ALBERT

Description: Abstract 4474 Introdution: Previously, leukemic stem cells in Chronic Myelogenous Leukemia could only be identified indirectly by using culture techniques. Jeroen J.W et al have developed a new flow cytometric approach that allows a direct discrimination of CML stem cells from their normal counterparts within single patient samples. CD34+CD38- leukemic stem cells could be discriminated from normal stem cells by high CD90 expression, which is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. Material: A total of 8 patients and 3 health controls were included in this study. Result: Health controls did not present CD90 molecular expression in stem cells. 100% of the patients presented different levels of CD90 in CD34+/CD38- cells. Our patients seemed to show an asociation between CD34+/CD38- stem cells which express CD90+ and bcr-abl levels. We have evolutive studies of 4 patients who are under treatment with imatinib; in those patients we apreciate decrease in the level of CD90 related to good response. Conclusion: Our results confirm Jeroen's data; we think that this new technique will expand our possibilities to identify CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures: García: Celgene: Research Funding.

Description: Abstract 3194 Poster Board III-131 Background Immunosuppressive therapy (IST) is considered to be the first-line treatment in patients with severe aplastic anemia (AA) who are not eligible for hematopoietic stem cell transplantation (HSCT). Most IST schemes are based on the combination of anti-thymocyte globulin (ATG) plus cyclosporine A (CsA). Differently from other countries, two ATGs have been approved in Spain for AA from 2003 to 2007: Lymphoglobuline (LG) (raised in the sera of horses) and Thymoglobuline (TG) (produced in rabbits). So, during this period of time, the standard therapeutic protocol of the Spanish study group for AA included both options, and the physicians chose LG or TG based on their own wishes. Most published studies in AA are with LG, which is no longer manufactured. Recent limited data have confirmed therapeutic efficacy of TG, but no randomized studies have been performed comparing both products' activity. The aim of this report is to communicate the outcomes of a group of patients with AA who received either a LG- or TG-based scheme as first-line treatment. Patients and methods we retrospectively investigated the outcome of 110 patients with AA treated with IST at front line between 2003 and 2008. Thirty-five patients (32%) got LG (15 mg/kg/day/x5 days), and 75 patients (68%) got TG (2.5 mg/kg/day/x5 days). All patients also received methylprednisolone and CsA. Response rate (RR) was assessed at post-IST day +90, day +180, and day +365. If complete response (CR) was not reached, patients received a second course of IST, a second-line therapy (HSCT or androgens), or no treatment. When a second course of IST was employed, it included LG at the same dose as in the first course (15 mg/kg/day/x5 days), or TG at a higher dose (3.5 mg/kg/day/x5 days). CR was defined as a neutrophil count 〉1.5×109/L, a platelet count 〉100 ×109/L, and a hemoglobin level 〉120 g/L. Partial response (PR) was defined as a neutrophil count 〉0.5×109/L, a platelet count 〉20 ×109/L, and a hemoglobin level 〉80 g/L. Subgroup analyses were conducted and differences in response were tested using the chi-square statistic test. Results After the first course of IST, CR was achieved in 31 patients (28%) (group A), and PR in 20 patients (18%). Overall response (OR) was similar for both globulins (LG: 49%, TG: 45%). Thirty-five of the patients who did not reach CR after the first IST course, received a second course of IST (6 with LG and 29 with TG) (group B), and 44 patients underwent a different approach (second-line therapy or no treatment) (group C). After the second course of IST, 14 patients achieved CR (40%) and 11 patients PR (31%). OR was similar for LG (67%) and TG (72%). If we exclude patients in group C, the RR among the remaining 66 patients (who underwent 1 or 2 courses of IST) was 85% (68% CR, and 17% PR). No major drug-related toxicities were reported in the whole group of patients. Conclusions ATG-based schemes with both LG and TG were well tolerated as treatment of patients with AA. OR after first course of IST was similar in the LG and in the TG group (49% versus 45%). RR after second course of IST was also similar when LG and TG were employed (67% versus 72%). After excluding those patients who, not having reached CR after the first course, underwent an approach different from a second course of IST, RR to IST was 85%, with 68% of CR. No statistical differences were found based on the type of ATG administered. The results of this study show that TG is, at least, as effective as LG for the treatment of AA patients. Based on these and other recently published data, the current standard therapeutic protocol of the Spanish study group for AA includes TG at the dose of 3.75 mg/kg/day/x5 days for both the first and, if necessary, second course of IST. To our knowledge, no reports are available in the medical literature comparing the outcome of patients with AA who received LG or TG as the first-line therapy for AA. So, in spite of the fact that our study is retrospective and not randomized, we think our data are unique and very useful for helping physicians in switching from LG to TG. