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We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter–driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell–derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene–modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Background: Thrombophilia in children is characterized by hypercoagulability and increased frequency of thrombotic events (Young G, et al. Circulation. 2008;118:1373-1378; Kenet G, et al. Circulation. 2010;121:1838-1847). Recently, phase IIb/III clinical trials in children with venous thromboembolism (VTE) have reported the non-inferiority of dabigatran etexilate (DE) versus standard of care (SOC) for the treatment of acute VTE (Albisetti M, et al. ISTH 2019, Abstract OC 57.3), and a favorable safety profile for DE in secondary VTE prevention in children with persistent VTE risk factor(s) (Brandão LR, et al. Blood. 2020;135:491-504). Aims: To perform a subgroup analysis evaluating the efficacy and safety of DE for the treatment and secondary prophylaxis of VTE in children with thrombophilia in the phase IIb/III DE clinical trials. Methods: In the open-label, phase IIb/III DIVERSITY trial (NCT01895777), children aged from birth to < 18 years (yrs) with an objectively confirmed VTE diagnosis (by imaging studies) initially treated with unfractionated heparin or low-molecular-weight heparin were randomized (2:1) to receive up to 3 months of DE or SOC. Primary composite efficacy endpoint: complete thrombus resolution and freedom from VTE recurrence, or VTE-related death. Safety endpoints included bleeding events (BEs). The open-label, phase III, secondary VTE prevention trial (NCT02197416) treated children aged from > 3 months to < 18 yrs with DE for up to 12 months or less, if the identified VTE clinical risk factor resolved. Eligible children had an objectively confirmed diagnosis of VTE treated with SOC for ≥ 3 months, or had completed DE or SOC treatment in DIVERSITY and had an unresolved clinical VTE risk factor requiring further anticoagulation. Primary endpoints included VTE recurrence and BEs. Thrombophilia status was confirmed according to the definitions used by the local experts. Results: In DIVERSITY, 23.2% of children had thrombophilia; demographics were comparable to the overall population, although they were slightly older (mean [standard deviation] age 13.2 [4.9] vs 11.1 [6.1] years in the overall population; p = 0.005). In children with thrombophilia, DE was found to be non-inferior to SOC for the primary endpoint (similar to the overall population), and more children treated with DE achieved the composite primary endpoint than with SOC (SOC 21.7% vs DE 35.9%; Mantel-Hänszel-weighted difference in rates for SOC minus DE [90% CI] −0.14 [−0.32 to 0.05]; p for non-inferiority = 0.0014), similar to the overall population (Table). Regardless of treatment, VTE recurrence appeared higher in children with thrombophilia than in the overall population, with numerically fewer VTE recurrences reported by children with thrombophilia treated with DE (7.7%) versus SOC (21.7%), although this was not significantly lower (p = 0.13). Numerical differences in residual thrombotic burden between SOC vs DE seen in the overall population appeared to be amplified in children with thrombophilia. Numerically fewer children with thrombophilia treated with DE reported thrombus progression (SOC 13.0% vs DE 5.1%) or stabilization (21.7% vs 10.3%), while more reported partial (34.8% vs 43.6%) or complete thrombus resolution (21.7% vs 35.9%). In the thrombophilia subgroup, BEs appeared to be lower in children treated with DE (SOC 26.1% vs DE 17.9%), while in the overall population BEs with SOC and DE were comparable. In the secondary VTE prevention trial, children with thrombophilia were also slightly older versus the overall population (mean [standard deviation] age 14.1 [3.6] vs 12.8 [4.6] yrs; p = 0.006). In this larger subgroup of children, rates of recurrent VTE at 12 months appeared to be higher in the thrombophilia group (2.8%) compared to the overall population (1.4%) (Table), with BEs largely comparable (27.4% and 22.5%, respectively). Conclusions: Unsurprisingly, numerically more children with thrombophilia appeared to report VTE recurrence, and in DIVERSITY thrombus progression/stabilization also seemed higher compared with the overall population. Compared with the overall populations, these subgroup analyses showed consistent results for DE in children with acute VTE and thrombophilia in the DIVERSITY trial, along with a favorable safety profile of DE for secondary VTE prevention. Disclosures Brandao: Boehringer Ingelheim: Other: Member of a paediatric expert working group. Tartakovsky:Boehringer Ingelheim: Current Employment. Albisetti:Boehringer Ingelheim: Other: Member of a paediatric expert working group; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bomgaars:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Chalmers:CSL Behring: Honoraria; Shire/Takeda: Honoraria; Boehringer Ingelheim: Other: Member of a paediatric expert working group; Grifols: Honoraria; Roche: Honoraria; Sobi: Honoraria; Bristol-Myers Squibb: Honoraria. Mitchell:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Luciani:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Lvova:Boehringer Ingelheim: Honoraria. Simetzberger:Boehringer Ingelheim: Current Employment. Sun:Boehringer Ingelheim: Current Employment. Gergei:Boehringer Ingelheim: Current Employment. Brueckmann:Boehringer Ingelheim: Current Employment. Halton:Boehringer Ingelheim: Other: Member of a paediatric expert working group. OffLabel Disclosure: dabigatran etexilate in paediatric VTE

