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International audience; Background Richter syndrome (RS) refers to the onset of aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL), in patients with chronic lymphocytic leukemia (CLL). The outcome of RS patients is usually very poor with short survival (typically 2. Tocilizumab was administered to 9 (75%) patients. Five (42%) patients had ICANS, 3 (25%) with grade > 3. Regarding hematotoxicity, 6 (50%) patients presented with grade > 2 thrombocytopenia, 5 (42%) with grade > 2 anemia, and 7 with (58%) grade > 2 neutropenia. One case of macrophage activation syndrome was reported. Three patients were admitted to intensive care. A total of 5 (42%) patients had infections. After a median follow-up of 1.6 months (range, 0-23), 8 (67%) patients were alive, 4 (33%) patients died (2 from CRS and 2 from disease progression).Conclusions CD19-directed CAR T-cell therapy showed high response rates in our series of heavily pretreated RS patients. Frequency of CAR T-cell-specific adverse events was in the range of what is observed in de novo DLBCL while severity appeared higher (Schuster et al., NEJM 2019; Neelapu et al., NEJM 2017). Larger cohort with longer follow-up and prospective trials are warranted to confirm these observations.

Acute myeloid leukemia (AML) intensive chemotherapy combined with broad-spectrum antibiotics, leads to gut microbiota dysbiosis promoting pathological conditions and an increased incidence of complications. Here we report findings from a phase II single-arm, multicenter study evaluating autologous fecal microbiota transfer (AFMT) in 25 AML patients treated with intensive chemotherapy and antibiotics (ClinicalTrials.gov number: NCT02928523). The co-primary outcomes of the study are to evaluate the efficacy of AFMT in dysbiosis correction and multidrug-resistant bacteria eradication. The main secondary outcomes are to define a dysbiosis biosignature, to evaluate the effect of dysbiosis correction on patient clinical status, to assess the short and mid-term safety of AFMT in this immunocompromised population, and to evaluate the feasibility of the AFMT procedure and acceptability by the patient. Intensive induction chemotherapy induces a dramatic decrease of α-diversity indices, and a microbial dysbiosis with a significant shift of the microbial communities and domination of pro-inflammatory families. After AFMT treatment, α-diversity indices return to their initial mean levels and the similarity index shows the restoration of microbial communities. The trial meets pre-specified endpoints. AFMT appears to be safe and may be effective for gut microbiota restoration in AML patients receiving intensive chemotherapy and antibiotics, with an excellent gut microbiota reconstruction based on both richness and diversity indices at the species level., The combination of chemotherapy and broad-spectrum antibiotics induces gut microbiota (GM) dysbiosis in acute myeloid leukaemia (AML) leading to additional complications. Here, the authors report the efficacy in GM restoration and safety of autologous faecal microbiota transfer in treated AML patients in a phase II clinical trial.

Objectives To assess population pharmacokinetics (PK) and pharmacodynamics (PD) of both piperacillin and tazobactam in neutropenia patients and examine dosage requirements related to the MIC distribution for Gram-negative bacteria involved in bloodstream infections (BSIs). Methods We conducted a prospective study including adult haematological malignancy patients with febrile neutropenia receiving piperacillin/tazobactam as short (30 min) or prolonged (4 h) intravenous infusions. Concentration data were analysed using a population approach. Dosing simulations with the final model investigated factors influencing the PK/PD of piperacillin/tazobactam quantified by fT>MIC or PTA for piperacillin and tazobactam, respectively. In parallel, the local MIC distribution of β-lactams was documented for Gram-negative bacteria involved in BSIs. Results Over 10 months, 31 patients were enrolled, with 11 (35.5%) short and 20 (64.5%) prolonged infusion regimens. A one-compartment model adequately described the data for both drugs. Prolonged infusion, increased serum alkaline phosphatase (ALP) values and renal function impairment were associated with increased piperacillin fT>MIC. For patients with normal or augmented renal CL, dosing regimens q8h or q6h with 30 min of infusion were insufficient to achieve acceptable PTA for piperacillin/tazobactam at the median MIC value of 8 mg/L. Prolonged infusion of large doses was associated with the best PTA for both piperacillin and tazobactam. Conclusions In a population of haematological malignancy patients with neutropenia, renal function and ALP influenced the PK of piperacillin/tazobactam. Prolonged intravenous infusion would optimize the PK of piperacillin/tazobactam, especially in the case of augmented renal CL and/or low-range bacterial susceptibility.

Summary: Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr−/− cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34+ cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation. : Paubelle et al. show that targeting the vitamin D receptor has anti-leukemic activity by acting on cell differentiation and by decreasing stemness of AML cells. Keywords: acute myeloid leukemia, vitamin D receptor, leukemic stem cell

Background Outcome of patients with mutation of the FLT3 tyrosine kinase domain (FLT3-TKD) in acute myeloid leukemia (AML) remains controversial. Patients and Methods Herein, we present a retrospective study of 126 newly diagnosed patients with AML performed in 2 French centers. Results FLT3-TKD mutations represented 12.7% of patients, whereas FLT3-internal tandem duplication (ITD) mutation was observed in 20.6% of AML cases and 1.6% of patients harbored both anomalies. At diagnosis, FLT3-TKD and FLT3-ITD were associated with higher peripheral leukocytes count and a higher blast count in bone marrow (P Conclusion Our data suggest that FLT3-TKD mutations should be routinely determined at the time of diagnosis. In association with NPM1 mutations, patients should follow the therapeutic schedule of favorable-risk patients with AML.