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4414 Introduction Discovery of novel non cytotoxic drugs for cancer focuses on targets selectively expressed in malignant cells, only testing at the end if they are toxic to patients. We have developed a novel approach to discover these drugs starting at the end; we screen 2.000 approved drugs with proven safety, directly on freshly extracted (ex vivo) blood samples of patients with Chronic Lymphocytic Leukemia (CLL). These screens are enabled by a novel technology platform based on automated flow cytometry we call ExviTech for ex vivo technology. Patients and Methods All screening studies were performed directly on either peripheral blood or bone marrow samples from 44 patients diagnosed with various subtypes of B-cell malignancies, after informed consent. Patient samples were diluted and plated with each of the 2.000 drugs individually, retaining the erythrocyte population and serum proteins to enable clinically relevant concentrations. The experimental assay was setup the same or a day after sample extraction. Each sample was diluted to achieve a leukemic cell concentration of approximately 3,000 cells/μl; then 45μl of the suspension is added to each well of 96-well plates that contain the pharmacological agents (final concentration of 30μM). The compound plates were then sequentially incubated for 24 hours at 37°C with 5% CO2 for screening (sterile conditions). After incubation, the erythrocytes were lysed and the leucocytes incubated with Annexin V-FITC, anti-CD45-APC and anti-CD19-PE added to each well. The plates were then transferred to an automated flow cytometry system where the contents of each well were aspirated and analyzed by a CyAn flow cytometer. Candidates from the primary screens were validated in additional samples with dose-responses, combinations with approved drugs, multiple incubation times, etc… Results Analyzing primary screens from 24 CLL patients, three related compounds (Vivia007, Vivia008 and Vivia009) were found to consistently induce apoptosis of nearly all leukemic B-cells from most of the patient samples diagnosed with B-cell chronic lymphocytic leukemia at levels equal to or greater than known CLL active cytotoxic agents. Notably, these candidates are equally effective against samples of p53 mutated patients. These 3 drugs are pharmacologically me-too drugs sharing the same target and mechanism of action, and are non cytotoxic drugs with a known and good safety profile, administered to millions of patients over many years. Validation experiments were done on 20 additional CLL patients and Vivia009 emerged as the most effective agent with an average EC50 of 18.2μM. The mechanism of action is different than the known mechanism of Vivia009 and its class members for their approved indications. Consistent with this observation, only 3 of 15 members of the same pharmacological drug class were efficacious against CLL malignant cells. All 3 Vivia′s candidates were equally efficacious against other B-Cell Malignancies such as B-ALL (pediatric and adult), and Multiple Myeloma. These drugs are not effective in their current oral formulation and require a novel intravenous formulation. Interestingly, kinetics of induction of apoptosis were faster for Vivia009 than for fludarabine, cyclophosphamide and mitoxantrone. Vivia009 requires only 1 hour of incubation with fresh cells to induce maximal apoptosis. This timeline is less than the 3 hours in which Vivia009 was found present at high concentrations in bone marrow of rats using a single intravenous bolus. Thus, Vivia009 seems to fulfill the pharmacokinetic criteria to eliminate all leukemic cells with a single intravenous bolus, which would be a major advantage over current treatments (5-days fludarabine or 3 days FCR). Animal models are ongoing to confirm the non cytotoxic nature of the candidates in the novel IV formulation and the fewer days needed to reach remission, both compared with fludarabine monotherapy. Conclusions In summary, our results demonstrate the potential of the ExviTech technology platform as a successful model for the systematic search of new uses for already existing approved drugs directly on patient samples of hematological malignancies. A new drug candidate with excellent safety profile has been identified with similar efficacy ex vivo as the best approved cytotoxic drugs, which is a non-cytotoxic drug with fast kinetics that might enable significantly safer and shorter treatments. Disclosures: Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Ana Espinosa, Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. González:Vivia Biotech SL: Consultancy. Dominguez-Gil:Vivia Biotech SL: Consultancy. Troconiz:Vivia Biotech SL: Consultancy. Rodriguez de Fonseca:Vivia Biotech SL: Consultancy. Saunders:Vivia Biotech: Consultancy. Montejo:Vivia Biotech SL: Consultancy. Caveda:Vivia Biotech SL: Employment. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Description: Abstract 1346 Poster Board I-368 Introduction The interaction of leukocytes, platelets and vascular endothelial plays a fundamental role in the development of the arteriosclerotic injuries. This initial interaction is caused by P-selectin. This P-selectin is expressed on the surface of activated platelets and its recipient, ligand 1 of the glycoprotein P-selectin (PSGL-1) of the monocytes and granulocytes. In this interaction the microparticles derived from platelets, leukocytes and endothelial cells play a role fundamentally too. Platelets microparticles are formed by a phenomenon of vesiculation of the activated platelets by agonists as the thrombin, the collagen, among others. They induce the adherence of monocytes and granulocytes to endothelial. Besides, they amplify all signs that will give rise to an increase of the activity of adhesion molecules which will culminate in the expression of the tissue factor and its transformation into a procoagulant surface. As we know, the iron contributes to oxidative changes in tissues through the Fenton's reaction, which generates free radicals that would affect the activation of platelets through the reduction of types derived from the nitric acid. Objective To evaluate the effect of desferasirox in the activation of platelets and the development of atherothrombotic factors in patients with MDS and iron overload secondary to transfusions. Patients and methods 16 patients with MDS and iron overload secondary to transfusion, as well as 15 healthy controls were included. Chelation treatment was administered with 20 mg / kg / a day of desferasirox to 8 patients, 30 mg / kg / a day to 4 patients, 10 mg / kg / a day to 3 of them (2 for gastrointestinal toxicity and 1 for renal toxicity) and one withdrew the treatment due to ocular toxicity. They all received treatment at least one month. They were studied at the beginning of the treatment with desferasirox and after the treatment (ferritin 1000-1200), biomarkers of platelets activation (expression of anexin-V and P-selectin), the expression of adhesion molecules in monocytes and granulocytes (VCAM-1, ICAM-1, E-selectin, PSGL-1 and b1-integrin) and the percentage of interactions monocytes-platelets. Results The patients with MDS and iron overload presented basal values of platelets activation and of expression of adhesion molecules above the healthy controls. The patients treated with desferasirox that obtained a decrease in their ferritin figures (10/16) presented statistically significant reductions of the scoreboards of platelets activation with the consistent improvement of the membrane damage in the vascular endothelial. Conclusions It seems that a suitable chelating of the iron in the patients with MDS and iron overload reduces platelets activation and different parameters of activation in monocytes and granulocytes that would contribute to the tissue damage in the vascular endothelial. Other additional studies will be necessary to correlate these changes with the long-term morbidity, the mortality and quality of life in this population of patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4473 Introduction: The BCR/ABL kinase alters the oxidative environment in chronic myelogenous leukemia (CML) cells, but the consequences of the increased reactive oxygen species (ROS) levels on signaling pathways remain unknown. Increased intracellular peroxides in progenitors cells have been linked to DNA damage, and promote self-mutation which subsequently render imatinib to resistance and failure to eliminate all leukemia cells. Material and Method: A total of 8 patients and 3 health controls were included in this study. 1 patient only at diagnosis, 3 patients at diagnosis and 3 months after treatment with Imatinib, 1 case after 3 and 6 month with Imatinib, 2 cases are long follow-up resistant patients and 1 with long follow-up and good response. Oxidative stress biomarkers were studied by flow cytometry in the bone marrow progenitor cells. Result: Not only levels of BCR-ABR and the number of CML stem cells were lower with the treatment, the oxidative stress biomarkers were lower too in normal controls and in CML cells in patients with response to imatinib but in the resistant ones oxidative stress biomarkers were higher in all stem and progenitor cells. Conclusion: The levels of oxidative stress are significantly higher in CML cells than in healthy subjects and correlate with tumor burden as well as the response to imatinib. This data demonstrate a role of oxidative stress in restore normal hematopoiesis in CML patients and probably in imatinib resistance. Disclosures: García: Celgene: Research Funding.

