in HT complicated by severe platelet transfusions refractoriness. Our data encourages the realization of a randomized prospective study with this drug in HT. Graphic evolution of platelet count over time is depicted in the next figure: Days after Romiplostim administration. Number of platelets x109/L. Disclosures: Ojeda: Amgen Inc.: Consultancy, Honoraria. Off Label Use: Romiplostim (Nplate)is an agonist of thrombopoietin receptor (TRAs) that have demonstrated to increase the platelet production in different pathological situations, like in the ITP and MDS patients.

Description: Background and objectives Protocols for acute myeloid leukemia (AML) 1st line patients are centered on the combination of Cytarabine and an anthracycline; Idarubicin (IDA), Daunorubicin (DNR), or Mitoxantrone (MIT). Patients may be treated with IDA, DNR, or MIT depending on the country of residence, because multiple clinical trials have not found significant differences among them. A new Personalized Medicine (PM) test developed by Vivia Biotech based on pharmacological responses in patient samples (ex vivo) is uncovering individual responses to these treatments. Our objective is to explore whether a significant % of individual patients may respond differently to IDA vs DNR vs MIT treatments, in spite that of their “on average” similar response shown by clinical trials. Patients and Methods Multicenter, prospective, non-interventional study of the PETHEMA group for treatment of AML. Bone Marrow (BM) samples were collected at diagnosis for 160 AML patients. Samples were incubated for 48 hours in 96-well plates, each well containing different drugs or drug combinations, each at 8 different concentrations, enabling calculation of dose response curves for each single drug (CYT, IDA, DNR, MIT) and combination used in treatments (CYT-IDA, CYT-DNR, CYT-MIT). Drug response was evaluated as depletion of AML malignant cells in each well after 48 hours incubations. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis. Malignant cells were identified with monoclonal antibodies and light scatter properties. 1) We use the whole bone marrow sample, retaining the erythrocyte population and serum proteins, during the entire incubation period; and after 48 h leukocytes are isolated prior to evaluation by flow cytometry. 2) We have pioneered development of a proprietary automated flow cytometry platform called ExviTech. 3) Pharmacological responses are calculated using pharmacokinetic population models. Results Figure left panel shows dose responses for both IDA (red) and DNR (blue) in 125 AML patient samples. Although their average curves (thick red & blue) are similar, the interpatient variability of either drug is quite large. We hypothesized that some patients could show very differential sensitivities to both drugs, as illustrated by the green arrow where a patient sample is resistant to DNR (right shifted dose response curve) but sensitive to IDA (left shifted dose response curve). To identify these cases Figure right panel shows a comparison of the potency IDA vs DNR. Potency is represented by their EC50 (concentration that kills 50% of the cells). Most dots tend to line up, but red dots represent patient samples with a difference in potency between these drugs 〉30%. Repeating this exercise for IDA-MIT and DNR-MIT to cover all alternatives among the 3 anthracyclines identifies 40% of patients samples with 〉30% different potency among IDA-DNR-MIT. Repeating this exercise with the combination treatments CYT-IDA, CYT-DNR, CYT-MIT increases to 58% the population of patients whose samples have a differential sensitivity to these anthracyclines. A fraction of this 57% of patients may benefit in if treatment selection among these 3 treatments were to be aided by this ex vivo testing sensitivities. To identify which fraction would benefit we would need a trial specifically designed. Conclusions This preliminary results show that Vivia's PM test seems able to identify a subset of AML patients who's ex vivo pharmacological response to anthracycline drugs is significantly different. Because this ex vivo test accurately predicts the clinical response to CYT-IDA, if these selective anthracycline ex vivo responses translate to clinical responses, a fraction of this 57% subpopulation could benefit significantly from receiving 1st or 2nd line treatments based on either IDA, DNR, MIT, and their combinations. Hence this approach stands for European integration of treatment protocols, based on ex vivo individual responses data rather than nationality. Disclosures: Primo: Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Bennett:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership.

Description: Introduction The rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively. Methods All experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van’t Veer, C., and Mann, KG, 1997 JBC). Results Extrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p

Description: Background To aid in the identification of effective treatments for individual patients, ex vivo assays for detecting cell death inducible by drugs for hematological malignancies have been in development for over 20 years. We have developed a novel approach incorporating 4 key innovations; incubating drugs in whole bone marrow sample without isolating leukocytes, using flow cytometry enables identification of the malignant cells selectively, an automated flow cytometry-based platform (ExviTech) decreases errors and enables full pharmacological characterization, and analyzing the data using pharmacodynamic population models. Aim The purpose of this study is to derive the ex vivo pharmacological profiles across the AML patient population of single drugs and combination treatments as a tool for individualized treatment selection. Patients and Methods Bone-marrow samples from 160 patients diagnosed with AML were sent to Vivia from 24 hospitals across Spain within 24 hrs. The plates were incubated for 48-hours prior to analysis with ExviTech, The percentage of leukemic cell death was determined via labeling with monoclonal antibodies and AnnexinV-FITC. A survival index is computed for each drug, the lower the survival index, the more effective the drug. Dose-response curves of cytarabine, idarubicin, daunorubicine, etoposide, mitoxantrone, fludarabine, clofarabine, and 6-thioguanine were measured in 160 patient samples. The added benefit of combining these drugs into 12 combination treatments was assessed by measuring their synergy in each individual patient. In 39 patients treated with CYT IDA we had clinical data of response, and then we performed a blinded interpretation of this in vitro test by an expert hematologist, to predict the clinical response based in this test result. Results There was a large range of interpatient variability in the response to a single drug and even larger in the synergism between drugs. The Population Pharmacological Profiles for an individual patient is shown on the figure below. The relative drug potency in terms of their percentile ranking within the population is shown in the left panel from 0 (weakest) to 100 (most potent). Green lines represent the individual patient potency relative to the population ranking, with confidence intervals. Third column lists when a drug leaves a significant % of leukemic cells alive, potential resistant clones. The panel on the right side shows the synergism of the drug combinations treatments shown as box-plots at 10-25-75-90% to highlight their distribution. The synergism value for an individual patient in each combination is shown in green, with confidence interval as parallel dotted green lines. This representation of the Pharmacological Profile of an individual patient sample quickly identifies extreme values, when a drug or combination is very sensitive (rightward shift green lines, green boxes) or very resistant (leftward shift green lines, red boxes). This patient showed average sensitivities for most drugs though highly resistant to Clofarabine (red box) that leaves 45% alive. However this patient showed lack of synergism in multiple treatments (right, red boxes). CYT and IDA show average potencies but lack of synergism, suggesting CYT-DAU might be a more efficient treatment. These representations lead to clear guidelines in 〉90% samples, and based on hematologist's interpretation of these guidelines show a clinical correlation with clinical responses to CYT-IDA of 84%. Conclusion We have developed an improved a methodology to measure the pharmacological activity of drugs and drug combinations in AML patient samples as well as modeling their pharmacological behavior. This information may be useful in selecting the optimal treatment for the individual patient, especially relapse/refractory patients in need of therapeutic alternatives. By testing the drugs used in the treatment protocols for AML directly on patient samples, a pharmacological based model has been developed to infer drug resistance or sensitivity, patient by patient. Disclosures: Ballesteros: Vivia Biotech: Equity Ownership. Primo:Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Iñaki:Vivia Biotech: Consultancy. Bennett:Vivia Biotech: Employment.

