Search

Only proven pathogenic mutations associated with myeloid neoplasms are key to establish the clonal nature of the bone marrow fibrosis. In cases with genetic variants of uncertain meaning, the clinical picture may be required to rule out secondary causes.

Introduction The European LeukemiaNet (ELN) 2017 classification for acute myeloid leukemia (AML) stratifies patients in 3 risk categories, according to genetic features of the disease. ELN classification is commonly used to guide post-remission treatment; favorable risk patients do not seem to benefit from allogeneic hematopoietic transplantation (alloHCT) in first complete remission (CR1) while this procedure is highly recommended in adverse risk patients. Post-remission treatment in intermediate risk patient is still debated. This classification has not been prospectively validated. Herein we analyze the prognostic impact of ELN 2017 classification in patients treated with the same protocol (CETLAM-12), including a well-defined preplanned alloSCT policy according to genetic risk. Methods We analyzed characteristics and outcome of patients diagnosed with de novo AML, included in CETLAM-12 protocol for patients up to 70 years, with an available genetic characterization at diagnosis allowing an accurate ELN 2017 stratification. Genetic risk allocation was based on cytogenetic analysis and pre-specified RT-PCR of determined markers (including CBF-rearrangement, NPM1, FLT3, CEBPA, and MLL-PTD) performed in all patients, and targeted Next Generation Sequencing testing (available in 143 patients). CETLAM-12 protocol defines a genetic risk stratification in three groups, closely similar to that proposed by the ELN 2017 classification, and recommends a post-remission strategy based on this risk assessment. Patients from the favorable group received three courses of consolidation with high-dose cytarabine (HiDAC), whereas alloSCT in CR1 is strongly recommended for intermediate and adverse risk patients following one HiDAC-based consolidation course. Results We included in the study 813 patients (400F/413M; median age 56, 17-76); with a 37 months median estimated follow-up (range 0.1-92). The outcomes of the entire cohort are shown in table 1. Out of all patients, 641 could be classified according to the ELN 2017 classification, due to the presence of risk defining genetic features identified by any of described methods. In the group of .atients allocated in the favorable risk category (n=316; 49%), twenty-seven patients died during induction. Fifty-eight relapses were observed, mostly in patients with NPM1 mutation (n=41). AlloSCT was finally performed in CR1 in 84 patients (27%) due to MRD persistence or reappearance (n=40), overt hematological relapse (n=27), persistent aplasia following chemotherapy (n=3) and protocol deviation (n=14). In the group of patients allocated in the intermediate risk category (n=95; 15%), twelve patients died during induction. AlloSCT in CR1 was performed in 62 patients (67%), with 5 patients receiving an alloSCT in CR2. In the group of patients allocated in the adverse risk category (n=230; 36%), twenty-five patients died during induction. AlloSCT could be finally performed in CR1 in 138 patients (60%) and 88 relapses occurred (42 before alloSCT, 46 after alloSCT). Amongst ELN adverse risk patients, a subgroup with a significant worse outcome has been identified, defined by AML with complex karyotype +/- TP53 mutation or chromosome 3q26/MECOM-GATA2 rearrangement. This group (ELNadv+) presented a lower CR rate, with a higher relapse rate and fewer proportion of patients who receive a pre-planned alloSCT in CR1. OS and EFS of ELNadv+ is significantly lower than ELN adverse patients. In the multivariate analysis for OS including age, sex, WBC count at diagnosis and number of cycles to achieve CR, only age and ELNadv+ status showed independent prognostic value. Outcome data is summarized in table and figures attached. Conclusions The initial risk-adapted post-remission assignment planned in CETLAM-12 could be performed in the majority of patients. Despite this different proposed post-remission treatment, ELN risk classification was able to identify three groups of patients with a markedly different outcome. Interestingly, within the unfavorable ELN category, a very high-risk ELNadv+ subgroup can be distinguished, with a dismal outcome with current approach, warranting the implementation of innovative pre and post-transplant strategies aimed to prevent treatment failure. Figure Disclosures Tormo: MSD: Honoraria; Janssen: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Honoraria; Servier: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Salamero:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria; Daichii Sankyo: Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Sierra:Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction. Mutations in genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 are found in 10-20% of patients with acute myeloid leukemia (AML). Recently, IDH inhibitors have shown good clinical response in patient's refractory to standard treatments, providing evidence for a new treatment paradigm. Comprehensive real-world studies are needed to explore genotype-to-phenotype correlations and prognosis of IDH mutated AML, which may influence targeted treatment strategies. Patients. From a retrospective, European, real-word population (ClinicalTrials.gov Identifier: NCT04369287) we studied 477 IDH mutated patients and 954 IDH wild type patients matched for age, sex and type of treatment with a 1:2 ratio. Results. Median age of IDH mutated patients was 67 years; IDH1 mutations were found in 202 patients (89% carried R132 mutation), while IDH2 mutations were found in 275 cases (51% and 28% carried R140 and R172 mutations, respectively). At diagnosis, IDH mutated patients had lower neutrophil and higher platelet count and higher percentage of marrow blasts (P Considering cytogenetic risk according to ELN criteria, the great majority of IDH1 and IDH2 mutated patients had an intermediate cytogenetic risk (84% and 86%, respectively, P We then analysed the most common co-mutational patterns in IDH mutated patients. A total of 53% of IDH1 mutated patients carried NPM1 mutations (without FLT3 mutations), while the majority of IDH2 mutated patients had wild type NPM1 gene (P Median overall survival from diagnosis (OS) was 14 months for IDH1 mutated patients, 23 for IDH2 mutated patients and 19 for IDH wild type patients (P IDH mutated patients receiving hypomethylating agents (n=211) had a lower response rate vs. wild type patients (56% vs. 36% of treatment failure, respectively, P=.04), while no significant different probability of response to intensive chemotherapy was noticed. In patients who received allogeneic transplantation (n=345), IDH1 mutated patients shower higher relapse rate vs. wild type and IDH2 mutated patients (53% vs. 34%, P Conclusion. In a real world context, AML patients with IDH1 and 2 mutations have high marrow blasts percentage, frequently present normal karyotype and show specific co-mutational patterns with respect to NPM1, FLT3 and ASXL1 genes. IDH1 mutations were an independent predictor of unfavorable outcome with high rate of disease recurrence under currently available treatment options, and could be considered as an additional marker to improve personalized prognostic assessment within ELN risk groups. Dissection of prognosis of IDH mutated AML may influence targeted treatment strategies in clinical practice. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heibl:Takeda: Honoraria; AOP orphan: Consultancy, Honoraria, Research Funding; BMS/celgene: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Honoraria. Metzeler:Astellas: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharma: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding. Thiede:AgenDix GmbH: Other: Co-owner and CEO. Fracchiolla:ABBVIE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau. Todisco:Jannsen, Abbvie, Jazz: Membership on an entity's Board of Directors or advisory committees. Passamonti:Novartis: Speakers Bureau; BMS: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support.