Background: In children, current standard of care (SOC) with heparins or vitamin K antagonists is limited by the need for parenteral administration and frequent monitoring. Dabigatran etexilate (DE), a direct oral anticoagulant, is an effective oral treatment for venous thromboembolism (VTE) in adults and may be an effective and safe alternative to SOC for treating acute VTE in children. Aims: The DIVERSITY trial evaluated the efficacy and safety of DE versus SOC, and the appropriateness of a DE dosing algorithm, in children with VTE aged from birth to < 18 years. Methods: This was an open-label, randomized, active-controlled, multicenter, phase IIb/III trial (NCT01895777; consent obtained and ethics committee approved). Children (12 to < 18 years, 2 to < 12 years, birth to < 2 years) with an objectively confirmed VTE diagnosis and initially treated with unfractionated heparin or low-molecular-weight heparin were randomized (2:1) to DE (capsules, and child-friendly pellets and oral suspension, dosed using an age- and body-weight-adjusted dosing algorithm) or SOC, and treated for up to 3 months. The primary composite efficacy endpoint was the proportion of children with complete thrombus resolution, and freedom from recurrent VTE or VTE-related death. Safety endpoints included bleeding events and adverse events (AEs), and pharmacokinetic/pharmacodynamic (PK/PD) relationships. Results: Of 267 children in the randomized set, 35 were aged < 2 years (13 SOC, 22 DE; of these, 14 were < 6 months [3 SOC, 11 DE]), 64 were 2 to < 12 years (21 SOC, 43 DE), and 168 were 12 to < 18 years (56 SOC, 112 DE). Overall, similar proportions of children treated with SOC and DE met the composite efficacy endpoint (38/90 [42.2%] vs 81/177 [45.8%]; Mantel Hänszel-weighted difference -0.04; 90% confidence interval [CI] -0.14 to 0.07; p < 0.0001 for non-inferiority), and major bleeding events were comparable (2/90 [2.2%] and 4/176 [2.3%]; hazard ratio 0.94; 95% CI 0.17-5.16; p = 0.95). Across the three age groups (12 to < 18 years, 2 to < 12 years, birth to < 2 years), DE was comparable with SOC for the combined efficacy endpoint, which was achieved by 33.9-57.1% of children treated with SOC and 42.0-59.1% treated with DE. Similar numbers of children experienced major bleeding events, reported by between 0.0-3.6% of children treated with SOC and 1.8-4.5% treated with DE. In the overall population, the incidence of AEs in children treated with SOC and DE was 66.7% and 76.7%, respectively, and serious AEs were reported by 20.0% and 12.5% of all children, respectively. Across the three main age groups, the frequency of serious AEs was comparable for SOC and DE (reported by 19.0-23.1% and 9.1-14.4% of children, respectively). Dabigatran PK/PD relationships for three laboratory coagulation parameters (activated partial thromboplastin time [aPTT], diluted thrombin time [dTT], and ecarin clotting time [ECT]) were comparable across the three main age groups and were also similar in infants aged < 6 months (Figure). There was a linear relationship between total dabigatran plasma concentration for both dTT and ECT, and a nonlinear relationship with aPTT. These PK/PD relationships were similar to those observed in adults (data not shown). Conclusions: This randomized trial confirmed that an age- and weight-adjusted dabigatran dosing algorithm was appropriate in children aged from birth to < 18 yrs. Dabigatran, given as either capsules or child-friendly pellets or oral suspension, was comparable with SOC in terms of efficacy for acute VTE treatment across all age groups and, notably, in vulnerable children aged < 2 yrs. Disclosures Halton: Boehringer Ingelheim: Other: Member of a paediatric expert working group. Brandao:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Luciani:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Bomgaars:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Chalmers:Shire/Takeda: Honoraria; CSL Behring: Honoraria; Bristol-Myers Squibb: Honoraria; Sobi: Honoraria; Roche: Honoraria; Grifols: Honoraria; Boehringer Ingelheim: Other: Member of a paediatric expert working group. Mitchell:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Sharathkumar:Takeda: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Kedrion: Consultancy. Svirin:Takeda: Consultancy, Other: Personal fees; CSL Behring: Consultancy, Other: Personal fees. Tartakovsky:Boehringer Ingelheim: Current Employment. Simetzberger:Boehringer Ingelheim: Current Employment. Huang:Boehringer Ingelheim: Current Employment. Sun:Boehringer Ingelheim: Current Employment. Kreuzer:Boehringer Ingelheim: Current Employment. Gropper:Boehringer Ingelheim: Current Employment. Brueckmann:Boehringer Ingelheim: Current Employment. Albisetti:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Other: Member of a paediatric expert working group. OffLabel Disclosure: dabigatran etexilate in paediatric VTE

Introduction: Epigenetic regulatory mechanisms such as the modification of histone residues are perturbed in myeloid neoplasms. Amongst others, the methylation status of Lysine residue 4 on Histone 3 (H3K4) represents one of the central dysregulated histone marks in acute myeloid leukemia (AML). Demethylation of H3K4 is mediated by the Lysine (K)-specific demethylase 1A (LSD1/ KDM1A). Remarkably, LSD1 expression is de-regulated in AML and therefore represents an attractive therapeutic target. In fact, pharmacologic LSD1 inhibition was shown to induce AML cell differentiation and apoptosis in combination with all-trans-retinoic acid (ATRA) exposure. Recently, a phase 1 trial with the irreversible LSD1 inhibitor IMG-7289 (IMG) ± ATRA in patients with advanced myeloid malignancies commenced (ClinicalTrials.gov Identifier: NCT02842827). However, the effect of a LSD1-inhibitory therapy on normal human progenitors has not been studied in detail. Methods: Experiments were performed with mobilized CD34+ cells of deceased multiple myeloma and lymphoma patients. The usage of these cells was approved by the local ethics committee. The following experimental approaches were employed: (1) studying in vitro colony forming unit (CFU) potential of human CD34+ progenitors in the presence of increasing IMG concentrations, (2) investigating the CFU potential in re-plating experiments of primary IMG-7289 exposed CD34+ cell CFU cultures and (3) studying the impact of IMG on in vitro cytokine (SCF, IL-3, IL-6, Flt3L and TPO for 7 days)-driven CD34+ progenitor expansion. Results: Performing CFU assays in the presence of increasing IMG concentrations revealed a shift from diverse colony types in DMSO control treated CFU assays to increasing phenotypic monocytic (CFU-M) and decreasing granulocytic (CFU-G) and erythroid (BFU-E) colony types (Fig. 1A). Accordingly, increased CFU-M and decreased CFU-G and BFU-E colony numbers were observed (Fig. 1B). In contrast, sole ATRA treatment abrogated CFU-M colony formation, increased CFU-G and slightly decreased BFU-E colony numbers (Fig. 1B). To study the impact of LSD1 inhibition in synergy with ATRA-driven differentiation therapy we treated CFU assays with increasing IMG concentrations in combination with a constant ATRA concentration (10 nM). Strikingly, IMG and ATRA co-exposure severely depleted CFU-M and BFU-E colonies while preserving CFU-G colony formation (Fig.1B). In order to study the impact of IMG on progenitor self-renewal we re-plated IMG-exposed CFU assays in the absence of IMG. In the re-plated dishes we observed a 2.9- and 3.7-fold increase of CFU-G colony numbers compared to the primary CFU-G IMG exposed (100 nM and 1µM, respectively) colony numbers. Finally, we investigated the impact of IMG exposure on in vitro cytokine-driven CD34+ progenitor expansion. In DMSO treated cultures 7 day cytokine incubation led to a 37-fold expansion of CD34+ cell numbers while increasing the total CFU numbers 1.8-fold. Compared to the DMSO control cultures, IMG exposure (100nM and 1µM) boosted CFU-M colony numbers 1.7- and 2.3-fold, respectively. Strikingly, also CFU-G colony numbers were slightly increased by IMG exposure (1.3- and 1.6-fold at 100nM and 1µM, respectively). To investigate how IMG exposure during cytokine-driven CD34+ progenitor expansion alters the transcriptional program of human CD34+ progenitors we analyzed the expression of the hematopoietic regulatory genes GFI1, GFI1B, IRF8 and RARA by real-time PCR. IMG exposure (100 nM) increased the expression of these genes 2.2-, 1.7-, 1.9- and 1.6-fold, respectively. De-repression of GFI1 and GFI1B expression by LSD1 inhibition was expected and the observed upregulation of the monocytic regulator IRF8 is in accordance with the observed increased monocytic colony numbers under LSD1 inhibition. Lastly, RARA upregulation suggested that LSD1 inhibition promotes retinoic acid receptor signaling. Conclusions: Our findings reveal that pharmacologic LSD1 inhibition increases human monocytic progenitor numbers. Furthermore, in vitro CFU-re-plating and also progenitor expansion experiments point to LSD1 inhibition not depleting granulocytic progenitors. Collectively, our data suggest that expected side effects of a LSD1-targeted therapy such as neutropenia will be reversible. Disclosures Rienhoff: Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dührsen:Janssen: Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Research Funding; Gilead: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Göthert:AOP Orphan Pharmaceuticals: Other: travel support; Proteros Biostructures: Consultancy; Novartis: Honoraria; Pfizer: Consultancy; Incyte: Consultancy, Honoraria, Other: travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support.

Introduction. It is known that imatinib can be safely discontinued in patients (pts) with Chronic Myeloid Leukemia (CML) with minimal residual disease. Here we report an update of the Imatinib Suspension And Validation (ISAV) study at 79 months (mts) from study initiation to provide long term follow up data. Aims. The ISAV study aims to validate the capability of digital PCR (dPCR) to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR and to evaluate relapse rate, time to recurrence, survival and the impact of imatinib treatment on Quality of Life (QoL). Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (chronic or accelerated phase) treated with imatinib for more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 mts, with a minimum of 3 Q-RT-PCR performed at their own sites. After discontinuation of imatinib therapy, Q-RT-PCR was performed monthly (mts 1-6), bimonthly for 36 mts and then every 6 mts for additional 2 years, to assess the maintenance of the molecular remission. The loss of molecular remission was defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resumed imatinib treatment at the same dosage used before interruption. dPCR was performed at screening and at 36 mts for those pts who were still in remission. Patients' QoL during imatinib discontinuation/resumption was evaluated through the EORTC QLQ-C30 questionnaire. Results. The ISAV study enrolled 112 pts with a median follow-up time of 60.0 mts [95% CI: 59.6-60.6] for pts who do not relapsed; 66.1% of them completed the study as per protocol. The 58.9% of pts were male and 37.4% were aged 65 or older; median duration of imatinib treatment was 103.2 mts with median duration of CMR of 25.6 mts before imatinib discontinuation. At 79 mts from imatinib discontinuation, 56 pts of the 107 eligible ones relapsed and resumed imatinib with a relapse rate of 52.3% [95%CI: 20.4-32.6]; 69.6% of them relapsed in the first 9 mts. Of the 52 not-relapsed pts, 40 (76.9%) regained Q-RT-PCR positivity without losing MMR. In this latter group 2 pts experienced late relapses, at 30.6 and 45.5 mts respectively. A loss of CCyR occurred in 13 pts (23.6%): 10/13 CCyR losses were recovered, the remaining 3 were not assessed for response. No case of CML progression or resistance to imatinib was observed. After the resumption of imatinib the median time to MMR/CMR was 1.8 [95% CI: 1.0-2.0] mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR, Sokal score or duration of CMR was identified, while an inverse relationship between pts age and risk of relapse was evident. dPCR results before imatinib discontinuation showed that 23.4% of pts were positive and 76.6% negative at the time of discontinuation, with a Negative Predictive Value ratio (dPCR/Q-RT-PCR) of 1.1 [95%CI: 0.99-1.22]. At 36 mts from imatinib discontinuation 80.4% [95%CI: 30.6-50.4] of the pts tested were positive in dPCR. Moreover, the results of dPCR performed at imatinib discontinuation and age together can predict the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥45 years and with negative dPCR (36.1%). The analysis of QoL evidenced a statistically significant improvement in the general well-being and symptoms scales at 1 month after imatinib discontinuation, particularly with regard to nausea, diarrhea, fatigue and insomnia (p Conclusions. At 79 mts from the beginning of the study, 52.3% of pts relapsed, with 24% loosing CCyR. The majority of relapses occurred in the first 9 mts after discontinuation however late relapses were also observed, up to the 4th year. Therefore, pts who discontinue imatinib should be monitored for a long period of time, especially if they show positive PCR values after discontinuation. All relapsed pts including those who lost CCyR regained their original response after restarting TKI. Age Disclosures le Coutre: Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria; Novartis: Honoraria. Abruzzese:BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Assouline:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding. Kim:Pfizer: Research Funding; BMS: Research Funding; Ilyang: Research Funding; Novartis: Research Funding. Gambacorti-Passerini:Pfizer: Consultancy, Honoraria, Research Funding; BMS: Consultancy.