Description: Abstract 2323 Graft-versus-host disease (GVHD) remains a major complication after allogenic hematopoietic stem-cell transplantation (alloHSCT) from HLA-identical donor. It is due to donor T-cell responses against minor histocompatibility antigens (mHags) of the recipient. Nevertheless, there is a complete lack of studies addressing the B-cell response to mHags in GVHD. Glutathione S-transferase T1 (GSTT1) is a drug metabolizing enzyme that is involved in detoxification processes. It is highly expressed in liver, kidney and erythrocytes. The GSTT1 gene is absent in 20% of the Caucasian population. In liver and renal transplantation, we have reported that some GSTT1-negative patients who received a GSTT1-positive graft produce anti-GSTT1 antibodies (abs) and present signs of chronic rejection. The aims of this study were to analyze the effects of GSTT1 donor/recipient mismatches in the development of hepatic GVHD and to detect production of anti-GSTT1 abs after alloHSCT. For that purpose, we have studied a group of 40 patients that received an alloHSCT from HLA-identical donors between January 2004 and July 2009 in HU Virgen del Rocío, whose DNA samples and their corresponding donor samples were available. All patients and their donors gave written informed consent. GSTT1 genotyping was performed by PCR and a commercially available ELISA test with the GSTT1 human recombinant protein was used to detect anti-GSTT1 abs. We studied abs in serum samples corresponding to different pre and post transplant phases. The distribution of the four possible GSTT1 genetic combinations was as follows: 25 donor+/recipient+, 6 donor+/recipient-, 5 donor-/recipient+ and 4 donor-/recipient-. Anti-GSTT1 antibodies were detected in 5 patients all of them with a donor that carries the null genotype. Four of the 5 patients included in the donor-/recipient+ group developed anti-GSTT1 abs (of the IgG class) after the infusion and were diagnosed with acute hepatic GVHD. In all of these cases the donor was a multiparous female. The presence of anti-GSTT1 abs is significantly associated with hepatic GVHD (p=0,01, Table 1). The fifth recipient within this group had a non-transfused male donor and did not produce abs. In all the cases, anti-GSTT1 abs were found in post-transplant serum samples of the recipient (never in pre-transplant samples) under a condition of 100% chimerism. Our hypothesis is that the immune system of the null donors has encountered the GSTT1 antigen during pregnancy of a positive-fetous and memory B-cells would be in the peripheral blood stem-cell infusion. These cells, once in a positive recipient, would be activated when contacted with the antigen present in the recipientxs liver. In the patients, the B-cells produced high levels of anti-GSTT1 abs that were associated with GVHD episodes. In conclusion, we describe that the presence of anti-GSTT1 abs is associated with the development of hepatic graft-versus-host-disease and that the null genotype of the donor is a necessary condition for antibody-production. These results confirm the GSTT1 gene as new minor histocompatibility antigen. With Hepatic GVHD Without Hepatic GVHD With 4 1 5 anti-GSTT1 abs Without anti-GSTT1 abs 6 29 35 10 30 n = 40 Disclosures: No relevant conflicts of interest to declare.

Description: Introduction The rapid coagulation response to vascular injury is mediated by the formation of the extrinsictenase, intrinsictenase, and prothrombinase complexes. The prothrombotic response to injury is down-regulated by the presence of circulating active protease inhibitors as well as the protein C pathway. Protein C is activated by the thrombin-thrombomodulin complex; activated protein C (APC) then regulates thrombin generation by proteolytically inactivating factors Va and VIIIa, cofactors of the procoagulant prothrombinase and intrinsic tenasecomplexes, respectively. Previous reports have analyzed the biochemistry of the protein C system in closed systems. Our goal is to characterize the behavior of the protein C system under flow as well as the impact of circulating cells on the activation of protein C. Methods Experiments were conducted in phospholipid (3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine) coated capillaries containing rabbit thrombomodulin (TM) that were preloaded with α-thrombin (αIIa) or recombinant meizothrombin (rMZ). Protein C (PC) activation was evaluated under flow at pH 7.4 and 37°C in either a buffered solution containing 2 mM CaCl2 and PC at its mean physiological concentration (65 nM) or in a mock blood mixture containing 60% of buffer containing 65 nM PC and 40% freshly prepared washed red blood cells; shear rates ranged from 100-1000 s-1. Capillary effluents were collected and then assayed for APC levels using a modified aPTT assay. To establish whether PC activation is under dilutional or diffusional control, the steady state concentrations of APC achieved at different shears were normalized to the residence time of one capillary volume specific for each shear rate. The efficiency of PC activation was also analyzed by normalizing the amount of APC generated to the amount of PC present in the mixture (1.3 pmol PC in buffer only vs. 0.78 pmol of PC in mock blood trials). Results At low shear rates (100 s-1 and 250 s-1) in the buffer only system the rMZ•TM complex generates 42-55% higher levels of APC than the αIIa•TM complex. Protein C activation by the αIIa-TM complex appears to be dilutionally controlled at shear rates ≥ 500 s-1, while diffusionally controlled at lower shear rates (≤ 250 s-1). The inclusion of red blood cells in the reaction system under flow resulted in a broader range of dilutional control (≥ 250 s-1) compared