Description: Introduction: Asciminib is a new BCR-ABL1 inhibitor that differs from previous tyrosine kinase inhibitors (TKIs) in that it does not bind to the ATP-binding site of the kinase. Data from different clinical trials has shown an adequate safety and efficacy profile in chronic myeloid leukemia (CML) patients failing previous TKIs. However, no findings have been communicated in real life experience. The aim of our study is to present first results of asciminib in CML patients failing previous TKIs under the current compassionate use program. Methods: We retrospectively collected data from 31 patients treated with asciminib in 25 centers under compassionate use program. Data collecting was performed between October 2018 and June 2020. Patients baseline characteristics are shown in table 1. Most patients were heavily pretreated with 28 patients receiving 3 or more TKIs previous to asciminib. Eleven patients (35.5%) had been treated with ponatinib at some point throughout the disease. Twelve patients showed BCR-ABL1 mutations (only 1 case with T315I mutation). Switch to asciminib was due to intolerance in 22 patients and due to resistance in the remaining 9. Median dose of asciminib was 80mg per day (40mg every 12 hours). Treatment responses were evaluated according to European Leukemia Net recommendations. Data compilation and analysis were performed with REDCap Software and IBM SPSS (Version 25.0). Results: Median time on asciminib for the entire cohort was 35 weeks. Regarding toxicities, 13 patients (42%) experienced mild extra-hematological side effects (grade 1-2) being the most frequent fatigue (19%), joint pain (16%) and nausea (9%). Four patients (12,9%) showed severe (grade 3-4) extra-hematological events: fatigue, hepatotoxicity, hypertension and pericardial effusion (1 patient each). Three patients (9,7%) suffered from grade 4 thrombocytopenia, 2 of them associating grade 4 neutropenia. All toxicities according to previous TKIs adverse effects as well as cross-intolerance data is shown in table 2. Dose reduction had to be carried out in 9 patients (29%), 7 of those with temporary treatment interruptions; most owing to hematological adverse effects. In terms of efficacy (Graph 1), probability of reaching or at least maintaining previous response was 100%, 61.3% and 35.5% for complete hematological response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMR), respectively. Regarding probabilities to improve previous responses, rates of CCyR and MMR were, respectively, 22,2% (2/9) and 22,2% (2/9) for resistant patients and 44% (4/9) and 62,5%. (10/16) for intolerant group. Amid the 11 patients previously treated with ponatinib, 3 patients (27,3%) showed improvement of response achieving at least MMR, 2 of them from the TKI-intolerant group and 1 from the TKI-resistant group. The median follow-up time was 40 weeks, after which 27 patients (87.1%) continued with asciminib. Treatment cessation happened in 2 patients due to progression to blastic phase and in 2 patients due to lack of efficacy. No patients discontinued due to side effects. Conclusion: The data presented, similar to that known from clinical trials, supports the use of asciminib in routine clinical practice in CML patients failing to previous TKIs. Disclosures Garcia-Gutiérrez: Novartis: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Pfizer: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Incyte: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding.