ALBERT

Description: Background: The trombopoietin receptor agonists (TRAs) romiplostim and eltrombopag are effective and safe in the treatment of chronic immune thrombocytopenia (ITP). However, when no response is achieved or when adverse events occur with one TRA the value of the sequential use of romiplostim and eltrombopag has not been clearly established. Here we have evaluated the efficacy and tolerance of using eltrombopag after romiplostim in ITP. Methods: Fifty-one primary ITP patients (aged 18 years or more) who had been sequentially treated first with romiplostim and then with eltrombopag in the Spanish Eltrombopag Registry were retrospectively evaluated. In accordance with the usual standards, complete response was defined as a platelet count of 100x109/L and a response as a platelet count of 30x109/L or a count of at least twice the initial (pre-treatment) value. This study was performed in accordance with the standards of the Helsinki declaration and approved by the Hospital Universitario de Burgos Ethics Committee. Results: The median age of our cohort was 49 [range, 18–83] years. There were 32 women and 19 men. According to the standard definition, patients were allocated to newly diagnosed (n=2), persistent (n=5) and chronic (n=44) ITP groups. The median number of therapies prior to administration of eltrombopag was 4 [range, 2–9], including splenectomy (39%), rituximab (33%) and romiplostim (100%). The median duration of romiplostim use before switching to eltrombopag was 12 (IQR 5–21) months. The reasons for switching from the romiplostim to eltrombopag were: lack of efficacy of romiplostim (n=25), patient's preference (n=16), platelet-count fluctuation (n=6), and side-effects (n=4). The initial response rate to eltrombopag was 41/51 (80.5%), including 67% (n=34) of cases with complete remission. After a median follow-up of 13 months with eltrombopag, 39 patients maintained their response. When eltrombopag was used for patients who were refractory to the maximum romiplostim dose the initial response rate of eltrombopag was 25%. However, 83% of patients who relapsed after their initial response to romiplostim responded to eltrombopag. Sixteen romiplostim responders requested their physicians to switch them to eltrombopag because they preferred an oral drug. The efficacy was maintained after switching in all 16 patients. In the platelet-count fluctuation group, the initial response rate was also 100%. All 4 patients who were switched to eltrombopag because they experienced side-effects of romiplostim achieved complete remission with eltrombopag and their adverse events were resolved. 16 / 51 (33%) patients experienced one or more adverse event during treatment with eltrombopag. The frequency of grade 3–4 adverse events during treatment with eltrombopag was 9.8%. Conclusion: The use of eltrombopag after romiplostim for treating ITP is effective and safe. The reason for discontinuing romiplostim was associated with the response to eltrombopag. Disclosures No relevant conflicts of interest to declare.

Description: HOX homeobox genes are important regulators of normal and malignant hematopoiesis. Abdominal-type HOXA genes like HOXA9 are highly leukemogenic. However, little is known about transformation by anterior HOXA genes. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5, all HOXA genes caused a block or delay of hematopoietic differentiation and cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all active HOXA genes immortalized mixed granulocytic/monocytic populations, HOXA13 preferentially specified monocytoid development. The anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells, generating permanent cell lines, although they did so less potently than HOXA9. Upon transplantation these lines induced myeloproliferation and acute myeloid leukemia in recipient animals. Kinetic studies with inducible HOX derivatives demonstrated that anterior HOXA genes autonomously contributed to cellular transformation. This function was not mediated by endogenous Hoxa9, which was persistently expressed in cells transformed by anterior HOX genes. In summary our results demonstrate a hitherto unexpected role of anterior HOXA genes in hematopoietic malignancy.

Description: Abstract 2903 Background: Despite improvements in chemotherapy protocols and bone marrow transplantation techniques, AML remains a lethal disease. Although patients with AML typically respond well to initial therapy, the majority relapse and eventually succumb to the disease. This relapse is thought to be the result of the resistance of leukaemic stem cells to the effects of chemotherapy. Some mechanisms have been discovered which contribute to resistance, but this phenomenon remains incompletely understood and is a significant barrier to improving patient outcomes. TEL is a member of the ETS transcription factor gene family and is essential for developmental processes such as haematopoiesis. TEL is frequently targeted by chromosomal translocations in human malignancies, resulting in the expression of oncogenic TEL gene fusions. A TEL-ARNT gene fusion was reported in a single case of AML, FAB subtype M2, in a patient whose leukaemic cells contained the balanced translocation t(1;12)(q21;p13). ARNT (also known as HIF-1β) is a nuclear protein and functions as part of a heterodimer, whose partners include HIF-1α and the aryl hydrocarbon receptor (AhR). HIF-1 expression is commonly deregulated in cancer and AhR signalling has been linked to leukaemogenesis. Materials and Results: Immunoblot analysis of patient bone marrow mononuclear cell lysates revealed that 5 of 29 AML specimens (∼17%) had elevated levels of ARNT protein, whereas 0 of 19 MDS specimens showed ARNT expression. In addition, AML specimens that had elevated ARNT protein levels also had elevated levels of phosphorylated Akt, while AML specimens that did not have elevated ARNT protein expression did not have activation of Akt signaling. Intriguingly, ARNT expression was noted in one patient who had progressed from MDS to AML. We then studied the expression of ARNT in HL60 cells, which are highly sensitive to induction of apoptosis by troglitazone (TG), and in U937 cells, which are TG resistant. In HL60 cells, ARNT mRNA levels remained constant following TG treatment and ARNT protein levels markedly decreased, while in U937 cells, ARNT mRNA levels increased and ARNT protein levels remained constant. We then tested the effect of exogenous expression of ARNT on the sensitivity of HL-60 cells to apoptosis induced by TG, daunorubin and hydrogen peroxide. HL-60 cells transduced with a retrovirus expressing ARNT became resistant to the induction of apoptosis by all these agents. These cells also had constitutive activation of Akt signaling, and treatment of these cells with a specific inhibitor of Akt signaling reversed their resistance to TG-induced apoptosis. Conclusions: The expression of ARNT confers resistance to apoptosis in AML cell lines and activates Akt signalling. In addition, ARNT is overexpressed in a significant subset of AML patients, in whom it activated Akt signaling, and ARNT expression correlates with progression of MDS to AML. ARNT may play an important role in chemoresistance and may be useful as a predictive or prognostic biomarker. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3560 Background: We have previously reported a high response rate with a conditioning regimen consisting of intravenous busulfan (ivBu) and melphalan (Mel) in patients with multiple myeloma (MM) undergoing autologous stem cell transplant (ASCT) (Blanes M et al, Leuk Lymphoma 2009). In this case-matched study, we analyze the efficacy and toxicity of ivBuMel as conditioning regimen before ASCT in 51 patients with newly diagnosed MM. Their clinical outcome was compared with that of a control group of 102 pair mates included in the Spanish PETHEMA/GEM2000 study of tandem transplant (control group). Patients and Methods: Patients in the control group were transplanted between 2002 and 2005, while patients in the ivBuMel group underwent ASCT between 2005 and 2009. Controls were matched with respect to sex, age, Durie-Salmon and ISS stage at diagnosis, and disease status at transplant. The conditioning regimen comprised iv Bu (3.2 mg/kg in a single daily dose, days -5 to -3) and Mel (140 mg/m2, day -2). Patients in the control group underwent a first ASCT after Mel 200 mg/m2 (Mel 200) in a single dose on day -2 followed in patients failing to achieve a complete response (CR) by a second autologous transplant. The conditioning regimen administered in the second transplant was either Mel 200 or a combination of cyclophosphamide, carmustine, and etoposide. Results: There were no differences in the hematopoietic recovery and mild or moderate mucositis was the toxicity most frequently observed in both groups of patients. Two (4%) patients died due to transplant-related complications in the ivBuMel group and 5 (5%) in the control group. The CR/near CR (nCR) rate was 51 % in both groups. After median follow-up of 