to the buffer only system (≥ 500 s-1). Normalization of the data to account for the differential amount of protein C present in a given volume indicate a two-fold greater efficiency of PC activation in the presence of red blood cells (14.7 ± 1.2 mol APC•mol-1 PC•min-1•cm-2) compared to buffer alone (6.7 ± 0.6 mol APC•mol-1 PC•min-1•cm-2). Conclusions In the presence of catalytically inert red blood cells the activation of protein C is regulated by diffusion only at the lowest shear rates tested (100 s-1). These data suggest that the dynamics and aggregation of red blood cell effects are shear dependent as red blood cells deform and migrate toward the center of the channel at increasing shear rates. We can hypothesize that at high shear rates (≥ 500 s-1), when the levels of APC generated in the red blood cell system and buffer only system are similar, the excluded volume created by the red blood cells agglomerated at the center of the capillary leaves a cell-free region adjacent to the wall which is large enough to accommodate the space needed for surface catalysis (depletion zone). Indeed the adjustment of PC concentration for excluded volume in red blood cell solutions yields the same concentration of APC generated as in the buffer solution. However, at low shear rates (100 s-1) the red blood cells do not create a distinct channel and the depletion zone extending from the capillary wall overlaps with red blood cells and maintains the diffusional control of the protein C system. These studies provide a foundation for studying the impact of circulating cells on the biochemistry of the coagulation cascade Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5049 Background. Treatment of frail or elderly patients with relapsing symptomatic/active Multiple Myeloma (MM) is very difficult due to concomitant diseases, impaired bone marrow reserve, systemic toxicity, relatively decreased renal function and general problems of old age. Dexamethasone and new agents (thalidomide, lenalidomide, bortezomib and bendamustine) have been used in this setting, in most cases with doses adapted to the clinical situation. Aims. To retrospectively analyze the management of frail and/or very elderly MM patients with relapsed and active disease treated with reduced doses of the aforementioned agents in five hospitals in Madrid, Spain. Methods. The files of this group of MM patients were studied. The most common treatment has been the combination of low doses of lenalidomide (len) and of dexamethasone (dex), whereas treatment with reduced doses of other agents has been anecdotal; therefore we analyzed the results of len/dex combinations. Len and dex have been used in lower than standard doses, adapted to the individual initial situation of the patients and tailored according to effect and toxicity throughout treatment. There was no specific protocol and the management of the patients has depended exclusively on the practice and criteria of the treating physicians. Patient risk was stratified following the Salmon and Durie (S&D) score and the International Staging System (ISS). Response was assessed with the IMWG criteria. The study has been approved by the Ethics Committee of Hospital Ramon y Cajal, as coordinating center. Results. 38 patients were included in the study. Mean age was 79 years (range 68–90). 30 pts (79%) were older than 75 years and 10 pts had over 85 years. More than half of the patients (21) had two or more comorbidities. Patients had previously received 1 to 5 (m=1. 8) different treatment modalities, including steroids, melphalan (25), bortezomib (20), thalidomide (6) (or their combinations), and others or even APBSCT (3). 23 pts (60%) had IgG (m=4087 mg/dl, range 868–13000); 13 (34%) IgA (m=2115, range 355–4930) and 2 (5%) only light chains. 22 had κ and 15 λ light chains. 19 (50%) had BJ proteinuria. Mean Hemoglobin level was 10. 7 gr/dl (7. 5–14. 1) and mean creatinine level 1. 3 mg/dl (0. 4–12. 9); 28 (74%) had bone disease. 3 pts had S&D stage I, 22 stage II, and another 13 stage III. 13 pts had ISS stage I, 17 had stage II and 7 stage III. Patients received between 1 and 30 cycles of len/dex (m= 8). Median initial Len dose was 10 mg, the majority between 5 and 15mg, although 4 received 25 mg that were rapidly reduced. Mean initial dex dose was 20mg/day for 4 days. 4 pts (10. 5%) achieved Complete Remission (CR) (3 with negative IF), 27 (71%) Partial Remission (PR) (5 with VGPR) and 2 (5%) a significant, but lesser than 50%, reduction of the M-component (Stable Disease, Std). Altogether, overall response (CR+PR+Std) occurred in 33 pts (86%). The best response occurred after 2 to 9 cycles (m=4) of len/dex. Treatment was stopped in 15 patients due to neurological (4) or hematological (1) toxicity, pulmonary embolism (1), unrelated causes (4) and after achieving a plateau response (5). Time to next treatment was 1–30 months, (m=8 mo). 7 pts relapsed after 3–21 months (m=7). 10 patients died, 5 of related (disease progression, cardiac amyloidosis, renal progression to ESRF) and 5 of unrelated (cancer, sepsis, myocardial infarction, congestive heart failure) causes. Grade III-IV bone marrow toxicity occurred in 9 pts and neurological toxicity (PNP) in 5 (all of them had previously been treated with bortezomib or thalidomide). Conclusions. Personalized low doses of len/dex have been the most common treatment for frail/very elderly patients with relapsed MM in our centers and it is an active and tolerable option in this setting. The haematological toxicity was expectable and manageable, but prior treatments with bortezomib or thalidomide were associated with limiting neurotoxicity. Disclosures: No relevant conflicts of interest to declare.