BACKGROUND: AML risk classification is based on genetics (cytogenetics and molecular features) and more recently also on minimal residual disease (MRD) after chemotherapy. These two aspects allow predicting relapse and supporting or not the most anti-leukemia treatment that remains allogeneic hematopoietic cell transplantation (HCT). We prospectively investigated the combined use of the two predictive markers to allocate post-remission therapy with or without HCT. Objectives of the study were testing: a) if this approach was feasible in a multicenter setting; b) the proportion of patients who were allocated to an allogeneic HCT and finally received the procedure; c) the final distribution into the risk categories and their outcome; d) to analyze the outcome of patients with favorable or intermediate genetics moved to the high risk category because of positive MRD. METHODS: Adult patients with primary AML treated at 15 academic hospitals were included between February 2012 and December 2018. Induction chemotherapy consisted of idarubicin 12 mg/m2 days 1-2-3 and cytarabine 200 mg/m2 days 1 to 7. Consolidation courses were high-dose cytarabine (3 g/m2 or 1.5 g/m2 if ≥60 y/o). The number of consolidation courses was based on genetic risk: 3 in favorable genetics category (FGC) (CBF, NPM1mut/FLT3-ITDwild or ratio0.1%) and/or quantitative PCR of the specific transcripts (RUNX1/RUNX1T1, CBFβ/MYH11 and NPM1). RESULTS: Seven hundred forty-five patients (median age: 55, range18-70 y/o, 51% male) were enrolled. Cytogenetics according the revised MRC classification in 707 informative cases was: CBF AML 12%, intermediate 65% (75% of them normal karyotype), and adverse 23%. FLT3-ITD was detected in 28% of patients with intermediate risk cytogenetics and NPM1 mutation in the same group was present in the 48%. Complete remission (CR) was achieved in 81% (n=603) of patients, 82% and 80% in patients up to and above 60 yrs, respectively. Induction death occurred in 9% of patients, 7% and 11% the two age groups, and 10% of patients had refractory leukemia; 542 (90%) of the 603 CR patients completed the consolidation phase and were risk allocated taking into account genetics and MRD. The remaining CR patients were not allocated because of early relapse (n=22), death in CR (n=5), severe toxicity (n=22) or others (n=12). After risk allocation, 208 (38%) patients were in the genetics-MRD combined favorable group (CFG), 103 (19%) in combined intermediate group (CIG) and 231 (43%) in the combined adverse group (CAG). In the latter, 185 (80%) of patients received an allogeneic HCT in first CR. Fifty-seven patients (11%) moved from the genetically FGC or IGC to the CAG because of high MRD at the end of consolidations. Median follow-up in survivors was 25 months. Overall 4-years survival (OS) of the whole series is 48±2%; event-free survival (EFS) is 77+3% in the CFG group, 45+6% in the CIG and 34+4% in the CAG (p CONCLUSION Risk adapted therapy for primary AML based on genetics and MRD is feasible in a cooperative group setting. The proportion of CR was high (>80%) even in patients older than 60 y/o. MRD assessment at the end of consolidation moved 57 patients with favorable or intermediate genetics to the CAG. Avoiding HCT in first CR in the FGC patients associated to EFS above 75% at 4 years. Allogeneic transplantation feasibility was 80% when this was the intended treatment because of adverse genetics and/or MRD positivity. Risk assessment based on genetics and MRD continues separating three groups of patients with different outcomes. Since relapses remain frequent when adverse AML features are present, further approaches after transplantation, such as targeted agents and immune therapies deserve investigation. Disclosures Sierra: Astellas: Honoraria; Pfizer: Honoraria; Daiichi-Sankyo: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau. Salamero:Daichii Sankyo: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Esteve:Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Daiichi Sankyo: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Pfizer: Consultancy.

// Josep Maria Cornet-Masana 1 , Daniel Moreno-Martinez 1 , Maria Carmen Lara-Castillo 1 , Meritxell Nomdedeu 1,2 , Amaia Etxabe 1 , Niccolo Tesi 1 , Marta Pratcorona 1,2 , Jordi Esteve 1,2 and Ruth M. Risueno 1 1 Josep Carreras Leukaemia Research Institute, Barcelona, Spain 2 Department of Hematology, Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Correspondence to: Ruth M. Risueno, email: // Keywords : AML, emetine, anti-leukemia drug Received : February 24, 2016 Accepted : February 28, 2016 Published : March 15, 2016 Abstract Acute myeloid leukemia (AML) is an hematologic neoplasia characterized by the accumulation of transformed immature myeloid cells in bone marrow. Although the response rate to induction therapy is high, survival rate 5-year after diagnosis is still low, highlighting the necessity of new novel agents. To identify agents with the capability to abolish the self-renewal capacity of AML blasts, an in silico screening was performed to search for small molecules that induce terminal differentiation. Emetine, a hit compound, was validated for its anti-leukemic effect in vitro , ex vivo and in vivo . Emetine, a second-line anti-protozoa drug, differentially reduced cell viability and clonogenic capacity of AML primary patient samples, sparing healthy blood cells. Emetine treatment markedly reduced AML burden in bone marrow of xenotransplanted mice and decreased self-renewal capacity of the remaining engrafted AML cells. Emetine also synergized with commonly used chemotherapeutic agents such as ara-C. At a molecular level, emetine treatment was followed by a reduction in HIF-1α protein levels. This study validated the anti-leukemiceffect of emetine in AML cell lines, a group of diverse AML primary samples, and in a human AML-transplanted murine model, sparing healthy blood cells. The selective anti-leukemic effect of emetine together with the safety of the dose range required to exert this effect support the development of this agent in clinical practice.

The outcome of allogeneic hematopoietic stem cell transplantation (HCT) in patients with myeloid malignancies is better in those without minimal residual disease (MRD) than in those with MRD+, as assessed by multiparametric flow cytometry (MPFC). WT1 quantitation also has been used to assess the probability of relapse in acute myelogenous leukemia (AML) treated with chemotherapy. We analyzed the clinical value of normalized bone marrow WT1 levels as a measure of the expanded myeloid progenitor compartment in a consecutive series of 193 adult patients with myeloid malignancies who underwent HCT. Bone marrow WT1 levels before the HCT, at the first bone marrow aspirate after infusion, and in the follow-up samples after HCT were determined by means of real-time PCR using the European LeukemiaNet normalized method. We sought to clarify the prognostic relevance in terms of overall survival (OS), progression-free survival (PFS), and cumulative incidence of relapse (CIR). Based on earlier experience in AML, we selected a threshold of 100 copies, defining 2 groups: patients with 100 copies also included patients who were negative for MRD as assessed by MPFC (19 of 32). During the HCT follow-up, patients with sustained WT1 levels 100 copies (mean, 68 +/- 11 versus 26 +/- 7 days, P < .001; 63 +/- 11 versus 20 +/- 8 days, P < .001; and 20 +/- 8 vs. 71 +/- 8 days, P

BACKGROUND Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL). METHODS This study analyzed via multiplex ligation–dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL. RESULTS The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)–positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively). CONCLUSIONS Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL. Cancer 2015;121:3809–3817. © 2015 American Cancer Society.

In normal B-cells, B-cell antigen receptor (BCR) signaling can be negatively regulated by the low-affinity receptor FcγRIIb (CD32b). To better understand the role of FcγRIIb in chronic lymphocytic leukemia (CLL), we correlated its expression on 155 samples from newly-diagnosed Binet A patients with clinical characteristics and outcome. FcγRIIb expression was similar in normal B-cells and leukemic cells, this being heterogenous among patients and within CLL clones. FcγRIIb expression did not correlate with well known prognostic markers [disease stage, serum beta-2 microglobulin (B2M), IGHV mutational status, expression of ZAP-70 and CD38, and cytogenetics] except for a weak concordance with CD49d Moreover, patients with low FcγRIIb expression (69/155, 44.5%) required therapy earlier than those with high FcγRIIb expression (86/155, 55.5%) (median 151.4 months vs. not reached; p=0.071). These results encourage further investigation on the role of FcγRIIb in CLL biology and prognostic significance in larger series of patients.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous neoplasia with poor outcome, organized as a hierarchy initiated and maintained by a sub-population with differentiation and self-renewal capacities called leukemia stem cells (LSCs). Although currently used chemotherapy is capable of initially reducing the tumor burden producing a complete remission, most patients will ultimately relapse and will succumb to their disease. As such, new therapeutic strategies are needed. AML cells differentially expressed serotonin receptor type 1 (HTR1) compared with healthy blood cells and the most primitive hematopoietic fraction; in fact, HTR1B expression on AML patient samples correlated with clinical outcome. Inhibition of HTR1s activated the apoptosis program, induced differentiation and reduced the clonogenic capacity, while minimal effect was observed on healthy blood cells. In vivo regeneration capacity of primary AML samples was disrupted upon inhibition of HTR1. The self-renewal capacity remaining in AML cells upon in vivo treatment was severely reduced as demonstrated by serial transplantation. Thus, treatment with HTR1 antagonists showed antileukemia effect, especially anti-LSC activity while sparing healthy blood cells. Our results highlight the importance of HTR1 in leukemogenesis and LSC survival and identify this receptor family as a new target for therapy in AML with prognostic value.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

The prevalence, the prognostic effect, and interaction with other molecular markers of DNMT3A mutations was studied in 415 patients with acute myeloid leukemia (AML) younger than 60 years. We show mutations in DNMT3A in 96 of 415 patients with newly diagnosed AML (23.1%). Univariate Cox regression analysis showed that patients with DNMT3Amutant AML show significantly worse overall survival (OS; P = .022; hazard ratio [HR], 1.38; 95% confidence interval [CI], 1.04-1.81), and relapse-free survival (RFS; P = .005; HR, 1.52; 95% CI, 1.13-2.05) than DNMT3Awild-type AMLs. In a multivariable analysis, DNMT3A mutations express independent unfavorable prognostic value for OS (P = .003; HR, 1.82; 95% CI, 1.2-2.7) and RFS (P < .001; HR, 2.2; 95% CI, 1.4-3.3). In a composite genotypic subset of cytogenetic intermediate-risk AML without FLT3-ITD and NPM1 mutations, this association is particularly evident (OS: P = .013; HR, 2.09; 95% CI, 1.16-3.77; RFS: P = .001; HR, 2.65; 95% CI, 1.48-4.89). The effect of DNMT3A mutations in human AML remains elusive, because DNMT3Amutant AMLs did not express a methylation or gene expression signature that discriminates them from patients with DNMT3Awild-type AML. We conclude that DNMT3A mutation status is an important factor to consider for risk stratification of patients with AML.

Introduction The association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio ( We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications. Methods and patients A total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1. Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used. Results Out of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234) When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A). In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B). Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012). Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B). Conclusions In this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. Disclosures No relevant conflicts of interest to declare.

Introduction Patients diagnosed with AML with NPM1mutation (NPM1mut AML) included in the European LeukemiaNet favorable genetic risk category (ELNfav, i.e., without FLT3-ITD or with a low allelic burden FLT3-ITD comutation [FLT3-ITD/FLT3wt Methods All patients diagnosed with ELNfav NPM1mut AML treated according to CETLAM-12 protocol who achieved CR after 1 or 2 courses of induction chemotherapy were included in the study. Intended post-CR therapy consisted of 3 courses of high-dose cytarabine chemotherapy (HDAC). MRD was assessed in bone marrow samples after each chemotherapy course and thereafter, at regular 3-month interval during 3 years, in reference labs from CETLAM group following standard NPM1-mutation specific RQ-PCR. MF was defined as failure to achieve a molecular response (molCR) after consolidation therapy (i.e., NPM1mut/ABL·100 ratio > 0.05) or MRD reappearance after molCR. All MFs were confirmed in a second sample collected at least 4 weeks apart from previous sample. For patients who present a MF, alloSCT was recommended, without a predefined indication of debulking previous salvage chemotherapy. Results Out of 145 patients with ELNfav NPM1mut AML (75 M/70 F; median age, 56, 18-74), 132 (91%) achieved CR after 1 (n=124) or 2 courses (n=8). After a median follow-up of 25 months, 2-year overall survival (OS), event-free survival (EFS), leukemia-free survival (LFS) of the entire cohort were 79.9% (±3.6), 71.8% (±4) and 79.4% (±3.8), respectively. Among patients with available complete MRD information (n=89), 33 patients developed an hematological and/or molecular failure (Mol/Hem failure), resulting into a Mol/Hem failure-free survival at 2 years of 62% (±5.6%); median time from CR to Mol/Hem failures was 8.2 months (2-43). Fourteen patients presented with an overt hematological relapse (hREL), preceded by a detectable MF in 8. Overall, 14 received salvage therapy in morphological CR, MRD(+) status (MF), and 14 For hREL. Among MF, 5 patients received HDAC-type salvage treatment (followed by alloSCT in 3) and 9 proceeded directly to alloSCT. In 5 additional MF patients with MRD persistence after consolidation, subsequent MRD monitoring showed decreasing MRD levels until complete clearance (n=4) or intermitent detection (n=1) and have not received further therapy. After salvage therapy, 12/14 (86%) patients with MF achieved molCR, and 10/14 (71%) treated for hREL achieved CR2 (MRD negative in 7, 50%), followed by alloSCT in 9. OS after Mol/Hem failure was 64.8% (± 8.4) at 2 years, 88.8 (±7.5%) in patients treated for MRD(+)-status (MF) and 32.1% (±13.6%) in patients treated with overt hREL (p Potential predictive factors of Mol/Hem failure were investigated. Interestingly, a ratio of NPM1mut/ABL*100 ≥1 after induction allowed distinction of two patient subpopulation with strikingly different Mol/Hem failure risk: Mol/Hem failure-free survival at 2 years of 84±6% vs. 33±10% in patients with a lower ( Conclusion Despite a significant proportion of Mol/Hem failures, NPM1mut AML patients allocated in the ELN favorable risk group presented a favorable overall outcome. NPM1mut-based MRD surveillance is able to anticipate most hematological relapses, and a MRD-driven early intervention policy, at time of MF, allowed a successful rescue of a significant proportion of patients. Moreover, an early measure of residual tumor burden, after induction therapy, might identify those patients with a high risk of molecular or hematological subsequent failure, allowing the potential implementation of preemptive intensification strategies. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (>= 60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P = 0.0025), shorter leukemia-free survival (P = 0.026) and higher cumulative incidence of relapse (P = 0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P = 0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML., Marina Diaz-Beya is supported by ISCIII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia y Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (PI: JE), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding), and grants AGAUR 2014SGR-1281, ISCIII RD12/0036/0071 and PI014/00450 (JS). Anna Cordeiro is an APIF fellow of the University of Barcelona.

Introduction In the last years genome wide profilings have identified recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of Acute Lymphoblastic Leukemia (ALL). These studies have identified deletions in B-cell development genes (IKZF1, EBF1, PAX5, TCF3, etc.), cell cycle regulation genes (CDKN2A/B, RB1, TP53, etc.), glucocorticoid resistance genes (BTG1, CREBBP) and growth factor receptors genes (CRLF2, CSF2RA, IL3RA) among others. Some of these CNA (i.e. IKZF1, CDKN2A, CRLF2) have been reported to have prognostic significance in several pediatric series but there are very few data regarding their impact in B-lineage adult ALL. Our aim was to analyze the frequency and prognostic significance of CNA in a series of 125 B-lineage adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods Bone marrow or peripheral blood (with significant blast burden) samples from 125 B-lineage adult ALL patients enrolled in risk-adapted protocols from the PETHEMA Group were analyzed at diagnosis. MLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284). Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Results The median age [range] was 40 [15-74] years, 71 (57%) males, median WBC count 12.11 x109/L [0.4-388]. Immunophenotype: pro-B 14 (11%), common 71 (58%), pre-B 26 (21%), mature-B 10 (8%), unavailable 2 (2%). Cytogenetics: normal 16 (13%), hyperdiploid 6 (5%), hypodiploid 2 (2%), t(9:22) 20 (16%), t(1;19) 8 (6%), 11q23/MLL 11 (9%), 8q24/C-MYC 7 (5%), complex 1 (1%), iAMP21 2 (2%), other translocations or deletions 31 (25%), no growth 20 (16%). CNA frequencies of the 125 patients are shown in the table. IKZF1 deletions were significantly associated with EBF1 deletions, high WBC count and Philadelphia (Ph) chromosome. In the IKZF1 deleted cohort whole gene deletions were as frequent as Ik6 isoforms (28% each). A high codeletion rate was detected in genes located in 9p (CDKN2A/B with PAX5, CDKN2A/B with hsa-miR-31 and PAX5 with hsa-miR-31). CDKN2A/B also showed concomitant deletions with ETV6 while PAX5 showed codeletions with BTG1. CDKN2A/B and PAX5 deleted patients had higher WBC counts than non-deleted individuals. Clinical follow-up data was available for 123 patients of the whole series and for the 105 patients of the Ph-negative cohort. Multivariate analysis showed that advanced age, BTG1 deletions and EBF1 deletions were negative prognostic factors for achieving Complete Remission (CR) and WBC count and IKZF1 deletions significantly reduced CR duration in both cohorts. Interestingly, there were significant differences in relapse rates between whole and partial gene IKZF1 deletions. IKZF1 haploinsufficient patients had a probability of CR duration at 3 years of 83% ± 30% vs. 6% ± 12% of partial gene deletion carriers. Advanced age and IKZF1 deletions were predictors for overall survival in the Ph-negative cohort and age>30 years, IKZF1 deletions and hsa-miR-31 deletions were associated with poor prognosis in the whole series. Conclusions In B-lineage adult ALL, deletions of IKZF1, EBF1, BTG1 or hsa-miR-31 are markers with prognostic significance in addition to age and WBC count. Patients with partial IKZF1 gene deletions have a significantly higher probability of relapse than those with whole gene loss. These genetic abnormalities could help to better define prognostic subgroups in adult patients with B-lineage ALL. Supported by the grants PI10/01417 and RD12-0036-0029 from Instituto Carlos III and a grant from the Spanish Society of Hematology and Hemotherapy (2012). Disclosures: No relevant conflicts of interest to declare.