To formally establish the risk of lupus anticoagulants and anticardiolipin antibodies for arterial and venous thrombosis, we ran a MEDLINE search of the literature from 1988 to 2000. Studies were selected for their case-control (11), prospective (9), cross-sectional (3), and ambispective (2) design. They provided or enabled us to calculate the odds ratio with 95% confidence interval (CI) of lupus anticoagulants and/or anticardiolipin antibodies for thrombosis in 4184 patients and 3151 controls. Studies were grouped according to the antibody investigated. Five studies compared lupus anticoagulants with anticardiolipin antibodies: the odds ratio with 95% CI of lupus anticoagulants for thrombosis was always significant. None of them found anticardiolipin antibodies were associated with thrombosis. Four studies analyzed only lupus anticoagulants: the odds ratio with 95% CI was always significant. The risk of lupus anticoagulants was independent of the site and type of thrombosis, the presence of systemic lupus erythematosus, and the coagulation tests employed to detect them. Sixteen studies served to assess 28 associations between anticardiolipin antibodies and thrombosis: the odds ratio with 95% CI was significant in 15 cases. Anticardiolipin titer correlated with the odds ratio of thrombosis. In conclusion, the detection of lupus anticoagulants and, possibly, of immunoglobulin G (IgG) anticardiolipin antibodies at medium or high titers helps to identify patients at risk for thrombosis. However, to take full advantage of the conclusions provided by the available evidence, there is an urgent need to harmonize investigational methods.

P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate–binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein–actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump–related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin–P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein–actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.

Background: The incidence of venous thromboembolism (VTE) in children is increasing. The current standard of care is unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) and Vitamin K antagonists (VKAs), all of which have limitations. Dabigatran, which is orally administered as the prodrug, dabigatran etexilate (DE), is effective for the treatment of VTE in adults and may overcome some of the limitations associated with standard care. Objective: The objectives of this phase IIa study were to obtain pharmacokinetic (PK) and pharmacodynamic (PD) data and to evaluate the tolerability and safety of an oral liquid formulation (OLF) of DE in patients aged 1 to < 12 years. Methods: The open-label, multinational, multicenter, non-randomized, single-arm study was conducted in patients aged 1 to < 2 years and 2 to < 12 years with a diagnosis of VTE, who had completed planned treatment with LMWH or oral anticoagulants (VKAs) for VTE. DE was administered as an OLF at a weight and age-adjusted dose (calculated using a nomogram) equivalent to 150 mg twice daily (bid) in adults. Originally, all patients were to receive multiple dosing (bid for 3 days). However, this was replaced with single dosing (SD) in the course of the study. A 30-day follow-up was included. Here we report data from SD patients only. The primary PK endpoints were plasma concentration of total dabigatran, free dabigatran, unchanged DE and intermediate metabolites. To compare the exposure to DE in pediatrics with that in adults with VTE, gMean concentrations at 10 hours after SD of DE were extrapolated to steady-state trough concentration. An accumulation ratio was calculated based on the terminal half-life (t1/2) and the dosing interval (for a bid dosing regimen) to project the steady-state dabigatran plasma concentration. Primary PD endpoints were activated partial thromboplastin time (aPTT), ecarin clotting time (ECT), and diluted thrombin time (dTT). Primary safety endpoints were incidence of all bleeding events and incidence of all adverse events (AEs). Results: Six patients aged 1 to < 2 years and 9 patients aged 2 to < 12 years received SD DE; 3 patients aged 2 to < 12 received multiple doses (bid for 3 days). In the SD groups, plasma concentration of total dabigatran increased at 2 hours (peak) after dosing then decreased until 10 hours after dosing. The projected steady-state dabigatran trough concentrations of both age groups of children studied were largely comparable to those observed in adults with VTE, i.e. the projected results for children fell between the first and the third quartile of adult trough concentrations (Table). Data from the SD groups indicated that there was a linear PK/PD relationship for ECT and dTT (see Figure) and a non-linear PK/PD relationship for aPTT. The PK/PD relationships were similar to those seen in adults (for all coagulation parameters analyzed) and adolescents (dTT and ECT) with VTE. During the on-treatment period there were no study drug related AEs; one patient had AEs (leukopenia and dizziness), which were mild in intensity and unrelated to the study drug. No AEs were reported during the post-treatment or post-study periods. No bleeding events were reported. Conclusion: The OLF of DE was well tolerated in pediatrics, with no drug-related AEs or bleeding events. Projected steady-state dabigatran trough concentrations were largely comparable to those seen in adults with VTE. The observed PK/PD relationship was similar to that seen in adults and adolescents with VTE. Disclosures Halton: Boehringer Ingelheim: Other: Pediatric Expert Working Group for Boehringer Ingelheim. Albisetti:Boehringer Ingelheim: Other: Pediatric Expert Working Group. Luciani:Boehringer Ingelheim: Other: Member of Pediatric Expert Working Group for Boehringer Ingelheim. Huang:Boehringer Ingelheim: Employment. Biss:Boehringer Ingelheim: Employment. Brueckmann:Boehringer Ingelheim: Employment. Gropper:Boehringer Ingelheim: Employment. Maas:Boehringer Ingelheim: Employment. Tartakovsky:Boehringer Ingelheim: Employment. Mitchell:Boehringer Ingelheim: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy.

Introduction. Chronic myeloid leukemia (CML) patients (pts) treated with imatinib first line achieve complete cytogenetic response (CCyR) in > 70% of cases and major molecular response (MMR) in 18-58%. These pts have a life expectancy similar to the general population. However even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been recently developed (Goh HG et al., 2011). dPCR corresponds to a 100 fold increase in sensitivity as compared to Q-RT-PCR. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The Imatinib Suspension And Validation (ISAV) study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible for this study. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples are obtained for dPCR and the pts discontinue imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is evaluated through the EORTC – C30 Quality of Life questionnaire. Results. The enrolment in ISAV began in November 2011 and ended in July 2013. The study enrolled 112 pts: Italy 69.6%, Germany 21.4%, Canada 5.3%, Spain 2.6% and Israel 0.9%. Among the 112 pts, 59.3% were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.8 mts before imatinib discontinuation. To date, the median follow-up (FUP) time is 16.6 mts [95% CI: 14.9-18.2]. Forty-seven pts (43.5%, 95% CI: 34.0-53.4) of the 108 eligible pts relapsed and resumed imatinib; 38/47 (80.9%) of them relapsed in the first 9 mts and the last relapse occurred 19.6 mts after imatinib discontinuation. A loss of CCyR occurred in 11 pts (23.4%): 10/11 CCyR losses were recovered; 1 patient withdrew the consent shortly after obtaining a partial cytogenetic response. No case of CML progression was observed. After the resumption of imatinib the median time to either MMR or CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 61 not-relapsed pts, 43 (39.8% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 3.6 mts [95% CI: 3.0-4.8] and the range of duration of Q-RT-PCR positivity (below 0.1%) was between 5.7 and 29.2 mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR or duration of CMR was identified. An inverse relationship between pts age and risk of relapse is evident: 90% of pts < 45 years relapsed vs 37.5% in the class ≥ 45 - < 65 years and 27.5% of pts ≥ 65 years, p(χ2) Conclusions. After 32 mts from the beginning of the study, with a median FUP of 16.6 mts, 43.5% of pts relapsed; the majority of relapses developed in the first 9 months after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. Funded by Regione Lombardia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