Objectives We previously reported the prognostic value of serum ferritin in younger patients with intermediate-risk acute myeloid leukemia (AML). The aims of this study were to confirm this finding in a larger cohort regardless of age and prognostic subgroups, to explore the expression and functional role of ferritin in AML cells as well as the regulation of serum ferritin levels in AML patients. Patients/materials/methods Serum ferritin levels at diagnosis were collected in a cohort of 525 patients treated by intensive chemotherapy. In silico, in vitro, and in vivo analyses were conducted to assess the pattern of expression and functional role of FTH1 and FTL in AML. Results We confirmed the independent prognostic value of serum ferritin. In transcriptomic databases, FTH1 and FTL were overexpressed in AML and leukemic stem cells compared to normal hematopoietic stem cells. The gene signature designed from AML patients overexpressing FTH1 revealed a significant enrichment in genes of the immune and inflammatory response including Nf-KB pathway, oxidative stress, or iron pathways. This gene signature was enriched in cytarabine-resistant AML cells in a patient-derived xenograft model. FTH1 protein was also overexpressed in patient's samples and correlated with the in vitro cytotoxic activity of cytarabine. Lastly, we demonstrated that chemotherapy induced an inflammatory response including a significant increase in serum ferritin levels between day 1 and 8 of induction chemotherapy that was blocked by dexamethasone. Conclusion Ferritin is deregulated in most AML patients likely through inflammation, associated with chemoresistance, and could represent a new therapeutic target.

Acute myeloid leukemia (AML) represents a disease with a very poor outcome and remains an area of significant unmet need necessitating novel therapeutic strategies. Among novel therapeutic agents, vosaroxin is a first-in-class anticancer quinolone derivative that targets topoisomerase II and induces site-selective double-strand breaks in DNA, leading to tumor cell apoptosis. Areas covered: Herein, the authors provide a comprehensive review of the preclinical development of vosaroxin. This includes coverage of vosaroxin's mechanism of action in addition to its pharmacology and of the main studies reported over the past few years with vosaroxin when used to treat adult AML. Expert opinion: Given that vosaroxin is associated with fewer potential side effects, it may be of benefit to elderly patients with relapsed/refractory AML and to those with additional comorbidities who have previously received an anthracycline and cytarabine combination. Furthermore, vosaroxin also was seen to be active in multidrug-resistant preclinical models. However, further studies have to be performed to better evaluate its place in the armamentarium against AML.

To the editor: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), characterized by the t(15;17) translocation resulting in the PML-RARA fusion transcript. The use of all-trans retinoic acid (ATRA) and/or arsenic has revolutionized the treatment of this disease, which

Although the outcome in younger adults with acute myeloid leukemia (AML) has improved, the benefit associated with standard intensive chemotherapy in older patients remains debatable. In this study, we investigated the incidence and the prognostic significance of genetic characteristics according to treatment intensity in patients aged 60 years or older. On the 495 patients of our cohort, DNMT3A R882 (25.2%), NPM1 (23.7%) and FLT3-ITD (16.8%) were the most frequent molecular mutations found at diagnosis. In this elderly population, intensive chemotherapy seemed to be a suitable option in terms of early death and survival, except for normal karyotype (NK) NPM1&minus, FLT3-ITD+ patients and those aged over 70 within the adverse cytogenetic/molecular risk group. The FLT3-ITD mutation was systematically associated with an unfavorable outcome, independently of the ratio. NK NPM1+/FLT3-TKD+ genotype tends to confer a good prognosis in patients treated intensively. Regarding minimal residual disease prognostic value, overall survival was significantly better for patients achieving a 4 log NPM1 reduction (median OS: 24.4 vs. 12.8 months, p = 0.013) but did not reach statistical significance for progression free survival. This retrospective study highlights that intensive chemotherapy may not be the most appropriate option for each elderly patient and that molecular markers may help treatment intensity decision-making.

French National Cancer Institute (INCA) and the Fondation Francaise pour la Recherche contre le Myelome et les Gammapathies (FFMRG), the Intergroupe Francophone du Myelome (IFM), NCI R01 NCI CA167824 and a generous donation from Matthew Bell. This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. Research reported in this paper was supported by the Office of Research Infrastructure of the National Institutes of Health under award number S10OD018522. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank the Association des Malades du Myelome Multiple (AF3M) for their continued support and participation. Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization

Introduction EVI1 (MECOM)-positive acute myeloid leukemia (AML) cells have shown in vitro sensitivity to all-trans-retinoic acid (ATRA) by inducing differentiation, cell death, and decreased leukemic engraftment. Methods In this pilot study, we investigated the response to ATRA in 13 high-risk AML patients with overexpression of EVI1. Results Seven of the 13 patients (53.8%) achieved complete remission (CR), and response can be combined with a decreased of the leukemia stem cell pool. Conclusion These primary results tend to confirm in vitro results and suggest that addition of ATRA might be of benefit in the treatment of patients with EVI1-positive AML.

International audience; Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34+, NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4− iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4− iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4− iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P

Introduction. AML standard intensive induction chemotherapy ("3+7" or equivalent) combined with wide spectrum antibiotics can dramatically alter the composition of the gut microbiota, leading to dysbiosis which is characterized by loss of microbial diversity. Such dysbiosis status can promote a pathological condition involving uncontrolled local immune responses, systemic inflammation and increased incidence of adverse events. The development of FMT-based drugs to restore microbial communities could offer novel therapeutic possibilities to reduce such adverse events and potentially improve outcomes in AML. We therefore conducted this single arm prospective phase I/II multicenter trial (NCT02928523) to evaluate the use of a FMT-based drug in association with AML induction treatment to restore the gut microbiota diversity. Patients and methods. A total of 62 consecutive patients aged between 24 and 69 years old with a diagnosis of de novo AML were screened in 7 French sites. At time of admission and AML diagnosis (Step 1=S1), patients' faeces were collected, rigorously screened, prepared following a standardized process, and stored at -80°C until later administration. The drug was administered as an enema after hematopoietic recovery (S2) and before consolidation chemotherapy (Conso). The primary endpoint was the recovery of at least 70% of microbiota diversity (based on the Simpson index) after drug administration and the reduction of multidrug resistant bacteria carriage. Blood and feces samples were collected at S1, S2, and around 10 days post-FMT before Conso (S3). Microbiome diversity restoration was assessed by metagenomics analysis through Illumina HiSeq shotgun sequencing. Antibiotic resistance gene carriage (ARGC, also known as resistome) was evaluated through mapping of readouts on the MEGARES database. Secondary objectives included safety and analysis of host response with assessment of blood and fecal markers by ELISA and Luminex. Results. Overall, 25 patients were actually treated with FMT, and 20 were included in the per-protocol population. Induction Chemotherapy (IC) induced a dramatic shift in microbial communities, with a significant 42.3% decrease of mean α-diversity Simpson index between S1 and S2 at species level (0.85 to 0.50; p No serious adverse events (SAE) were observed within 30 days after FMT and all post FMT SAEs were not related to the FMT procedure. Moreover, FMT did not induce any local or systemic inflammatory reaction as measured by fecal and blood markers (fecal neopterin and IgA; plasmatic CRP, IL-6 and sCD14). Interestingly, restoration of the microbiome diversity was associated with a significant reduction of CRP and fecal neopterin levels, suggesting a potential anti-inflammatory impact of FMT. Overall, FMT was well tolerated and had an excellent safety profile. The one-year overall survival estimate in the whole cohort was 84% (4 deaths among 25, none of which were related to FMT: 2 multiple organ failures, 1 heart attack and 1 grade IV resistant GVHD). The median time to death from the second FMT was 182.5 days (113-225 days). Conclusions. This is the first prospective trial testing the safety and efficacy of FMT in AML patients receiving intensive induction chemotherapy. The trial achieved its primary endpoint and established the capacity of FMT to restore a diverse microbiome with high levels of similarity to baseline, as well as reducing ARGC and intestinal inflammation. A controlled randomized trial with repeated FMT administrations is currently planned to further evaluate the impact of FMT on clinical outcomes and long-term survival. (This trial was funded by MaaT Pharma whose product was tested in this protocol). Disclosures Mohty: MaaT Pharma: Consultancy, Honoraria. Doré:MaaT Pharma: Consultancy, Honoraria.

Cellular immunotherapy with autologous or allogeneic T cells, genetically engineered to express chimeric antigen receptors (CARs) or T-cell receptors, in order to redirect their cytotoxic specificity toward malignant cells, is emerging as a promising new treatment modality. The most advanced approach in clinical development is the use of anti-CD19 CAR T-cells for the treatment of CD19

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients’ bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients’ outcome.

Introduction Secondary acute myeloid leukemia (sAML) remains a therapeutic challenge. In elderly patients with AML, it is unclear whether sAML displays an inferior outcome compared with de novo AML. Patients and Methods We studied AML with an antecedent of hematologic disease, treatment-related AML, or AML occurring concurrently to another malignancy in a single-center cohort of patients aged 70 and older with AML. The study included 169 patients who were compared with a cohort of patients with de novo AML, without any prior history of malignant disorders, seen during the same period of time. Results Hematologic antecedents or presence of prior/concurrent solid malignancy did not impact complete remission rates and overall survival. In multivariate analysis, sAML appeared without independent prognostic value in the elderly. Conclusion Our results support that sAML and de novo AML in elderly patients are not prognostically distinct entities. They should therefore not be considered separately when investigating outcomes and new treatment strategies.

International audience; Outcome of patients with primary refractory/relapsed (R/R) acute myeloid leukemia (AML) remains dismal. Herein, we present a retrospective monocentric study of 24 very high-risk AML patients who received a combination of fractionated gemtuzumab ozogamicin (GO) with intermediate-dose cytarabine and daunorubicin as salvage therapy. Median age was 55.3 years. Diagnostic was secondary AML for 33% of them. Seven patients had favorable risk, 8 had intermediate-1 or intermediate-2, and 6 had unfavorable risk of AML according to the European LeukemiaNet prognostic index. Complete remission was achieved in 50% of cases (46% in refractory and 55% in relapsed AML) without excessive toxicity. Thirteen patients could be referred for transplant. Only allogeneic hematopoietic stem cell transplantation provided a benefit in this patient cohort with a 1-year overall survival of 50.7 versus 18.1% in the absence of transplantation. Patients treated with reduced intensity conditioning (RIC) showed a longer survival as compared to those undergoing myeloablative conditioning regimen mainly because of decreased toxicity.Our data suggest that salvage therapy with fractionated GO combined with intermediate-dose cytarabine and daunorubicin in very high-risk patients may serve as a potential bridge therapy to RIC transplant

We evaluated the positivity of (1-3)-β-D-glucan, a serum marker of invasive fungal diseases (IFD), at diagnosis and during treatment. (1-3)-β-D-Glucan may not be an early marker but could prove useful for diagnosis of chronic IFD., Background. Early diagnosis and treatment are crucial in invasive fungal diseases (IFD). Serum (1-3)-β-d-glucan (BG) is believed to be an early IFD marker, but its diagnostic performance has been ambiguous, with insufficient data regarding sensitivity at the time of IFD diagnosis (TOD) and according to outcome. Whether its clinical utility is equivalent for all types of IFD remains unknown. Methods. We included 143 patients with proven or probable IFD (49 invasive candidiasis, 45 invasive aspergillosis [IA], and 49 rare IFD) and analyzed serum BG (Fungitell) at TOD and during treatment. Results. (1-3)-β-d-glucan was undetectable at TOD in 36% and 48% of patients with candidemia and IA, respectively; there was no correlation between negative BG results at TOD and patients' characteristics, localization of infection, or prior antifungal use. Nevertheless, patients with candidemia due to Candida albicans were more likely to test positive for BG at TOD (odds ratio = 25.4, P = .01) than patients infected with other Candida species. In 70% of the patients with a follow-up, BG negativation occurred in >1 month for candidemia and >3 months for IA. A slower BG decrease in patients with candidemia was associated with deep-seated localizations (P = .04). Thirty-nine percent of patients with rare IFD had undetectable BG at TOD; nonetheless, all patients with chronic subcutaneous IFD tested positive at TOD. Conclusions. Undetectable serum BG does not rule out an early IFD, when the clinical suspicion is high. After IFD diagnostic, kinetics of serum BG are difficult to relate to clinical outcome.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and has significant morbidity and mortality as 20–40% of patients progress to end-stage renal disease (ESRD) within 20 years after disease onset. We aimed to gain insight into the molecular mechanisms involved in IgAN progression. A systematic evaluation of renal biopsy specimens from IgAN patients revealed that the MAPK/ERK signaling pathway was activated in mesangial areas of patients presenting with >1 g/day proteinuria and elevated blood pressure, but was absent in biopsy specimens from IgAN patients with modest proteinuria (

Differentiating agents have been proposed to overcome the impaired cellular differentiation in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective target in the treatment of AML. Iron chelating therapy induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a manner involving modulation of reactive oxygen species expression and the activation of mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the induction of VDR target genes. When used to treat one AML patient refractory to chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of pancytopenia and in blast differentiation. We propose that iron availability modulates myeloid cell commitment and that targeting this cellular differentiation pathway together with conventional differentiating agents provides new therapeutic modalities for AML.

Rationale: Acute promyelocytic leukemia (APL) is a curable subtype of acute myeloid leukemia. APL is currently treated with combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) resulting in the induction of apoptosis and differentiation of the leukemic cells. Differentiation syndrome (so-called ATRA syndrome) is the main life-threatening complication of induction therapy with these differentiating agents. Patient concerns: Herein, we report the case of a 49-year-old woman diagnosed with APL with, concomitantly, a bulky cutaneous lesion of 10 cm diameter with a red-to-purple background and a necrotic center, localized on her abdomen. Diagnoses: After 10 days of treatment, the cutaneous lesion became purulent. Fluorescence in situ hybridization (FISH) analysis performed on this pus confirmed the presence of malignant features in the involved granulocytes proving their origin from the differentiation of leukemic APL cells, as all the analyzed nuclei showed 2 promyelocytic leukemia (PML)–retinoic acid receptor-a (RARA) fusions signals. Intervention: The association by ATRA and ATO was continued. Outcome: Eventually, the evolution was favorable with healing in three weeks. Lessons: This case report therefore highlights the differentiation phenomenon of promyelocytic blasts within promyelocytic sarcoma with the ATRA–ATO combination and the efficacy of this drug association in resolving both the malignant sarcoma and a secondary local infection.

Synergy between FLT3 and Pim kinase inhibition in acute myeloid leukemia with FLT3-ITD mutation., Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

The prognosis of acute myeloid leukemia (AML) in elderly (≥65 years) patients is poor and treatment remains non-consensual especially for those who are not eligible for intensive therapies. Our group has shown that in vitro the iron chelator deferasirox (DFX) synergizes with vitamin D (VD) to promote monocyte differentiation in primary AML cells. Herein, we present results from a retrospective case-control study in which the association of DFX (1-2 g/d) and 25-hydroxycholecalciferol (100,000 IU/week) (DFX/VD) was proposed to patients following demethylating agents failure. Median survival of patients treated with DFX/VD combination (n = 17) was significantly increased in comparison with matched patients receiving best supportive care (BSC) alone (n = 13) (10.4 versus 4 months respectively). In addition, the only factor associated to an increased overall survival in DFX/VD-treated patients was serum VD levels. We conclude that DFX/VD treatment correlated with increased overall survival of AML patients in this retrospective cohort of elderly patients.