32 and 40.5 months in the ivBuMel and control group, respectively, median progression free survival (PFS) was 37.6 for patients who received ivBuMel and 26.5 months for those in the control group. At 4 years, overall survival and PFS was 65±10% and 42±9% in the ivBuMel and 62±5% and 32± 5% in the control group, respectively (P = NS). For patients achieving CR/nCR after ASCT, the 4-years PFS rate was 52±14% and 40±8% in ivBuMel and control group, respectively (P = 0.3). Finally, in patients achieving partial response or less after transplant, the 4-years PFS rate was 33±12% and 23±6% in ivBuMel and tandem transplant group, respectively (P = 0.3). Conclusion: Results of this case-matched study suggest that the use of ivBuMel conditioning regimen before ASCT in patients with newly diagnosed MM is associated with low transplant-related morbidity and mortality and a high anti myeloma efficacy that compares favourably with a tandem transplant strategy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5044 Introduction: Epidemiological studies are the base to evaluate the efficiency of medical interventions in the interest of the public health. Updated epidemiological data and effectivenes in the daily clinical practice are needed in Multiple Myeloma (MM). Materials and Methods: Epidemiological retrospective, longitudinal, multicenter nation wide Spanish study of an historical cohort of patients with MM. Data from patients aged ≥ 18 years, with a MM Stage II or III, who received a treatment on a daily clinical practice (not in clinical trials) for MM in the September'03-August'05 time frame were collected. The study protocol was approved by an Ethics Committee in 2009. Data were collected in 37 Spanish Centres during a 6 months period. Stratified effectiveness and survival analysis were performed (age

Description: Abstract 1866 Myelodysplastic syndromes (MDS) comprise distinct disorders characterized by dysplastic and ineffective hematopoiesis that seems to be related to an increased apoptosis of bone marrow cells (Nimer, Blood 111: 4841, 2008). Clinical manifestations in MDS range from a diverse degree of anemias, leuko- or thrombocytopenias to severe transfusion-dependent peripheral pancytopenias. Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS. Many of the features of apoptosis such as membrane fragmentation, microvesiculation and phosphatidylserine (PS) exposure are observed during platelet activation to a procoagulant state, raising the possibility that apoptosis may regulate platelet function. The aim of this work was to determine whether a correlation exists between apoptosis and activation processes in platelets from MDS patients. Twenty six patients diagnosed MDS and classified according to WHO-2008 were included: 6 with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), 6 with refractory RA with excess blasts-1 (RAEB-1) and 7 with cytopenia with multilineage dysplasia (RCMD) associated with isolated 5q deletion. Twenty six healthy donors were included as control group. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane PS exposed under basal conditions and after stimulation with a PAR-1 receptor agonist (TRAP, SFLLRN, thrombin receptor-activating peptide 6). Levels of pro- apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis of western blots performed with platelet lysates. Platelet activation was determined through FITC-fibrinogen, FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 mM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (aIIb and b3 subunits) was determined by flow cytometry with specific mAbs. Platelets from RA, RARS and RCMD patients expressed more PS than control ones under basal conditions (p

Description: Abstract 2524 WT1 monitoring is an almost universal target to follow de novo AML. Its exppression in myeloid malignancies is upregulated in parallel to the blast percentage. Recently, WT1 determination has been standardized as result of an European Leukemia Net initiative. Early reports have demonstrated that the best results are obtained when peripheral blood is used to establish clinical predictions. Pediatric studies in AML have shown that raised WT1 levels after induction associate with unfavourable outcome. Despite all the mentioned, WT1 quantitation has not yet gained widespread use, in part because some AML show normal WT1 levels at diagnosis. To investigate the prognostic impact of the normalized bone marrow WT1 levels at diagnosis and post-induction in a consecutive series of de novo AML patients enrolled in the CETLAM group trials. Available bone marrow samples at diagnosis (586 cases) and post induction (367 cases) were obtained in each participating center and sent to the CETLAM repository center at the Hospital de la Santa Creu i Sant Pau for complete immunophenotype and molecular analyses. One μg of RNA