The shift in sites of hematopoiesis during embryonic development leads to primitive hematopoiesis within the fetal liver at E12.5, which generates primarily erythrocytes. Definitive hematopoiesis, which occurs within the bone marrow, follows at birth. During the erythroid differentiation of fetal hematopoiesis, progenitor cell maturation is accompanied by global DNA demethylation, a process necessary for fetal liver erythrocyte formation and accompanied by diminishing expression of the de novo DNA methyltransferases, Dnmt3a and Dnmt3b. In our current study of hematopoietic cell specific Prmt5 knockout mice, we have identified Prmt5 as a master regulator of erythropoiesis; the cell-specific deletion of Prmt5 in fetal liver cells is embryonic lethal as Prmt5-null embryos have severe anemia and increased expression of Dnmt3a and Dnmt3b proteins. RNA-seq and pathway analysis studies revealed profound defects in several critical pathways that regulate normal hematopoiesis, including the tumor suppressor p53 pathway. Methyl-seq studies are currently being conducted to determine the effects of the enforced expression of key DNA methyltransferases on global DNA methylation and gene expression in erythroid progenitors and how it leads to a block in erythrocyte maturation. To decipher the extent of Dnmt3a or p53's involvement in the observed phenotypes, we have generated double knockout mouse models that are being analyzed. Mechanistically, p53 has been shown to be directly methylated by Prmt5, a modification that affects its tumor suppressor activity. Here we have found that Dnmt3a is also a substrate of Prmt5 and the effects of the di methylation of Dnmt3a on its function are currently under investigation. Thus, we have uncovered a potential functional interaction between DNA methylation and protein arginine methylation triggered by Prmt5 that regulates primitive erythropoiesis. Disclosures Levine: Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Introduction. A substantial proportion of patients (pts) affected by Chronic Myeloid Leukemia (CML) achieve complete negativity in Q-RT-PCR. In this situation, as already demonstrated in other STOP trials, it is possible to safely discontinue imatinib treatment but it is still not clear how to discriminate subjects who will relapse. In fact even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been developed. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The ISAV study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results and to evaluate relapse rate and timing of recurrence, survival and the impact of imatinib treatment on Quality of Life (QoL). Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples were obtained for dPCR and the pts discontinued imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients' QoL during imatinib discontinuation/resumption is evaluated through the EORTC QLQ-C30 questionnaire. Results. The study enrolled 112 pts with a median follow-up (FUP) time of 28.0 mts [95% CI: 25.5-30.1]. The 59.3% of pts were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.7 mts before imatinib discontinuation. The cumulative probability of survival is 97.8% [95% CI: 91.4-99.5]. dPCR results showed that 23.1% of pts were positive and 75.9% negative, with a significant Negative Predictive Value ratio (dPCR/Q-RT-PCR) of 1.112 [95% CI: 1.009-1.225]. At 24 mts from imatinib discontinuation, 53 pts (49.1%, 95% CI: 39.3-58.9) of the 108 eligible pts relapsed and resumed imatinib; 73.6% of them relapsed in the first 9 mts and the last relapse occurred 21.8 mts after imatinib discontinuation. A loss of CCyR occurred in 13 pts (34.2% of those tested): 10/13 CCyR losses were recovered, the remaining 3 were not assessed for response. No case of CML progression or resistance to imatinib was observed. After the resumption of imatinib the median time to MMR/CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 55 not-relapsed pts, 42 (38.9% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 2.9 mts [95% CI: 2.0-3.1] in the relapsed pts and 4.5 mts [95% CI: 2.9-6.9] in pts who developed only PCR positivity. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR, Sokal score or duration of CMR was identified, while an inverse relationship between pts age and risk of relapse is evident. Moreover, age and dPCR results together can predict the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥ 45 years and with negative dPCR (36.1%). The analysis of QoL evidenced a statistically significant improvement in the general well-being and symptoms scales at 1 month after imatinib discontinuation and in particular nausea, diarrhea and fatigue (p Conclusions. At 45 mts from the beginning of the study, with a median FUP of 28.0 mts, 49.1% of pts relapsed; the majority of relapses developed in the first 9 mts after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. QoL analysis showed a significant decrease in symptoms after imatinib discontinuation. Funded by Regione Lombardia. Disclosures Abruzzese: BMS, Novartis, Pfizer, Ariad: Consultancy. Assouline:BMS: Consultancy; Pfizer: Consultancy; Novartis: Consultancy. D'Emilio:Celgene: Research Funding. Dong-Wook:ll-Yang: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Introduction Outcomes for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have improved, but the prognosis for pts with R/R disease remains poor. Childhood AML and MLL-rearranged ALL (MLL r-ALL) often overexpress or have activating mutations in the FLT3 tyrosine kinase (TK). Midostaurin (PKC412) is an orally available TK inhibitor that targets FLT3, KIT, and other kinases. Data in adults for single-agent midostaurin, including from studies CPKC412A2104 and CPKC412A2106, showed that 100 mg bid (≈ 60 mg/m2 bid) was adequately tolerated, elicited leukemic blast reduction, and inhibited FLT3 autophosphorylation. Here, we present the safety and preliminary clinical activity of midostaurin in children with R/R acute leukemia. Methods In this multicenter dose-escalation study, children (3 mo to < 18 y) with FLT3 -mutated AML or MLL r-ALL refractory to standard induction therapy or in second or subsequent relapse received oral midostaurin solution (NCT00866281). The primary objective was to estimate the maximum tolerated dose (MTD) or recommended dose for expansion (RDE) for 2 age groups: younger (3 mo to 2 y) and older (> 2 y to < 18 y). The secondary objective was to evaluate response, time to relapse, overall survival (OS), pharmacokinetics (PK), pharmacodynamics, safety, and tolerability. In the dose-escalation phase (starting dose, 30 mg/m2 bid), each cohort received increasing doses until a maximum dose of 60 mg/m2 bid was reached. Dose escalation was determined using a Bayesian logistic regression design, allowing separate dose escalation in each age group. Dose-limiting toxicity (DLT) was defined as a grade 3/4 nonhematologic adverse event (AE), abnormal drug-related laboratory value, or drug discontinuation within 14 days of study start. Results This study enrolled 22 pts (7 at 30 mg/m2 bid [2 younger, 5 older]; 15 at 60 mg/m2 bid [9 younger, 6 older]) starting on Sep 21, 2009. The median age was 2 y (range, 0.5-17 y); 9 pts (9 older) had AML and 13 pts (11 younger, 2 older) had ALL. The dose-determining set included 17 pts. No DLTs occurred at 30 mg/m2 bid; thus, in each age group, the dose was escalated directly to 60 mg/m2 bid. One DLT (increased alanine aminotransferase [ALT]) occurred in a younger pt receiving 60 mg/m2 bid; no older pts had a DLT. The most common AEs regardless of study drug relationship (any grade/grade 3/4) were vomiting (68%/5%), pyrexia (41%/14%), and thrombocytopenia (41%/36%). Grade 3/4 AEs (particularly thrombocytopenia, elevated ALT, and anemia) were more common with the higher dose. Consistent with the safety profile of midostaurin in adults, the most frequent drug-related any-grade AEs were vomiting (55%), nausea (32%), and diarrhea (18%). The MTD was not reached. Median OS was 3.7 mo (95% CI, 2.7-8.3 mo) in pts with AML and 1.4 mo (95% CI, 1.0-2.9 mo) in pts with ALL. All 5 on-treatment deaths (ie, death while on study drug or within < 28 days of final dose) were due to underlying AML (n = 4) or ALL (n = 1); 16 additional pts (4 with AML, 12 with ALL) died ≥ 28 days after the final dose. One pt was alive at trial end. Midostaurin demonstrated limited single-agent clinical activity in this study. Five pts (56% [95% CI, 21%-86%]) with AML achieved a clinical response. The best response was a morphological complete remission with incomplete count recovery on day 14. The pt remained in remission until stopping treatment on day 64, received a stem cell transplant (SCT) on day 76, and was alive at day 960 after completing the per-protocol survival follow-up. Three pts (23% [95% CI, 5%-54%]) with ALL achieved a clinical response; all were peripheral blood blast responses. All pts discontinued treatment; reasons were disease progression (64%), start of new cancer therapy (including SCT [18%]), withdrawn consent (14%), or AEs (5%). Median duration of exposure was 15.5 days (range, 3-64 days). Time-dependent PK and exposures of midostaurin and its 2 active metabolites (CGP52421 and CGP62221) at the selected doses were similar to adult data. Conclusions Single-agent midostaurin was adequately tolerated at 60 mg/m2 bid, the highest dose evaluated. Only limited clinical activity was demonstrated; thus, future trials in children with FLT3 -mutated AML should be in combination with established chemotherapies. The RDE of midostaurin for such studies is 30 mg/m2 bid due to a higher frequency of grade 3/4 AEs in the 60 mg/m2 bid dose group and known toxicities of existing standard pediatric AML regimens. Disclosures Zwaan: Janssen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Söderhäll:Novartis: Other: Investigator in clinical trials within my duties or ordinary salary at the hospital; Merck: Other: Investigator in clinical trials within my duties or ordinary salary at the hospital. Sternberg:Novartis Oncology: Employment, Equity Ownership. Besse:Novartis: Employment. Dutreix:Novartis: Employment. Ho:Seattle Genetics (employment started after completion of involvement in presented study): Employment. Dufour:Pfizer: Consultancy.