Description: About 25% of severe hemophilia A patients undergoing factor VIII (FVIII) replacement therapy develop antibodies against FVIII (FVIII inhibitors). The formation of inhibitor is generally accepted as the most common and challenging complication of hemophilia treatment. In the circulation, FVIII is tightly bound to von Willebrand factor (VWF), forming a complex that plays a significant role in FVIII protection from premature degradation (Delignat S et al, Haemophilia 2012;18:248-54). Several laboratory and clinical studies suggest that the presence of VWF in FVIII concentrates might have some benefits due to the protective effect against antibodies (Shi Q et al, J Thromb Haemost 2012;10:2328-37; Mannucci PM, Haemophilia 2012;18 Suppl 2:2-7). Plasma-derived (pd) FVIII/VWF concentrates show higher residual FVIII activity and more thrombin generation after incubation with inhibitors compared to isolated FVIII concentrates (pdFVIII or recombinant, rFVIII). The protective effect of VWF observed in native pdFVIII/VWF complex is not completely restored for isolated FVIII even after mixing with VWF before analyzing the reaction with inhibitors (Bravo MI et al, Haemophilia 2014; in press). This study analyzes the impact of VWF on FVIII protection against inhibitors in an in vivo model, by evaluating the differences of FVIII in vivo recovery between FVIII concentrates with or without VWF in FVIIInull E16Knockout (KO) mice infused with inhibitors purified from hemophilic patients’ plasma. Inhibitor IgG was purified from a pool of human plasmas from hemophilic patients (Technoclone GmbH, Vienna, Austria) by using Protein G Sepharose chromatography as described elsewhere. IgGs were tail vein infused to achieve 3.2 BU/ml (or buffer as a control) prior to FVIII treatment. Five minutes after IgG infusion, animals (n=5-6 for each treatment group) were infused with FVIII therapeutic concentrates (100 IU/kg) from different sources, including native pdFVIII/VWF complex; isolated pdFVIII and full-length recombinant FVIII (rFVIII). In addition, some animal groups were treated with in vitro preformed complexes with pdVWF (therapeutic concentrate for von Willebrand disease) and FVIII (rFVIII+pdVWF and pdFVIII+pdVWF mixed at 1IU:1IU ratio; 15 min at 25ºC). After 5 min post-FVIII infusion, plasma samples were obtained by abdominal cave vein sampling and residual FVIII:C was measured using chromogenic assay. FVIII recovery was estimated according on the empirical finding that 1IUFVIII/kg body weight raises the plasma FVIII activity by 2.1±0.4% of normal activity. In the absence of inhibitor, in vivo FVIII recovery in FVIIInull E16 KO mice was similar for all FVIII concentrates as expected, with values ranging from 107% to 124% (see table). In contrast, in the presence of inhibitors the FVIII recovery showed marked differences among treatment groups. Recovery was higher for the native VWF-containing FVIII concentrate (pdFVIII/VWF: 71.1±17.4%) when compared to concentrates composed of isolated FVIII (rFVIII: 31.4±9.9%; pdFVIII: 25.0±2.7%). When the products containing isolated FVIII were premixed with VWF prior to infusion, FVIII recovery was only partially restored (rFVIII+VWF: 67.1±15.1%; pdFVIII+VWF: 45.2±8.8%). FVIII:C recovery and ratio FVIII:C, 5 min post-infusion of FVIII products (100 IU/kg), without inhibitor and with inhibitor (3.2±0.4 BU/ml) in severe hemophilic mice. Abstract 2826. TableFVIII productsFVIII:C Recovery (%) [mean±SD (n)]FVIII:C ratio (IU) in absence vs. presence of inhibitorWithout inhibitorWith inhibitorpdFVIII /VWF107.9 ± 6.1 (6)71.1 ± 17.4 (5)1.52rFVIII106.9 ± 8.3 (6)31.4 ± 9.9 (5)3.41pdFVIII120.5 ± 5.1 (6)25.0 ± 2.7 (5)4.77rFVIII+pdVWF124.3 ± 11.2 (6)67.1 ± 15.1 (6)1.84pdFVIII+pdVWF111.8 ± 5.9 (6)45.2 ± 8.8 (6)2.47 Our data indicate that VWF-containing FVIII concentrates are more efficient in the restoration of FVIII circulating levels in the FVIIInull E16 KO mice model with inhibitors. Moreover, results also suggest that this protection would be higher in native pdFVIII/VWF complex that in the complex of FVIII+VWF formed from the isolated proteins. Disclosures Bravo: Instituto Grifols: Employment. Da Rocha-Souto:Instituto Grifols: Employment. Grancha:Instituto Grifols: Employment. Jorquera:Grifols Bioscience: Employment.

