ALBERT

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: Background and objective Fludarabine in combination with cyclophosphamide (FC) plus rituximab (R) is an effective treatment for newly diagnosed as well as relapsed follicular lymphoma (Tam 2004; Keating 2005; Sacchi 2007). Maintenance treatment with R, after different induction treatments, improves overall and progression-free survival (Forstpointner 2006; van Oers 2006). Therefore, we aimed to evaluate the efficacy and safety of the FC-R regime followed by maintenance doses of R. Patients and Methods We present an intermediate report of the one-arm study in which 75 previously untreated patients with a diagnosis of follicular non-Hodgkin’s lymphoma in Ann Arbor stage II–IV were included between October 2004–2006. Seventy four were assessed for safety after receiving at least one FC-R dose (F: 3x25 mg/m2 and C: 1 g/m2; R: 375mg/m2), and 72 for response to treatment. Patients aged 53.4 years in average, one in five showed bulky disease and 72.2% Ann Arbor IV staging. FLIPI index determined 23.9% patients with low (0–1) score, 38% with intermediate (2) and 38% with poor score (3). A total of 47 patients presented some molecular alteration in PB or BM. Results Induction therapy was delivered throughout 4–6 courses, resulting in 91% complete responses (CR) and 9% partial responses (PR) (Table 1). From the patients who presented monoclonal population at diagnosis, 40 were evaluated for molecular response after induction and only 1 remained MDR positive for bcl2/IgH. Overall survival (OS) at 24 months was 87.5%, and two patients presented progressive disease within this period. The median OS has not been reached at this evaluation. To the date, 262 adverse effects grade 3–4 (32.6%) have been documented (80.9% neutropenias) and 80 infectious complications were recorded (23.8% grade 3–4). Three patients died from respiratory diseases, two from acute leukemia, and six from other causes. Table 1 EVOLUTION OF RESPONSE Evaluated Response at End of Induction Therapy Evaluated Response Post-Course 3 Assesable End Ind. (n=67) Missing End Ind. (n=5) CR: complete response; uCR: unconfirmed CR; PR: partial response; NE: not evaluated; WD: withdrawn; EX: exitus. Assesable PC3 (n=70) CRITERIA CR PR NE WD EX 14 CR 12 - 1 1 - 32 uCR 31 - 1 - - 24 PR 16 6 - - 2 2 Missing PC3 (NE) 2 - - - - 72 Total 61 6 2 1 2 Conclusions The FC-R has proven a potent antitumoral activity in untreated follicular lymphoma patients, rendering very high clinical and molecular responses. However, as reported in similar studies (Hochster 2007 ASCO), the high incidence of prolonged neutropenias and lymphopenias developed as consequence of the chemotherapy regime, questions the safety of the induction treatment.

Beschreibung: 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.

Beschreibung: 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

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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.