Risk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (= 0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio. (Blood. 2013;121(14):2734-2738)

Background & Objective: Acute Lymphoblastic Leukemia (ALL) is an aggressive neoplasia characterized by a high genetic heterogeneity both at diagnosis and at relapse. Due to the high incidence of relapse in adults and the dismal prognosis beyond recurrence, diagnosis and relapse samples of adult ALL patients were carefully analyzed in order to identify genetic alterations related with drug resistance and disease progression. Patients & Methods: Paired diagnosis-relapse bone marrow samples from 5 adult B-cell precursor ALL (B-ALL) patients were analyzed (Ph+ ALL [n=2], normal karyotype [n=1], t(1;19)(q23;p13) [n=1] and t(8;13)(p21-22;q12) [n=1]). Copy Number Alterations (CNA) were studied with Multiplex Ligation-dependent Probe Amplification (MLPA, kits P-335 and P-202 from MRC-Holland, Amsterdam, Netherlands) and Affymetrix CytoScan HD arrays (Affymetrix, Santa Clara, USA). In the array analyses, only the CNA that encompassed at least 25 markers were considered significant. Results: Regarding karyotype, 2 patients (1 Ph+ and 1 t(1;19) at diagnosis) showed the same chromosomal translocations within a complex karyotype at relapse. On the contrary, the other Ph+ patient showed a normal karyotype at relapse, while 2 patients did not experience any karyotypic change. Regarding immunophenotype, 3/5 patients showed changes on antigen expression from diagnosis to relapse such as expression of markers of immaturity (CD34, TdT positivity and CD38 negativity), loss of lymphoid markers (CD20 and CD22) and/or acquisition of myeloid markers (CD33 and CD66c). Concerning CNA, all relapse samples were genetically related to the diagnosis clone (common clonal origin). All relapsed populations lost CNA detected at diagnosis and/or acquired new CNA but retained some of the CNA showed at diagnosis revealing clonal evolution from ancestral clones. CNA in B-ALL key genes involved in lymphoid development (IKZF1, PAX5, EBF1,VPREB1 and BLNK), proliferation (CDKN2A/B, RB1, CRLF2, C-MYC and ERG), apoptosis (BTG1, TP53 and ATM), hematopoiesis transcription factors (ETV6 and MLL) and histone modifications (KDM6A) were detected, among others. Losses in 9p were the most recurrent event both at diagnosis and at relapse. CDKN2A/B deletions were observed in all relapse samples (3/5 in homozygosis) while PAX5 deletions were present in 4/5 relapsed cases. Interestingly, all relapse samples showed CNA favoring the activation and/or the transcription of proteins involved in the Akt/C-MYC signaling pathway. Another common feature (4/5 patients) were CNA affecting genes involved in drug transport such as several ABC transporter genes and genes related to drug resistance such as PRKDC and RUNX1T1 (in 3/4 of the cases, the CNA appeared exclusively at relapse or were already present at diagnosis and increased their frequency at relapse). CNA in genes that may confer stem cell characteristics (EGR1 and USP16) were another recurrent event at relapse (3/5 samples, 2 of them were not present at diagnosis). CNA affecting the X/Y PAR1 region (CRLF2, CSF2RA and IL3RA) or VPREB1 at 22q11.22 were detected in 3/5 relapse samples, respectively. An important apoptosis cluster at 11q21q24.2 (BIRC2/3, CASP1/4/5/12, hsa-miR-34b/c, ATM and BTG4) was lost in 2/5 relapse samples (one of them was not detected at diagnosis and the other increased its frequency at relapse). Finally, ETV6 deletion (12p13.2) and duplication of Xq26.2q28 (containing ABCD1, BCAP31 and genes coding for several cancer/testis antigens) were observed in 2 relapse samples. Conclusions: SNP arrays analysis of paired B-cell precursor ALL samples at diagnosis and at relapse allows the identification of genetic alterations potentially related with ALL progression. The systematic analysis of relapse samples could contribute to the identification of specific genetic targets with potential therapeutic impact for each patient (personalized medicine). Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Sole:Celgene: Membership on an entity's Board of Directors or advisory committees.

Significant progress in the understanding of the genetic basis of acute myeloid leukemia (AML) has been made during the last 30 years. The aim of the present study was to assess whether the detection of recurrent gene rearrangements by fluorescent in situ hybridization (FISH) studies and NPM1 and FLT3 gene mutations by molecular studies added clinically relevant information to the karyotype in 113 AML patients. Thus, FISH and molecular studies were found to add new information in 22 and 55% of the patients, respectively, particularly in cases with normal karyotype (NK) or when a cytogenetic analysis failed. Patients with NK changed their genetic risk group to favorable in 27 and 29% of cases using FISH and molecular biology studies, respectively. Our results demonstrate that molecular biology and FISH studies provide relevant information in AML and should be routinely performed.

Translocations involving nucleoporin 98kD (NUP98) on chromosome 11p15 occur at relatively low frequency in acute myeloid leukemia (AML) but can be missed with routine karyotyping. In this study, high-resolution genome-wide copy number analyses revealed cryptic NUP98/NSD1 translocations in 3 of 92 cytogenetically normal (CN)–AML cases. To determine their exact frequency, we screened > 1000 well-characterized pediatric and adult AML cases using a NUP98/NSD1-specific RT-PCR. Twenty-three cases harbored the NUP98/NSD1 fusion, representing 16.1% of pediatric and 2.3% of adult CN-AML patients. NUP98/NSD1-positive AML cases had significantly higher white blood cell counts (median, 147 × 109/L), more frequent FAB-M4/M5 morphology (in 63%), and more CN-AML (in 78%), FLT3/internal tandem duplication (in 91%) and WT1 mutations (in 45%) than NUP98/NSD1-negative cases. NUP98/NSD1 was mutually exclusive with all recurrent type-II aberrations. Importantly, NUP98/NSD1 was an independent predictor for poor prognosis; 4-year event-free survival was < 10% for both pediatric and adult NUP98/NSD1-positive AML patients. NUP98/NSD1-positive AML showed a characteristic HOX-gene expression pattern, distinct from, for example, MLL-rearranged AML, and the fusion protein was aberrantly localized in nuclear aggregates, providing insight into the leukemogenic pathways of these AMLs. Taken together, NUP98/NSD1 identifies a previously unrecognized group of young AML patients, with distinct characteristics and dismal prognosis, for whom new treatment strategies are urgently needed.