Introduction: Despite recent improvements in the treatment of adult acute myeloid leukemia (AML), many patients still fail to achieve complete remission (CR) or relapse early after response to initial induction chemotherapy. Outcome of patients with primary refractory/relapsed (R/R) acute myeloid leukemia (AML) remains dismal. Nevertheless, most of these patients undergo salvage chemotherapy. Gemtuzumab ozogamicin (GO) is an antibody-targeted chemotherapy agent consisting of a humanized murine anti-CD33 monoclonal antibody linked to calicheamicinϒ1, a potent antitumor antibiotic. Binding of GO induces cell death in CD33-positive cells by internalization of the calicheamicin drug and toxin release intracellulary leading then to then cleavage of double-stranded DNA. In relapsed AML, GO used alone with an unfractionated dose of 9 mg/m² on days 1 and 14 resulted in 13% of CR and a median OS of 5 months. Combined with intermediate-dose cytarabine and mitoxantrone, unfractionated dose of GO gave 50% of CR in patients with R/R AML, and a 2-year OS of 41%. However, early toxic deaths were reported related to sinusoidal obstruction syndrome (SOS). These results were confirmed by subsequent studies evaluating unfractionated GO combined with various agents in patients with R/R AML. The main objective of the present retrospective study was to evaluate the efficacy of the combination of fractionated GO with intermediate-dose cytarabine and daunorubicin in very high-risk patients with AML, and its potential use as bridge to transplant in this patient population. Methods: Herein we present a retrospective monocentric study of 24 very high-risk AML patients who received a combination of fractionated gemtuzumab ozogamicin (GO) with intermediate-dose cytarabine and daunorubicin as salvage therapy. Results: Median age was 55.3 years. Diagnoses were secondary AML for 33% of them. Seven patients had favorable, 8 had intermediate-1 or intermediate-2 and 6 unfavorable-risk AML according to the European LeukemiaNet prognostic index. Complete remission rate was achievied in 50% of cases (46% in refractory and 55% in relapsed AML) without excessive toxicity. Only allogeneic hematopoietic stem cell transplantation provided a benefit in this patient cohort with a one-year overall survival of 50.7% versus 18.1% in the absence of transplantation. Patients treated with reduced intensity conditioning (RIC) showed a higher survival as compared to those undergoing myeloablative conditioning regimen. CD33 expression was not associated with survival. In multivariate analysis in a model including the age older than 60 years, allo-HSCT, refractory AML and achievement of CR after GO, only the performance of allo-HSCT predicts patients' outcome with a Hazard Ratio (HR) of 0.25 (95% CI 0.07-0.86), p=0.02. Conclusions: Our data suggest that salvage therapy with fractionated GO combined with intermediate-dose cytarabine and daunorubicin in very high-risk patients may serve as a potential bridge therapy to RIC transplant. Data from studies published after withdraw of GO from the market, of which our present study, suggest that the licence status of GO might be reviewed, at least for certain subtypes of patients and certain situations of which R/R AML patients. Disclosures Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria; Ariad pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Gilead: Honoraria, Research Funding; Roche/Genentech: Consultancy, Honoraria, Research Funding; Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Thomas:Pfizer: Consultancy.

Background. Despite progress in the molecular and genetic classification of pediatric acute myeloid leukemia (AML), the prognosis remains heterogeneous. The ATP-binding cassette transporter A3 (ABCA3) seems specifically involved in the resistance of pediatric AML to intensive chemotherapy. However, studies having investigated the prognostic impact of ABCA3 expression have yielded conflicting results with respect to patient outcomes while the small sample size of these studies precluded the use of multivariate analysis. Here we investigated the prognostic impact of ABCA3 expression in a representative series of homogeneously treated pediatric AML. Methods. Samples derived from 233 patients with available high-quality RNA and enrolled in the ELAM2 protocol (NCT00149162). qRTPCR amplification of 2 conserved ABCA3 mRNA sequences was performed with GUS and ABL as reference genes. Primer sets were complementary to exons 6-7 and exons 19-20 junctions. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups (Rubnitz JE, Blood 2012;119:5980-5988, Creutzig U, Blood 2012;120:3187-3205). Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant (HSCT) in complete remission (CR) after 1 to 2 consolidation courses. Results. The discovery cohort included 120 patients. Median age, median WBC, CR rate, relapse rate, median follow-up, 5-years EFS, DFS, and OS were 9.4 years, 19.3 G/L, 95%, 29%, 60 months, 58±6%, 61±6%, and 71±5 months, respectively. The two primer sets yielded consistent results (R=0.9, p Conclusion. ABCA3 expression represents an independent prognostic factor in pediatric AML. As they indicate that the level of ABCA3 expression is significantly associated with survival for currently accepted cytogenetic and molecular prognostic categories, our findings suggest that assessing ABCA3 expression will permit a better assessment of disease risk. Finally our results suggest that inhibiting ABCA3 expression, such as with indomethacin, could be beneficial in order to overcome drug resistance in pediatric AML. Disclosures No relevant conflicts of interest to declare.