The association of antiphospholipid antibodies with thrombosis and obstetric events defines the antiphospholipid syndrome. A recent systematic review of the literature showed lupus anticoagulants to be risk factors of thrombosis, independent of the type and site of the event, the presence of

Immunoglobulin (Ig) and T-cell Receptor (TcR) gene rearrangements are used as patient-specific PCR targets for MRD detection in ALL. However, oligoclonality was reported in childhood ALL, and in those cases, the uncertainty of which clone is going to emerge at relapse may give rise to false negative MRD results. In particular, oligoclonality is a peculiar characteristic of Infant ALL (less than 1 year of age at diagnosis). So far, the prognostic relevance of MRD monitoring in Infant ALL has not been defined, and the assessment of oligoclonality and stability of PCR markers may have a profound impact in MRD monitoring. We successfully studied by PCR the frequency and stability of the currently used rearrangements of the Ig Heavy (IgH), Ig Kappa deleting element (IgK-Kde), TcR delta (TcRD), and TcR gamma (TcRG) gene rearrangements in 42/43 Infant ALL patients prospectively enrolled in Italy in the Interfant-99 International ALL protocol. Pro-B, common and pre-B ALL were 29 (67%), 8 (19%), and 6 (14%), respectively. Sixty-three percent of cases were prednisone good responders (PGR), 70% were MLL-rearranged, 44% were aged less than 6 months, and 21% of cases had more than 300.000 wbc at diagnosis. Overall, rearrangements of the IgH, IgK, TcRD, and TcRG genes were found in 91, 21, 40, and 21% of Infant ALL, respectively. The pattern of Ig/TcR rearrangements in immunophenotypic subgroups of Infant ALLs has been compared to the one of older children prospectively enrolled in Italy into the AIEOP-BFM ALL2000 protocol (n=649). While the overall frequency of IgH and TcRD are similar in the two age groups, IgK and TcRG are less rearranged in Infant cases, mainly due to the low percentage in the pro-B group (10% versus 41% for IgK VK-Kde, and 17% vs 36% for TcRG). In particular, none of the 29 pro-B cases showed rearrangement of the IgK intron. Sixteen Infant relapsed so far (37%), and 12 of them were successfully analyzed for Ig/TcR rearrangement at diagnosis and relapse. Ten were early (less than 18 months from diagnosis), and 2 were late relapses. None of the patients showed an identical Ig/TcR pattern at diagnosis and relapse; 7 (58%) had at least one, and 1 had at least 2 PCR targets preserved, while 4 cases (33%) presented completely different markers at relapse. This distribution was independent on age at diagnosis, site and time of relapse. Interestingly, we observed an increase of complete-IgH (from 10 alleles in 7 patients to 23 alleles in 11) and TcRG (from 4 alleles in 2 patients to14 alleles in 8) rearrangements at relapse. The stability of Ig/TcR markers was as it follows: IgH, 6/26; IgK, 3/5; TcRD, 1/9 and TcRG, 0/4. In conclusion, the oligoclonality feature of Infant ALL may potentially hamper the MRD predictivity in Infant ALL. An appropriate identification and selection of Ig/TcR MRD-PCR targets in Infant ALL is a crucial premise for obtaining clinically relevant MRD data and for preventing false-negative MRD results. Ig Kappa may be a preferential marker for MRD studies in Infant ALL, although its use is limited by the low frequency of the rearrangement. The leukemia-specific MLL genomic breakpoint may potentially overcome these limitations and improve the MRD analysis.