Background Cytogenetics at diagnosis is the most important prognostic factor in acute myeloid leukemia (AML). Of note, intermediate cytogenetic risk group (IR-AML) is a very heterogeneous subset including normal karyotypes and all the cytogenetic abnormalities not included in the favorable or the adverse groups. Molecular alterations affecting NPM1, FLT3 and CEBPA show a prognostic impact in IR-AML. MLL partial tandem duplications (MLL-PTD) have also been described in this group of AML, but their prognostic impact have not been well established. Aim To analyze the prognostic relevance of MLL-PTD in the subset of patients diagnosed with IR-AML since 2003, and included in the CETLAM protocols LMA-2003 and LMA-2012. Methods Between 2003 and 2004 MLL-PTD were analyzed by Southern Blot (enzymes employed BglII, EcoRI, BamHI). Since 2004, a long PCR strategy was used to identify this abnormality. Results NPM1 mutations (NPM1mut), FLT3 internal tandem duplications (FLT3-ITD) and MLL-PTD were available for 893 patients. No MLL-PTD was found among 111 and 161 patients of the good and poor cytogenetic risk groups, respectively. The IR-AML group included 621 patients, and 37 carried a MLL-PTD (6%), thus only this cytogenetic group of patients was analyzed. NPM1mut were found in a 41% of patients and none of them had a concomitant MLL-PTD (p Conclusions MLL-PTD is a genetic alteration found in a 6% of IR-AML. Patients with this abnormality have a worse LFS and OS than the rest of patients of the IR-AML group. Based on these results, patients with MLL-PTD should be considered as patients with poor cytogenetic risk AML for treatment allocation. Disclosures No relevant conflicts of interest to declare.

Introduction: Allogeneic stem cell transplant (alloHSCT) is the treatment of choice in patients with high-risk acute myeloid leukemia (AML). However, it is uncertain exactly how many of these patients actually undergo alloHSCT as planned initially. Moreover, the real efficacy of this therapeutic option remains controversial. Since few studies have addressed these questions, to shed light on these issues, we have analyzed the results of a policy of early donor search, the proportion of patients in which initially planned alloHSCT was performed, and the outcome of alloHSCT in our center. Patients and Methods: We included in the study 246 AML patients (median age, 51 years, 16-70; 51% males) considered fit for intensive chemotherapy, who were treated according to 3 consecutive protocols of the Spanish CETLAM cooperative group (CETLAM-99, CETLAM-2003, and CETLAM-2012). Patients were diagnosed between 2003 and 2013 in our center or referred to our center for alloHSCT; median follow-up of alive patients was 70.4 months (12.5-143.8). Indication of allloHSCT in CR1 was established according to the stratification risk of each protocol, based on AML origin (de novo vs. secondary), cytogenetics, number of courses to achieve CR, NPM1/FLT3-ITD/CEBPA mutational profile, and MRD status at end of consolidation. Statistical analyses were performed using R v3.1 and SPSS v20. The effect of alloHSCT was analyzed by Mantel-Byar test with SCT as a time-dependent covariable. Results: AlloSCT was planned in 167 (68%) patients deemed of high-risk and it was actually performed in 130 out of these high-risk AML patients (78%). Types of donor were: matched related donor (MRD), n=63; 7/8 or 8/8 matched unrelated donor (MUD), n=51 (HLA matching: 8/8 or 10/10= 32; 7/8 and 9/10, n=19); unrelated cord blood (UCB) n=14; and family haploidentical donor, n= 2. Status at SCT was first CR (CR1) in 63 patients (48%), ≥CR2 n=23 (18%), and non-CR AML in 44 patients (34%). The type of conditioning regimen was myeloablative in 66 (51%) of the patients. Ninety-eight patients out of 167 patients with an indication for alloHSCT, lacked a MRD (59%). An unrelated donor search was performed in 80 (82%) out of these 98 patients, and was successful in 54 (67.5%). An UCB unit was selected in 14 patients without a MUD in a timely fashion. The main reasons for not initiating an unrelated donor search were poor performance status (n=5), refractory disease (n=4), and age (n=4). Median time from start of search to finding of an adequate donor was 50 days, and median time from start of search to SCT was 131 days. The overall outcome of these 167 patients with an indication of alloHSCT was 2-year OS: 48±8% and 5-year LFS: 36%±8% with a markedly better outcome among patients in whom alloHSCT was finally performed (2-year OS: 57±8% vs. 11%±10%; Mantel Byar, p Conclusions: A policy of early search of MUD enabled alloHSCT to be performed in the majority of patients for whom it was planned. Performing of alloHSCT in patients improved outcome of this cohort of high-risk AML patients, compared to patients not achieving alloHSCT. An adequate MUD was identified in a two thirds of patients, and a similar outcome was found after alloHSCT for all donor sources (MRD, MUD, and alternative source) in this study. Disclosures Rosiñol: Celgene, Janssen: Honoraria.

Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet. Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes. Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters. Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure). Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p Conclusions: LncRNAs expression profile seems specific of several AML subtypes, including t(8;16) AML. Some of the lncRNAs of this distinctive signature in t(8;16) AML are located in the HOXA genomic region, and correlate with several of the characteristic microRNAs previously described in this entity. Interestingly, we have predicted in silico GATA2, which interacts with CREBBP, as the most significant TF that could potentially regulate this lncRNAs signature. Nonetheless, further investigation is warranted to determine the mechanisms leading to this lncRNA signature and to identify the specific targets of these lncRNAs. Río Hortega CM13/00205, FIS PI13/00999 Disclosures No relevant conflicts of interest to declare.

Introduction Younger patients with acute myeloid leukemia and intermediate cytogenetic risk (AML-IR) harboring NPM1 mutation (NPM1mut) without FLT3 internal tandem duplication (FLT3-ITD) have a relatively favorable prognosis, and are not deemed as candidates to receive an allogeneic hematopoietic stem cell transplantation in first complete remission (CR1). However, it remains uncertain if this favorable prognosis is also maintained in older patients within the same molecular features with some conflicting results. Patients and methods We analyzed the cohort of patients ≥60 year-old considered fit for intensive chemotherapy and included in the CETLAM protocols LMA-2003 and LMA-2012 for patients up to 70, consisting of a standard induction chemotherapy, HiDAC post-remission therapy, followed by alloHSCT in selected patients with high-risk features. Overall we identified 192 patients between 60 and 71 year-old diagnosed with AML-IR with known NPM1 and FLT3 mutational status. Results We identified 192 AML-IR patients (93♀, 99♂) aged >=60 (median age was 65 years old (range: 60-71)), with a median WBC count 10.6 x 109/L (range: 0.23-400 x 109/L), median bone marrow blasts 65% (range: 20-100%). Overall, CR rate was 78%, five-year overall survival (OS) was 30±4%, and leukemia-free survival (LFS) was 31±4%. Patients were classified in three molecular groups depending on NPM1mut and FLT3-ITD: 40 patients harbored NPM1mut/FLT3 without ITD (FAV group), 98 patients had NPM1wt/FLT3wt, and 54 had a FLT3-ITD. Five-year OS of these 3 groups were: 64±9%, 25±6%, and 19±6%, respectively (p Multivariate analysis for OS, including WBC count, bone marrow blasts and molecular group (NPM1mut/FLT3wt vs. the other groups) identified WBC and molecular subgroup showed significance for the WBC count (HR=1.003, 95% CI: 1-1.006) and the molecular group (FAV vs. UNFAV, HR=0.345, 95% CI: 0.192-0.621). Interestingly, when we compared the outcome of the FAV group with a cohort of younger patients (up to 60, n=99) with the same molecular features included in these 2 protocols, the outcome did not differ depending on age (5-year OS 69±5% for younger patients, and 64±9% for older patients, p=0.463, and 5-yr LFS 67±5% for younger patients, and 55±12% for older patients, p=0.567). Moreover, the outcome of a small cohort of FAV patients older than 65 years (n=16) included in these protocols was relatively favourable, with a 5-yr OS and LFS not differing substantially from that of younger patients allocated in the same molecular subgroup. Conclusions The favorable impact of NPM1mut/FLT3-ITDneg genotype was maintained in a cohort of patients >60 who received intensive chemotherapy, with a high proportion of long-standing responses after HiDAC-based post-remission therapy. Disclosures No relevant conflicts of interest to declare.