Background. We have recently investigated transcriptional and posttranscriptional changes in AML cells (Oncotarget, 2016, 7, 2889-909) and identified ABCA3 expression, TET2 expression and TET2 exon 2 skipping (TET2E2S) as prognostic factors in pediatric AML (See companion abstracts from Ceraulo et al.). The present study was conducted in order to test whether the combination of these prognostic factors might help stratifying patients according to their relapse risk. Methods. Samples derived from 120 patients with available high-quality RNA and enrolled in the French ELAM2 protocol. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant in complete remission (CR) after 1 to 2 consolidation courses. qRTPCR amplification of 2 conserved ABCA3 (exons 6-7 and exon 19-20) and TET2 (exon 4 and exon 2-3) mRNA sequences were performed with GUS and ABL as reference genes. TET2E2S was quantified as already described (Oncotarget, 2016, 7, 2889-909). The concordance index (c-index) was used to compare the relative statistical power of the new model compared with the standardized cytogenetic and molecular-based scoring system. Results. ABCA3 expression [HR 2.6 (95% CI 1.3-5), p=0.006], TET2 expression (HR 0.5 (0.2-0.9) p=0.04, and TET2E2S (HR 2.5 (1.2-5.1), p=0.01) represented independent prognostic factors for EFS. To design a convenient prognostic score, continuous variables were dichotomized using cutoff points that were based using the Maximally Selected Rank Statistics (maxstat R-statistics package, from the R-Project). One point was attributed for lower TET2 expression (n=85), higher ABCA3 expression (n=44), and TET2E2S (n=61). Three groups were formed: lower-risk patients (0 point, n=33), intermediate-risk patients (1 point, n=41), and higher-risk patients (either 2 or 3 points, n=46). The present score identified subgroups of patients with significantly different outcome (p Conclusion. We propose a new prognostic model that includes transcriptional and posttranscriptional data and can predict survival in pediatric AML. A validation in a larger, independent, multicenter, data set of patients is important to facilitate the translation of this model into clinical practice. Disclosures No relevant conflicts of interest to declare.

BACKROUND: The protein defective in type 1 hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins, associates with beta2-microglobulin and is implicated in membranous protein recycling. Gemtuzumab ozogamicin (GO), a monoclonal antibody directed against CD33 linked to a cytotoxic agent, has been used with controversial effects in acute myeloid leukemia (AML). Internalization of GO is required for its anti-leukemic effect. Therefore, we hypothesized that H63D or C282Y HFE gene mutations may impair GO activity by preventing its internalization. METHODS: Wild type, C282Y and H63D HFE leukemic cells and primary cell were used to assess effects of GO alone or in combination with cytarabine on cell proliferation and apoptosis. Flow cytometry, confocal and AMNIS stream analysis were used to evaluate GO internalization. HFE mutations were analyzed by PCR analysis on DNA from patients included in clinical studies. Post-hoc subgroup analysis was performed to assess in vivo the role of HFE status on GO efficacy and toxicity among the 280 patients of the ALFA-0701 study as a study cohort (patients aged 50-70 years; GO 3mg/m² on days 1, 4, and 7 of chemotherapy and on day 1 of the first and second induction ; total dose 15 mg/m² ) and then on the GOELAMS-LAM 2006 IR study (patients aged 18-60 years; GO 6 mg/m² on day 4 of chemotherapy during the induction and the first consolidation; total dose 12 mg/m²) and UK NCRI AML17 study (patients aged 18-81 years; GO 3 vs 6 mg/m² on day 1 of chemotherapy but not during consolidation) as validating cohorts. RESULTS: GO induced cell death by apoptosis in AML cell lines and primary cells in a dose-dependent manner and synergistically in combination with cytarabine. However, the IC5O of GO was significantly higher in HFE mutated cells (125 vs 10 ng/mL p HFE mutations were screened in 242 of the 280 ALFA-0701 patients with DNA available. There were 155 non-mutated patients (64%), 68 (28%) heterozygous for H63D, 11 (5%) heterozygous for C282Y, and 8 (3%) homozygous for H63D, which is consistent with the prevalence of the various mutations in the French population. Median age was 62 years (50-71) and the M/F ratio was 0.5, equally distributed among the different groups. No significant difference was observed with respect to the diagnosis of various hematological parameters, including white blood cell count, blasts number, cytogenetic subgroups, molecular mutation incidences (FLT3, NPM1, CEBPA, IDH, DNMT3A). In the ALFA-0701 study cohort, HFE wild-type (WT) patients had a higher overall survival (OS) when treated in the GO arm (median, not reached vs 19.5 months, p=0.0193). In contrast, OS was similar among patients with heterozygotes HFE mutations treated in the GO and the control arm (median, 19.9 vs 21.9, p=0.9675). In confirmatory cohorts, GO treatment led to a trend to increased OS in the GOELAMS-LAM 2006 IR cohort only in HFE WT patients (4-year OS with GO 62% vs 48%, p=0.08) but not in mutated patients (4-year OS with GO 73% vs 64%, p=0.45). Furthermore, in this cohort in the FLT3 WT patients subgroup, GO further improve OS in WT patients (4-year OS with GO 72% vs 50%, p=0.017), but not in patients with heterozygous HFE mutation (4-year OS with GO 80% vs 65%, p=0.23) In the UK NCRI AML17 cohort, which used GO only during induction, 245 patients were randomized between GO at 3mg/m2 and 6mg/m2 and evaluated for HFE status. Overall there was no effect of GO dose on outcomes, and no evidence of either any heterogeneity by HFE, nor any subgroup, which showed a differential effect of GO dose. CONCLUSIONS: Future studies should focus on optimising the fractionated schedule for GO at the 3mg/m2. Fractionated high doses (3x3mg/m2) during induction and single dose during consolidation seems to be the best schedule. Most importantly, the effect of GO treatment differed between HFE WT and heterozygote mutated AML patients. GO only increased OS among HFE WT patients. This is likely related to impaired internalization of the CD33 target. Our data suggest that HFE status should be used as a companion test to predict outcome of AML treated with GO. Disclosures No relevant conflicts of interest to declare.