The production of interleukin-1beta (IL-1beta), a powerful mediator of inflammation, is tightly regulated at several levels. However, in some pathologic conditions, a pharmacologic treatment is required to control the toxicity of excessive extracellular IL-1beta. Because of the heavy side effects of most therapies used in IL-1beta-mediated pathologies, a goal of pharmacologic research is the development of selective anti-IL-1beta drugs. We show here that the sulfonylurea glyburide, currently used in the oral therapy of noninsulin dependent diabetes, is an inhibitor of IL-1beta secretion from human monocytes and mouse macrophages. Glyburide reduces dramatically the recovery of extracellular 17-kD IL-1beta in the absence of toxic effects on the cells and without affecting the synthesis or processing of the IL-1beta precursor. IL-1beta belongs to the family of leaderless secretory proteins released from the cell by a nonclassical secretory route. In bacteria and yeast Atp binding cassette (ABC) transporters are involved in the secretion of leaderless secretory proteins. Interestingly, glyburide blocks the anion exchanger function of ABC1, a mammalian member of the family of ABC transporters. We thus investigated the involvement of ABC1 in IL-1beta secretion, through the analysis of the effects of drugs known to inhibit IL-1beta secretion, on the activity of ABC1 and in turn the ability of known inhibitors of ABC1 of blocking IL-1beta secretion. Our data show that IL-1beta secretion and the function of ABC1 as an anion exchanger are sensitive to the same drugs, therefore suggesting an involvement of the ABC1 transporter in the secretion of leaderless proteins in mammals.

Abstract 5143 The RVD regimen has shown a promising activity in patients with relapsed/refractory multiple myeloma (MM). In the present study, we report preliminary results on 14 patients in first relapse after autologous stem cell transplantation (ASCT) treated by VRD followed by a maintenance treatment with revlimid-dexamethasone (Rev-dex) in responder-patients. Treatment was as follows: RVD included lenalidomide 15 mg per day (d) d1 through d14, bortezomib 1 mg/m2 on d1,4,8 and 11 and dexamethasone 20 mg on d1,2,4,5,8,9,11 and 12 of 21-d cycles with the objective to deliver at least 6 cycles. A maintenance treatment by Rev-dex with lenalidomide 25 mg d1 to d21 plus dexamethasone 20 mg weekly of d-28 cycles, delivered until progression was planned in patients achieving at least partial response to RVD. Patients' characteristics at relapse: median age was 65 years (range 59 to 70), median time to relapse following ASCT before RVD was 26 months (range 4 to 86). All pts were naive for lenalinomide and 8 for bortezomib. Translocation t(4;14) was observed in 4 patients and was associated with del(17p13) in one case. Patients received a median number of 6 cycles of RVD (range 3 to 8). Overall response rate was 71% and 43% of the patients achieved very good partial or better response. Discontinuation of RVD was due to disease progression in 4 cases. During RVD treatment, 4 patients (29%) experienced grade 3–4 adverse events: neutropenia n =2, thrombocytopenia n=1, thrombosis n=1. Following RVD, 10 patients received Rev-dex maintenance. With a median follow-up of 15 months (range 6 to 29), 11 patients are alive free of evolutive disease. Four patients progressed during VRD: one with solitary plasmocytoma was efficiently treated by radiotherapy, the 3 other patients received anthracycline or platin based regimens but were refractory and 2 of them died. One patient progressed during maintenance and was efficiently treated by melphalan-cyclophosphamide-dexamethasone. Among the 4 patients with high-risk cytogenetic abnormalities, one CR, one PR and 2 progressions were observed during RVD. The first patient remains in CR 16 months after the beginning of salvage treatment. Conclusion: RVD followed by Rev-dex maintenance is a promising therapeutic option in MM patients in first relapse after ASCT with low toxicity profile. Disclosures: No relevant conflicts of interest to declare.

Abstract 4263 Introduction. Precise and effective measurements of iron overload in the liver, where iron deposition seems to be primarily noticeable (Noetzli LJ et al, Blood 2008), are important for the early diagnosis, treatment and follow-up of patients with hemochromatosis or hemosiderosi. T2* Magnetic resonance imaging (MRI) represents the most available noninvasive technique to assess hepatic iron content and shows a good correlation with biopsy results (Wood JC et al, Blood 2005). In the clinical practice, a single hepatic slice is acquired and T2* measurement is performed in a single region of interest (ROI) of the parenchyma (Pepe A et al, Eur J Haematol 2006). This approach may mask an heterogeneous iron distribution. Thus, the aims of this study were to set up a MRI acquisition technique for the detection of the iron burden in the whole liver and to evaluate the effectiveness of the single ROI technique. Methods. Five transverse hepatic slices were acquired by a T2* gradient-echo sequence in 101 thalassemia major (TM) patients (48 males, mean age 29 ± 8 years, mean serum ferritin level 1413 ± 1209 ng/mL, mean pre-transfusion hemoglobin 9 ± 1 g/dl.) and 20 healthy subjects. The T2* value was calculated in a single ROI defined in the medium-hepatic slice. Moreover, the T2* value was extracted on each of the eight ROIs defined in the functionally independent segments according to Couinaud classification. The mean hepatic T2* value was obtained by averaging all segmental values. Results. For patients the mean T2* values over segments VII and VIII were significantly lower than the other segments (P Conclusions. Hepatic T2* variations are low and due to artifacts and measurement variability. The single ROI approach can be adopted in the clinical arena, taking care to avoid the susceptibility artifacts, occurring mainly in segments VII and VIII. Disclosures: No relevant conflicts of interest to declare.