Background: Non-coding RNAs (ncRNAs) have recently emerged as key regulators of diverse cellular processes, including leukemia. ncRNAs are classified according to their size as short (eg, microRNAs) or long ncRNAs. lincRNAs are long ncRNAs located in intergenic regions and have multiple regulatory functions, including gene expression regulation. Interestingly, active crosstalk between microRNAs and lincRNAs has been observed. lincRNAs are known to be deregulated in some cancers but their importance in acute myeloid leukemia (AML) is so far unknown. HOX genes play an important role in hematopoiesis and are deregulated in AML. lincRNAs are especially abundant in the clusters of HOX genes. HOTAIRM1, a myeloid lineage-specific lincRNA, is located at the 3’end of the HOXA cluster and seems to play a regulatory role in myelopoiesis. However, to date the potential prognostic role of HOTAIRM1 expression in AML has not been examined. Aims: To investigate first whether the expression of the lincRNA HOTAIRM1 is associated with the clinical, cytogenetic and molecular characteristics and microRNA expression in AML patients. Secondly, since intermediate risk (IR) AML patients have a highly diverse prognosis, we analyzed the potential prognostic value of HOTAIRM1 expression in IR-AML patients. Methods: To explore the expression level of HOTAIRM1 among different AML subtypes, we analyzed samples from 244 AML patients including CBF-rearranged AML (n=5), APL (n=4), MLL-rearranged AML (n=3), EVI1-rearranged AML (n=3), t(6;9) AML (n=9), AML with monosomal karyotype (n=3), and a large cohort of IR-AML (described below). For the analysis of prognostic value of HOTAIRM1, we analyzed specifically the outcome of 217 IR-AML patients (median age, 52; 51% males) sequentially included in CETLAM trials during the period 1995-2009. Molecular genotyping of this group identified NPM1 mutation (NPM1mut), FLT3-ITD, and biallelic CEBPA mutation (CEBPA mut) in 99 (45%), 79 (36%) and 17 (11%), respectively. The expression of HOTAIRM1 was analyzed using TaqMan® Gene Expression Assays (Applied Biosystems). microRNA and mRNA expression data were obtained in previous studies (Díaz-Beyá, Leukemia 2013). Statistical analyses were performed with BRB Array Tools, SPSS v20 and R v3.0. MaxStat package from R software was used to determine the optimal cutoff point of HOTAIRM1 expression. Results: Among all 244 patients, HOTAIRM1 expression was significantly different among the 7 included genetic subgroups (ANOVA p=0.0024), with the lowest levels observed in APL-AML patients and the highest in the t(6;9)AML patients. Within the IR-AML group, HOTAIRM1 overexpression was observed in NPM1mut patients (p The prognostic study showed that high HOTAIRM1 expression was associated with shorter 5-year overall survival (OS) (27+11% vs.47+8%; p=0.009) shorter 5-year disease-free survival (LFS) (22+12% vs. 53+9%; p Supervised analysis by means of t-test based on multiple permutations revealed a distinctive 33-microRNA signature which correlated with HOTAIRM1 expression including miR-196b (p Conclusion: The expression level of the lincRNA HOTAIRM1 varied among different molecularly-defined AML. Interestingly, HOTAIRM1 expression level showed independent prognostic value within the IR-AML group. Moreover, HOTAIRM1 expression strongly correlates with its neighboring HOXA4 gene and harbors a distinctive microRNA signature. Our findings can pave the way for further studies of HOX-related lincRNAs and microRNAs regulatory networks and their influence on clinical outcome. Acknowledgments: ISCIII RH13/00205, SEHH, FIS13/00999 Disclosures No relevant conflicts of interest to declare.

Introduction Recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of ALL have been identified in genes involved in B-cell development, cell cycle regulation, proliferation, apoptosis and drug resistance. Their independent prognostic significance in adult ALL patients is controversial. The aim of this study was to analyze the prognostic significance of CNA in a series of 96 high-risk, Ph-negative, B-precursor adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods MLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284) . Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Univariable and multivariable analyses including the most relevant clinical parameters (age, WBC count, phenotype, cytogenetics, CNS involvement) were performed for CR attainment, CR duration and OS. Results The median age [range] of the 96 patients was 39 [15-72] years, 50 (52%) patients were males, with a median WBC count 14.3 x109/L [0.4-388]. Phenotype: early pre-B 19 (20%), common 51 (54%), pre-B 22 (24%), unknown 2 (2%). Cytogenetics: normal 18 (19%), hyperdiploid 5 (5%), hypodiploid 2 (2%), near haploid 6 (6%), t(1;19) 7 (8%), 11q23/ MLL 11 (12%), complex 1 (1%), other 27 (29%), no growth 17 (18%). The most frequent CNA deletions involved CDKN2A /B (43/96, 45%), PAX5 (34/94, 36%), IKZF1 (32/95, 34%), hsa-miR-31 (25/96, 26%), 14q32.33 region (18/96, 19%), RB1 (17/96, 18%), EBF1 (12/91, 13%) and X/Y PAR (10/96, 10%). The most frequent duplications involved X/Y PAR (11/96, 12%) and 14q32.33 region (7/96, 7%). The CR rate was 83% (80/96), the median (95%CI) of CR duration was 2.7 years (0-5.9) and the median (95%CI) of OS was 2.1 (1.0-3.2), being the median (range) follow-up of the series of 3.8 (0.6-8.0) years. Table 1 shows the results of univariable and multivariable analyses. By multivariable analyses advanced age and EBF1 deletions were significantly associated with less CR rate, WBC count and X/Y PAR duplication were associated with shorter CR duration, and advanced age and CDKN2A /B deletion were associated with shorter OS. Conclusions The CNA of EBF1 , X/Y PAR1 genes and CDKN2A/B have independent prognostic significance in adult patients with high-risk, Ph-negative, B-precursor ALL. This study suggests that these genetic studies should be added to the initial work-up of these patients for more accurate prognostic assessment Supported by grants PI10/01417, RD12-0036-0029 from Instituto Carlos III, 2014 SGR225 (GRE) from Generalitat de Catalunya and a grant from the Spanish Society of Hematology and Hemotherapy (2012). | Variable | CR rate | CR duration | OS | | ---------- | -------- | ------------------ | -------- | ------------------- | | | P (univ) | OR (95%CI) | P (univ) | HR (95%CI) | P (univ) | HR (95%CI) | | Age | 0.011 | 0.93 (0.89 - 0.98) | NS | - | 0.005 | 1.03 (1.01 - 1.05) | | WBC | NS | -