was reverse transcribed to cDNA in a total reaction volume of 20μl containing Cl2Mg 5mM, 10× Buffer, DTT 10mM, dNTP's 10mM each, random hexamers 15μM, RNAsin 20 units (Promega) and 200 units of MMLV enzyme. WT1 expression levels were determined by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI PRISM 7700® Genetic Analyzer (Applied Biosystems, Foster City, CA) using the primers and conditions described by the ELN group (Cilloni et al J. Clin. Oncol 2009;27:5195-201). For WT1 copy number titration, the IPSOGEN® (Marseille, France) plasmid was employed. Results were expressed as copies and four normal bone marrow samples were used as test controls. Patients were treated between 2004 and 2011 according to the CETLAM03 protocol. Adults up to 70 years of age received induction chemotherapy with idarubicin, intermediate-dose cytarabine and etoposide, followed by consolidation with mitoxantrone and intermediate-dose ara-C. Subsequently, patients with favourable cytogenetics at diagnosis received one cycle of high-dose cytarabine.G-CSF priming during induction and consolidation was used. Patients with favorable cytogenetics and high leukocyte counts at diagnosis were treated with autologous transplantation instead of high-dose cytarabine. Furthermore, patients with a normal karyotype but an adverse molecular profile (FLT3 mutations or MLL rearrangements) were allocated to the treatment for unfavorable cases; this included allogeneic transplantation from an HLA-identical donor. Overall survival (OS) was measured from the date of enrolment until the date of death. Leukemia-free survival (LFS) for patients who achieved a CR was calculated from the date of CR to relapse or death. OS and LFS were plotted by the Kaplan-Meier method; differences between curves were analyzed by the log-rank test. The probability of relapse was calculated using cumulative incidence estimates and taking into account the competing risk of death in remission. A WT1 cut-off value of 5065.2 copies at diagnosis was obtained. Two hundred and four samples had WT1 levels greater than this value, whereas 382 samples showed levels below this cut-off. These groups had statistically different OS 55±3 vs 33±5 p

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 4557 INTRODUCTION: Advanced low grade non-Hodgkin Lymphomas have many and varied treatment options. For patients with relapsed or refractory disease, outcome following only chemotherapy protocols remains poor. The standard Conditioning Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) might be an alternative for relapsed low grade non-Hodgkin lymphomas, but its high Transplant Related Mortality (TRM), varying from 24% to 54%, accounts for a lower overall survival in this group. Reduced Intensity Conditioning (RIC) HSCT has been shown to be a good alternative to myeloablative regimens, with lower TRM, Graft versus Lymphoma Effect, and appears to be associated with improved outcome, presenting a 3 years disease free survival and overall survival from 32% to 66% and from 32% to 73%, respectively, depending on factors such as disease stage before transplantation. PATIENTS, METHODS AND RESULTS: Sixteen heavily pretreated patients with low grade non-Hodgkin Lymphoma (NHL) were submitted to RIC HSCT with matched related donors at the Bone Marrow Transplantation Unit at HCPA between the period of September 2002 to October 2009. The mean age was 43 years and were 7 patients male and 9 female. Fifty percent (8) of the patients had refractory disease pre-HSCT, 43.8% (7) were at third complete remission (CR) and 6.2% (1) at second CR. Median of CD34/kg infused was 10.7 × 106 and the median of days for engraftment was 14.53. Conditioning regimens were Fludarabine + Cyclophosphamide in 3 patients, Fludarabine + Melphalan in 7, Fludarabine + Cyclophosphamide + Rituximab in 2, Fludarabine + Cyclophosphamide + Melphalan in 1, Cyclophosphamide + Total Body Irradiation (TBI) in 1 and Fludarabine + Cyclophosphamide + TBI in 2. Acute Graft versus Host Disease (aGVHD) occurred in 50%, with 37.5% of those were steroids resistant; and 62.5% of the patients evolved to chronic GVHD, with 80% of them classified as extensive. Chimerism from 9 patients was analyzed, and 89% of them were complete. Mean overall survival was 2.95 years; 62.5% of patients were alive within 1 year and 40% within 3 years. TRM was 6.66%. Eight patients died, five of them were transplanted with refractory disease. Only 2 patients died of relapse, 5 of infections and 1 of aGVHD. CONCLUSION: Low grade NHL patients treated at HCPA with RIC HSCT had an overall survival and TRM comparable to the observed at other centers, appearing as a good alternative for this heavily treated group of patients with less toxicity than myeloablative regimens. Disclosures: No relevant conflicts of interest to declare.