Virtually all recurrent molecular alterations in acute myeloid leukemia (AML) functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as patients, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, as demonstrated by western blotting, immunofluorescence, flow cytometry and electron microscopy. Blocking autophagy via shRNA-mediated knockdown of ATG5 and ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation in AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1 both directly (through raptor phosphorylation) and indirectly (through TSC2 phosphorylation), sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. This protective effect was mediated by mTORC1-dependent modulation of the ATF4/CHOP stress response pathway. The ultimate functional consequence was that, rather than diminishing GSK621-induced cytotoxicity, persistent mTORC1 activity was in fact synthetically lethal with AMPK activity in AML cells. This synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. It could also be enhanced in AML cells by mTORC1 overactivation induced by CRISPR-mediated deletion of TSC2. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. This finding may have important implications for AMPK and mTORC1 signaling pathways in cancer biology more broadly. Context-dependent permissiveness towards mTORC1 activation may amplify the response to cytotoxic stress, such as that resulting from AMPK activation by GSK621. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials. Disclosures Brusq: GSK: Employment. Nicodeme:GSK: Employment.

Abstract 247 Background: β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods: Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results: RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary: Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures: Daniel: Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.

Abstract 3622 Background: AML is a group of heterogeneous malignant diseases characterized by uncontrolled cell growth and differentiation arrest. Following the success of differentiating therapies in APL, great hopes were placed in Vitamin D (VD) and its ability to promote differentiation of non-APL AML cells. However, results of clinical studies were disappointing and trials were interrupted due to the occurrence of life-threatening hypercalcemia. Our group has shown that iron chelators such as deferasirox (DFX) are able to promote monocytes differentiation in both normal hematopoietic progenitors and AML cells (Callens et al Jexp Med 2010). Moreover, iron deprivation synergized with VD to promote cell differentiation on leukemic cells. Most elderly patients diagnosed with AML suffer from secondary iron overload because of in some cases ineffective erythropoiesis and iterative red blood cell transfusions. Furthermore, in myelodysplastic syndromes, retrospective studies have suggested that iron chelators may increase life expectancy and decrease the risk of transformation into AML. In AML of the elderly, the use of demethylating agents such as 5-azacytidine or decitabine may induce hematological response and increase life expectancy. However, response is often of short duration. Since VD deficiency and iron overload prevalence is high in the elderly, the association of VD and DFX was given to a subgroup of patients following demethylating agents failure. Methods: A retrospective chart review of 17 elderly AML patients after demethylating agents failure was performed in three French centers. Patients treated by the combination of DFX/VD were matched to patients treated with best supportive care (BSC). Based on ferritin, and creatinin levels the dose of DFX was adapted in each case. DFX dose was up to 2000 mg a day and VD was used at 100,000 units orally weekly. The tolerance and the overall survival (OS) were analyzed. Pre-clinical studies were conducted in vitro on cell lines (HL60, U937, OCI-AML3, THP-1, MOLM 13) to evaluate cell differentiation induced by DFX and a new VDR agonist (Inecalcitol) by cell morphology and flow cytometry (expression of CD11b and CD14 markers). VDR activity and expression were evaluated by flow cytometry, immunoblotting, luciferase reporter assays and qPCR to detect VDR-targeted genes. Results: Median age of DFX/VD patients and BSC control group were 76 (range 63–84) and 71 (58–85) respectively. Most patients were diagnosed with AML with multilineage dysplasia (cases 70%, controls 76%). Prognosis groups were distributed homogeneously between the treated patients and controls. There were no significant differences in blast infiltration, leukocytosis, neutropenia, systemic iron and phosphocalcium parameters. All patients received 5-azacytidine (median of 8 courses for the cases and 7 for the controls).No renal insufficiency, hepatotoxicity or hypercalcemia were observed in DFX/VD patients. At 3 months, 4 treated patients (23.5%) had significant monocyte level increase an evidence of the enhanced monocyte differentiation efficacy. The treatment did not decrease the need of transfusion. Most interestingly median survival of treated patients was significantly increased (10.4 m vs 4 m, p=0.002). In vitro studies were conducted in parallel aiming to characterize new potential alternative therapeutic associations, which could improve patients' response. We show that the use of a new highly potent VDR agonist (Inecalcitol) potentiated the effect of DFX in promoting terminal monocyte differentiation of leukemic cell lines. It also increased VDR activity evaluated by VDR expression and phosphorylation and expression of VDR-targeted genes. In vivo studies in mice model of AML using combined DFX/inecalcitol therapy will be presented. Conclusions: The prognosis of elderly patients diagnosed with AML after demethylating agents remains poor. Here we show that the differentiating therapy by the association of Deferasirox and Vitamin D was able to improve overall survival with low toxicity. New generation of highly-potent VDR agonists (which are devoided of hypercalcemic properties) significantly enhanced VDR activation and terminal monocyte differentiation of AML cells and represent potential therapeutic alternatives in the near future. These encouraging results should be verified in a large randomized prospective multicenter study. Disclosures: No relevant conflicts of interest to declare.