Prognosis of acute myeloid leukemia (AML) is mainly determined by presenting cyto- and molecular genetics, as systematized in the recent European LeukemiaNet (ELN) risk classification. Moreover, mutational profiling is currently used to assign post-remission therapy, especially in the category of intermediate-risk cytogenetics (IR-AML), given the observed benefit of allogeneic hematopoietic stem-cell transplantation (alloHSCT) in first complete response in patients harboring high-risk molecular features such as FLT3-ITD and the lack of benefit in patients with a favorable genotype (i.e., mutated NPM1 and CEBPA biallelic mutation). Nonetheless, the prognosis for patients with an IR-AML lacking mutations of these three genes (“triple negative” AML) is uncertain, and optimal post-remission strategy for these bona fide intermediate-risk patients is mostly unknown. In order to elucidate if alloHSCT could improve the prognosis of this molecular subgroup, we analyzed the outcome in a large series of patients with “de novo”, IR-AML (70% harboring a normal karyotype), diagnosed between 2000 and 2013, lacking mutations of NPM1, FLT3-ITD, and CEBPA (only patients with biallelic mutations were excluded), who had achieved a first complete remission after 1 or 2 courses of standard induction therapy. These 630 patients (median age, 52 years, range, 18-72; 58% male) have been included in the database of three different cohorts, the cooperative AML groups AMLSG (n=239) and Spanish CETLAM (n=146), and the HSCT registry of the ALWP-EBMT (n=245). The prognostic impact of alloHSCT in CR1 was analyzed as a time-dependent variable in univariable (Mantel-Byar method, Simon-Makuch plots) and multivariable (Cox with time dependent-variable) analyses. After a median follow-up of 37 months, 3-year survival (OS), leukemia free-survival (LFS) and relapse incidence (RI) in the pooled two subgroups of AMLSG and CETLAM cooperative groups, regardless of post-remission therapy, was 53% [95% CI: 47-58], 36% [95% CI: 31-41], and 51% [95% CI=45-56], respectively (57±3%, 37.5±3%, and 44±4% in patients up to 60 year-old, respectively), without significant differences between both cohorts. In order to investigate the potential benefit of alloHSCT, we analyzed the entire group, including also patients from the ALWP-EBMT cohort. Remarkably, the outcome of patients from national cooperative groups AMLSG-CETLAM compared to registry ALWP-EBMT who received an alloHSCT in CR1 was not statistically different (3-year OS, LFS, and RI: 57 vs. 68%, p=0.13; 61 vs. 73%, p=0.08; and 22 vs. 19%, p=0.67). Patients who received an alloHSCT in CR1 (n=396) had an improved outcome compared to other post-remission options (high-dose cytarabine based chemotherapy, n=189; autoHSCT, n=45) with a higher OS (3-yr: 65 vs. 49%, p=7x10-5) and LFS (3-yr: 60 vs. 26%, p In conclusion, IR-AML patients with a triple negative genotype constitute a subgroup with a high risk of relapse after postremission therapy with high-dose cytarabine based chemotherapy or autoHSCT. This large study, involving patients from two cooperative groups and the EBMT registry, indicates that an alloHSCT in first CR is associated with a marked relapse reduction and survival benefit in the two cooperative group cohorts with prospective treatment data as well as in the whole cohort including the EBMT registry data. Disclosures No relevant conflicts of interest to declare.

Background: The invention of Next Generation Sequencing (NGS) has spurred research into human diseases, especially in the field of malignancy. In acute myeloid leukemia (AML), a plethora of novel alterations have been identified, including mutations in epigenetic regulator genes (e.g. IDH1, IDH2, DNMT3A), genes coding for proteins of the cohesin complex (e.g. SMC1A, SMC3, STAG2) and spliceosome genes (e.g. SF3B1, U2AF1). Although the diagnostic and prognostic implications of many of these alterations are not yet clear, there is increasing evidence that several of them might have major implications for understanding the disease biology or for patient-treatment. Thus, there is increasing need to reliably detect these mutations in large patient groups in clinically relevant time-frames and at an affordable cost. Due to the large number of genes to be screened, amplicon-based NGS represents an attractive detection method. Although, several assays have been reported, integrating different numbers of genes, it is currently unclear whether they really allow reliable detection of alterations in a reproducible way. Here we report our results from a round robin comparison of the detection of known AML-variants using a highly multiplexed, single tube assay coamplifying a total of 568 amplicons covering 54 entire genes or hot spot gene regions involved in leukemia (TruSight Myeloid sequencing panel; Illumina), with respect to the sensitivity, reproducibility and quantitative accuracy. Material and Methods: Ten European laboratories routinely involved in molecular AML diagnostics participated in this study. All groups performed two sequencing runs, each containing 8 samples. These samples were centrally aliquoted and distributed, the analyses were done in a blinded fashion. Six out of the 8 samples on each run were derived from a set of 9 samples composed of DNA isolated from the blasts of 18 different newly diagnosed AML patients mixed at a 1:1 ratio, with 50 ng of DNA being used for the library preparation. Three of these 9 samples were analyzed in replicate in separate runs by each group. The remaining two samples were a commercial test DNA containing 10 known single nucleotide variants (SNV) or insertion/deletion (InDel) alterations with defined variant allele frequencies (VAF) between 4 and 25% and DNA derived from the OCI-AML3 AML cell line (mutant for DNMT3A and NPM1). Sequencing was performed on MiSeq NGS systems (Illumina) using 2x151 bp-runs. Sequencing data were analyzed by all laboratories using the VariantStudio software (Illumina), with the threshold for mutation calling set at 3%. Results: Analysis of data quality indicated that 85% of the samples met the predefined acceptance criteria (>=95% amplicons with at least 500 reads/amplicon), the median coverage was 7379 reads/amplicon (range 0-47403 reads). Of the 9 mutations present in the positive control, 7 were called at least once in the two replicates by all labs, two mutations with a VAF of 5% were missed by 1 and 4 participants, respectively. Overall, the VAF calls for this sample showed a high level of accuracy across the participants (median coefficient of variation 5%, range 0-22.5%) as well as excellent intra- and inter-laboratory reproducibility (Fig.1). In total, the 9 primary leukemic samples contained 43 known variants in 19 genes, including all commonly mutated genes in AML, i.e. CEBPA, DNMT3A,RUNX1, NPM1, FLT3, WT1. For these samples, the sensitivity was 95.7%. Based on the entire data set (positive control and leukemic samples), the calculated sensitivity of the assay for known variants with an expected VAF>=5% was 93.3%. The rate of non-calls was slightly higher for InDels (14/179; 7.8%) than for SNVs (25/407; 6.1%; P=.47). Two 57-bp long insertions in FLT3 exons 14/15 were not called, which is expected due to the specifications of the assay (max. detectable InDel length Conclusions: This inter-laboratory comparison shows a high sensitivity and impressive quantitative accuracy of NGS-based characterization for known variants down to a minor VAF of 5%. Although additional optimization in individual parameters might be necessary, these initial results clearly indicate that rapid comprehensive molecular characterization of patient samples appears feasible, even in the clinical setting. Figure 1 Figure 1. Disclosures Thiede: AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other. Bullinger:Illumina: Research Support Other. Hernández-Rivas:Illumina: Research Support Other. Heuser:Illumina: Research Support Other. Preudhomme:Illumina: Research Support Other. Lo Coco:Illumina: Research Support Other. Martinelli:Illumina: Research Support Other. Schuh:Illumina: Research Support Other. Enjuanes:Illumina: Research Support Other. Lea:Illumina: Research Support Other. Schlesinger:Illumina: Employment.

To the editor: The prognostic significance of multilineage dysplasia (MLD) in acute myeloid leukemia patients with intermediate-risk cytogenetics (IR-AML) presenting as de novo disease is unclear.[1][1]–[4][2] Falini et al have recently analyzed the biologic and prognostic significance of MLD in

Background The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC). Objective The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs. Methods and patients Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria. Results G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL). Conclusions G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients. Disclosures: No relevant conflicts of interest to declare.