Description: Introduction Pts with high-risk cytogenetics and/or relapsed multiple myeloma (MM) post autologous transplant have a particularly limited prognosis. Conventional allogeneic hematopoietic stem cell transplantation (allo HSCT) has been associated with unacceptably high rates of mortality and non-myeloablative allo HSCT has resulted in high rates of acute and chronic graft-versus-host disease (GvHD) and progression. Methods We report results of a phase II clinical trial of 34 pts, using T-cell depleted allo HSCT (allo TCD HSCT) from HLA compatible (matched related = 12, matched unrelated = 13, and mismatched unrelated = 9) donors. All 34 pts had relapsed myeloma within 15 mos following auto HSCT, and 26/34 pts also had high-risk cytogenetics at diagnosis [t(4;14), t(14;16), del17p by FISH and/or del13q by karyotyping]. All pts had to achieve at least a partial response from preceding salvage chemotherapy (n=26) or second salvage auto HSCT (n =8). Pts underwent allo TCD HSCT with busulfan (0.8mg/kg x 10 doses), melphalan (70mg/m2 x 2 days), fludarabine (25mg/m2 x 5 days) and rabbit ATG (2.5mg/kg x 2 days). T-cell depletion was performed by positive CD34 selection (Isolex) followed by rosetting with sheep erythrocytes for the initial 13pts (2008-09) and by CD34+ enrichment by the Miltenyi Device in 21pts thereafter, achieving 〈 104CD3+/kg for all grafts. None of these pts received immuno suppressive therapy post TCD HSCT. Pts with 10/10 HLA matched donors were also eligible to receive low doses of donor lymphocyte infusions (DLI) (5x10e5 – 1x10e6 CD3+/kg) no earlier than 5mos post allo HSCT. Results 34 pts with a median follow up of 31.6mos (range: 7.6 – 65.1 mos) of survivors are reported, median age 56 years (range 32 – 69). All pts engrafted promptly (median d+10, range d+9 to +12).TRM and acute GvHD (grade II-IV) at 12mos is 9% (95% CI: 2% – 23%) and 6% (95% CI: 1% – 17%). Chronic GvHD was not observed in any pt. The overall survival (OS) and progression-free survival (PFS) with their 95% confidence intervals (CI) are shown in Table 1. Factors associated with worse outcome were disease status and number of previous treatments prior to TCD HSCT. (Table1; Figure 1) 15/34 pts are alive, 10/15 pts are in complete remission (CR), 4 pts are have been in continued CR for 44, 53, 62 and 65mos post allo TCD HSCT. 5/15 pts alive have progressed and 4/5 pts are currently responding to salvage chemotherapy and/or DLI. 14/19pts pts died of disease progression, 3/19 died of infectious complications and 2 pts died of complications associated with acute GvHD. Conclusion Long-lasting disease control can be achieved with TCD HSCT in pts with multiply relapsed MM including those with high-risk cytogenetics in the absence of chronic GvHD. TRM and acute GvHD are low in these heavily pretreated pts. Outcome of TCD HSCT is influenced by numbers of regimens administered and disease status prior to allo BMT. Pts who failed to respond to standard chemotherapeutics pretransplant responded to reuse of this therapy post TCD HSCT. Based on these results, we are aiming to perform TCD HSCT for pts with MM who have high-risk cytogenetics at an earlier time point before multiple relapses develop and to integrate post transplant immunotherapeutic or immunomodulatory strategies to further reduce risk of relapse in these high-risk pts. Disclosures: No relevant conflicts of interest to declare.