Background. In AML, the frequency of TET2 mutations increases with age ranging from 1-5% in children to 6-27% in adults. These mutations have both been reported to exert unfavorable or neutral effects on patient outcomes. Besides mutations, fluctuation in TET2 expression has been evidenced in AML while we recently found that TET2 exon 2 skipping (TET2E2S) is associated with favorable outcome in adult AML (Mint-Mohamed et al., ASH 2014 and submitted). Here we investigated the effect TET2 expression and TET2E2S in homogeneously treated adult and pediatric AML patients with intensive chemotherapy (IC). Methods. Samples derived from 341 patients with available high-quality RNA and enrolled in the ELAM2 protocol (n=120) and the ALFA-0701 protocol (n=221).qRTPCR amplification of 2 conserved TET2 mRNA sequences was performed with GUS and ABL as reference genes. TET2 exon 2 skipping was quantified throughexon(E)-specific qRTPCR (qESPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2E2S was quantified by calculating the ratio E1E3/E2E3. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. In children, treatment consisted in 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant (HSCT) in complete remission (CR) after 1 to 2 consolidation courses. Adult patients were given a 3+7 induction course without (control group, n=110) or with fractionated intravenousgemetuzumab ozogamicin (GO) (n=111) as already described (Castaigne S, Lancet 2012; 379:1508-1516). Results. In pediatric AML, median age, median WBC, CR rate, relapse rate, median follow-up, 3-years EFS, DFS, and OS were 9.4 years, 19 G/L, 95%, 29%, 48 months, 60±5%, 63±6%, and 72±5%, respectively. Lower TET2 expression was associated with inferior EFS (3-years: 44±10%vs 69±6%, log-rank p = 0.01), DFS (51±8%vs 64±5% p=0.02), and OS (60±8%vs 83±3%, p=0,0003) while TET2E2S was associated with inferior EFS (44±8%vs 70±7%, p = 0.006) and OS (61±7%vs 81±4%, p=0.01). For TET2 expression, multivariate analysis identified WBC, age, risk group, and TET2 expression as independent prognostic factors for EFS and OS, and age, WBC, and TET2 expression for DFS (Table 1). For TET2 splicing, WBC and TET2E2S independently predicted EFS while TET2E2S alone independently predicted DFS (Table 1). In the low- (n=29) and intermediate-risk (n=62) groups, lower TET2 expression remained associated with shorter 3-years EFS. There was no significant correlation between TET2 expression and TET2E2S and TET2E2S retained its negative prognostic impact in the 85 patients with higher TET2 expression (3-years EFS 48±8vs 84±6%, p=0.004). In adult AML, median age, median WBC, CR rate, relapse rate, median follow-up, 3-years EFS, DFS, and OS were 62 years, 8 G/L, 76%, 59%, 47 months, 26±3%, 30±4%, and 44±4%, respectively. In the entire population, TET2 expression and splicing had no significant effect on outcome. In the 110 patients treated with IC alone, TET2 expression was associated with inferior OS (25±9vs 29±9%, p=0.02) while TET2E2S was associated with better EFS (17±5vs 7±6%, p=0.008), DFS (28±8vs 8±6%, p=0.02), and OS (46±8vs 29±8%, p=0.05). TET2E2S, age, and disease risk represented independent prognostic factors for EFS (Table 1). In the GO arm, TET2 expression had no significant effect on outcome whereas TET2E2S was associated with inferior EFS (27±5vs 60±12%, p=0.01), DFS (31±6vs 63±12%, p=0.05), and OS (39vs 72±11%, p=0.04). Conclusion. Inchildren, higher TET2 expression confers a favorable outcome whereas TET2E2S represents an independent unfavorable prognostic factor. In adult treated with IC alone, present results confirm the favorable prognostic impact of TET2E2S and the weak prognostic effect of TET2 expression. In contrast, in patients fulfilling the same inclusion criteria, TET2E2S loses its favorable prognostic effect and is even associated with unfavorable outcome when GO is associated to IC. While TET2 expression and splicing might represent useful biomarkers, the data highlight the strong difference in tumor biology between adult and pediatric AML and suggest distinct relationships between TET2E2S and the cytotoxic effect of GO plus IC versus IC alone. Disclosures Thomas: Pfizer: Consultancy.

AIM To evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival. METHODS Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia (AML) were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8C panels and FACS CANTO and Diva software (BD Bioscience). RESULTS We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38- population. Using a cut-off value of 1% of CD34+CD38- from total “bulk leukemic cells” we found that a high (> 1%) level of CD34+CD38- blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38- leukemic cells > 1% was an independent predictor of DFS [HR = 2.8 (1.02-7.73), P = 0.04] and OS [HR = 2.65 (1.09-6.43), P = 0.03]. CONCLUSION Taken together, these results show that a CD34/CD38 “backbone” for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.