Abstract 4615 INTRODUCTION Long-term blood transfusion is essential for patients with sickle cell disease (SCD) in case of cerebral vasculopathy, organ dysfunction, leg ulcer, failure or intolerance of hydroxyurea treatment. Secondary iron overload is a factor of morbidity and mortality by organ damage. In practice, three chelators are available: deferoxamine (DFO) which is administrated by subcutaneous infusion and therefore source of poor compliance in SCD. Deferasirox (DFX), the new oral chelator is the first line therapy since 2007. Deferiprone (DFP) is an option when DFX or DFO are contraindicated or inadequate. However, DFP has no approval for SCD. The purpose of this study is to describe the characteristics of SCD patients treated by DFP. METHODOLOGY The patients included in this study arise from the group of the patients with long-term blood transfusion by manual exchange in Henri Mondor's SCD center, and in whom iron overload is treated by DFP. The monitoring of iron overload is obtained by regular serum ferritin level, combined liver and heart MRI. Only one iron measure by MRI is available for each patient throughout the study. RESULTS Nine patients (8 SS and 1 Sβ0thal) are included: 5 men and 4 women. The mean age is 44.2 years (22 to 64 years). The median duration of chronic transfusion is 10 years (4 to 27 years). The average dose of DFP is 68 mg / kg / day (50 to 93 mg / kg / day). The median follow-up under DFP is 30 months (7 to 60 months). The median level of serum ferritin before the initiation of DFP is 5830 μg / l (1800 to 9300 μg / l); and the median level of serum ferritin at the end point is 7940 μg / l (4540 to 11300 μg / l). MRI shows an important hepatic iron overload (up to 320 μmol) in all patients and one cardiac iron overload (T2* = 12 ms). Three patients stopped DFP and switch to deferasirox (DFX) as soon as DFX was available. For the other patients, the reason of prescribing DFP instead of DFX was renal failure in 5 patients and DFX related GI symptoms in one patient. No agranulocytosis is observed. The weekly then monthly monitoring of blood count is insured for all patients. No cytolysis by drug's toxicity is observed, except for one patient with liver transplant and who has an active HCV infection. DISCUSSION Serum ferritin level is the easiest marker of iron overload follow-up, but is subject of important variations due to inflammation, hemolysis, and cytolysis. Indeed, MRI is the only one reliable measure. The evidence of cardiac iron overload is proved in one patient, and confirms the importance of this measure on SCD patients. This motivates the edition of guidelines concerning the prevention and monitoring of iron overload among these patients. The dosage of DFP remains reasonable compared to the mean dosage use in other pathologies. This dosage depends of the degree of iron overload and the individual tolerance. Data are not sufficient at these days to evaluate the efficacy of DFP to reduce or to stabilize the level of iron overload. However, we observe globally a good clinical and biological tolerance, even in patients who have organ transplant and therefore have several concomitant treatments. CONCLUSION DFP in patients with SCD is globally well-tolerated, but its efficiency is not proved yet. Approval of DFP for SCD is needed. As life expectancy improves in SCD, more patients will require long-term transfusion and thus iron chelation therapy. Cardiac Iron overload is possible in patients with SCD. So, it would be systematically looked after. Disclosures: No relevant conflicts of interest to declare.

Abstract 2643 Poster Board II-619 C/EBP is a transcription factor that regulates terminal granulocytic differentiation. CEBPA mutations have been associated with improved outcome in both adult and pediatric patients with acute myeloid leukemia (AML). However, the impact in different treatment protocols, as well as the different outcome between single and double mutants, is still to be definitively established. We evaluated the prevalence and prognostic significance of CEBPA mutations in children with de novo non promyelocitic AML treated in Italy with the AIEOP-LAM 2002/01 pediatric protocol. Among 205 patients enrolled in the protocol between December 2002 and December 2007, 103 were successfully analyzed for CEBPA mutations by PCR amplification of TAD and bZIP domains of the gene, and through sequencing of positive cases after DHPLC analysis. Characteristics of analyzed and non analyzed patients were not statistically different. Two types of CEBPA mutations, N-terminal (TAD) truncating mutations and in-frame bZip domain mutations, were detected in 13/103 (12.6%) patients tested; 8 of them (61.5%) harboured both mutation types. Laboratory, clinical characteristics and outcomes for patients with CEBPA mutations were compared to those of patients with wild-type CEBPA. CEBPA mutations were significantly more common in older patients (8/13 vs 30/90 children were older than 10 years), in patients with FAB M1 (7/13 vs 4/90), and in patients with normal cytogenetics (13/13). None of the CEBPA mutated cases carried either FLT3 or NPM1 mutations. Only 1 mutated case was found in Standard Risk patients (defined as children carrying isolated CBF abnormality and achieving complete remission after 1 cycle of induction therapy), while the other 12 patients belonged to the High Risk group. Although the values did not reach statistical significance because of the low prevalence of CEBPA mutations, with a median follow up of 39 months (range 4–86) the event-free survival probability at 5 years was 76.9% vs. 59.6% for children with or without CEBPA mutations, respectively. The values for Disease-Free Survival were 83.3% vs. 65.4% and those for Overall Survival were 90.0%. 66.4%, respectively. No difference in terms of outcome was found between patient with a single and those with double mutants, neither between those with TAD- and bZIP- mutations. Therefore, patients in the AIEOP-LAM 2002/01 pediatric trial carrying CEBPA mutations seem to have a lower relapse rate and improved outcome, their overall survival approaching that of children belonging to the Standard Risk group (90% vs. 97%). If confirmed in a larger cohort of patients and with longer follow-up, these data suggest that CEBPA mutation analysis could be usefully employed to identify patients at lower risk of treatment failure and for allocating them into different classes of risk. Disclosures: No relevant conflicts of interest to declare.