Introduction The improvements in genetic characterization of AML allowed the ELN to propose a prognostic classification into 4 categories: Favorable, Intermediate I, Intermediate II and Adverse. Several groups have tested the outcome of AML patients based on these genetic subgroups in registries and databases from prospective trials. Since ALLOSCT recommendations are being established based on the ELN risk categories, we considered of interest to investigate whether these also have an impact on the results of allogeneic transplants. This could be helpful to identify the patients that may benefit the most from ALLOHCT in the first complete remission (CR1). Objectives To investigate whether the ELN genetic groups of AML have any impact on the results of ALLOHCT performed in CR1. To identify the patients who are best candidates for transplantation, based on the ELN categories as well as in other characteristics. Methods Patients were transplanted between 1990 and 2012. In all cases, treatment prior to transplant had consisted of anthracycline based induction and intermediate-dose cytarabine consolidation according to the CETLAM AML-88, AML-94, AML-99 and AML-03 trials. Upper age limit for transplantation was 60 years until 2002 and 70 years thereafter. Most patients above 50 years received either CD34 selected grafts or reduced intensity conditioning. GVHD prophylaxis after transplant was usually based on cyclosporine and prednisone or methotrexate. The main characteristics of patients and transplants were included in the exploratory analysis of variables influencing survival; those with a p- value up to 0.1 were incorporated as covariates to the multivariable model. Results One hundred ninety two patients in CR1 received an ALLOHCT from HLA-identical siblings (n=140), adult unrelated donors (n=26) or umbilical cord blood (n= 26). Age distribution of the patients was as follows: 16-35 years-old (y-o) n=65 (34%), 36-50 y-o n= 55 (29%), 51-60 y-o n= 54 (28%), >60 y-o n=18 (9%). Twenty-three patients (12%) were classified as in the favorable ELN category, 54 (28%) in the intermediate I, 41 (21%) in the intermediate II and 74 (39%) in the adverse. One-hundred fifty-seven patients (82%) had achieved CR1 after a single course of chemotherapy. Conditioning was myeloablative (MA) in 139 (72%) patients and reduced-intensity (RIC) in 53 (28%). In 76 cases of the MA regimens total body irradiation was included. Thirty-eight patients (20%) died due to transplant complications other than relapse and 47 (24%) experienced a leukemia recurrence. Overall survival (OS) and leukemia-free survivals at 8-years were 55±4% and 53±4%, respectively. Multivariate analysis of factors influencing OS included as covariates age at diagnosis of AML, ELN categories, courses to CR1 and conditioning regimen (MA versus RIC), since they had a p-value 60, p=0.01) and courses to CR (1 vs more, p=0.03). Conclusions The ELN genetic categories have impact on OS of AML patients who receive an ALLOHCT in CR1. The results were very good in the favorable category and, most important, in intermediate II patients. Even patients in the adverse category had a substantial probability of long-term survival. This is remarkable, since the outcome of patients with adverse genetic features when treated with CT only is very poor. Disclosures: No relevant conflicts of interest to declare.

Abstract 1375 Introduction: Acute myeloid leukemia (AML) is a highly heterogeneous disease, with diverse genetic and epigenetic variables determining sensitivity to current standard therapy. A disruption of the normal DNA methylation pattern, which can result in altered gene and microRNA (miRNAs) expression, has been observed in different AML subtypes. Hydroxymethylation of 5-methylcytosine (5-mC) has recently been described as an intermediate key step in the process of DNA demethylation. Nonetheless, the correlation of DNA methylation and hydroxymethylation levels with clinical and biological characteristics and clinical outcome in AML is mostly unknown. Aim: To investigate the prognostic impact of overall methylation and hydroxymethylation levels in patients with intermediate-cytogenetic risk AML (IR-AML) and to identify miRNAs correlated with methylation and hydroxymethylation in these patients. Patients and Methods: We have analyzed 86 IR-AML patients (median age, 53 [range, 17–74]; 52% males) who received intensive therapy from 1994 to 2009 in a single institution. The level of overall methylation and hydroxymethylation in total DNA was estimated after determining the percentage of 5-mC and hydroxymethylcytosine (5-hmC), using anti-5-mC and anti-5-hmC monoclonal antibodies (MethylFlash Methylated or Hydroxymethylated DNA Quantification Kit, Epigentek). The expression of 670 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays (Applied Biosystems). The statistical analysis was performed with SPSS version 15.0.1 and R software version 2.9.0. MaxStat package of R was used to determine the optimal cutoffs. Results: The univariate analysis for overall methylation showed that patients with lower levels of methylation (cutoff < percentile 75) had shorter overall survival (OS) than those with higher 5-mC levels (5-year OS: 30±6% vs 52±11%; p=0.03) and a trend for shorter leukemia-free survival (LFS)(p=0.06). Overall methylation levels did not show any correlation with clinical features at diagnosis or with gene mutations, including DNMT3A. Concerning hydroxymethylation, patients with lower 5-hmC levels had a worse prognosis than those with higher 5-hmC levels, with a lower complete response rate (79% vs. 96%; p=0.04), shorter OS (5-yr OS: 21± 6% vs. 55± 6%; p=0.008), and shorter LFS (5-yr LFS: 24±8% vs. 52 ±10%; p=0.03). Interestingly, when analyzed as a continuous variable, 5-hmC levels retained their prognostic value as a marker of response rate (T-test, p=0.007), OS (Cox, p=0.015), and LFS (Cox, p=0.041). Moreover, 5-hmC levels were inversely correlated with FLT3-ITD (p=0.001) and the FLT3-ITD/FLT3wild-type ratio (Pearson correlation:-0.6; p=0.01). The multivariate analyses, including the main clinical and biological variables, identified older age, wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=3.072; 95% CI: 1.096–3.917; p=0.025) as independent prognostic markers of shorter OS and wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=2.002, 95% CI: 1.032–3.881, p=0.040) as independent prognostic markers of shorter LFS. Of note, lower 5-hmC levels retained their value as a marker of worse prognosis in the subgroup of IR-AML patients with unfavorable molecular markers (wild-type NPM1 and CEBPA and/or FLT3-ITD; p= 0.037). Finally, we have identified a 3-miRNA signature (miR-378*, p=0.02; miR-493, p=0.02; and miR-181, p=0.02) associated with global methylation levels, and a 12-miRNA signature associated with hydroxymethylation, including miR-183* (p=0.001), miR-125a-3p (p= 0.01), miR-586 (p=0.02), and miR-513–3p (p=0.02). Conclusions: Hydroxymethylation levels appear as an independent prognostic factor in IR-AML and maintain their prognostic value in the subset of patients with unfavorable molecular markers. Moreover, methylation and hydroxymethylation are associated with specific miRNA profiles. Further studies are warranted to confirm the clinical impact of these findings and to clarify the underlying molecular mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 235 The prognosis of AML patients within the intermediate cytogenetics category is mainly determined by the mutational status of some relevant genes, such as NPM1 mutations (NPMmut), or biallelic CEBPA mutations (CEBPAmut), associated with a favorable outcome, and with the presence of FLT3 internal tandem duplication (FLT3-ITD), which correlates with an adverse prognosis. Nonetheless, additional biological features such as microRNA (miRNA) expression pattern might contribute to refine prognosis and guide therapy in this setting. The aim of the present study is to investigate whether miRNA expression is associated with molecular characteristics and clinical outcome in intermediate-risk AML patients (IR-AML). We have analyzed samples from 85 IR-AML patients (median age, 52 [range, 18–71]; 52% males) who received intensive therapy from 1994 to 2009. Forty-three patients (51%) harbored NPMmut, 37 (44%) harbored FLT3-ITD (including 23 with NPMmut), and 11 (13%) harbored CEBPAmut, including 7 with biallelic mutations. The expression of 670 mature miRNAs was analyzed by multiplex Real Time PCR using TaqMan Human MicroRNA Arrays (Applied Biosystems). All PCR reactions were performed using an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2−DDCt method, using RNU48 as endogenous control. Statistical analysis was performed with BRB Array Tools, SPSS version 15.0.1 and R software version 2.9.0. Supervised analysis by means of t-test based on multiplex permutations (class comparisons analysis, p In this series of patients of intermediate-risk cytogenetic AML, measurement of expression levels of several miRNAs such as miR-409-3p, miR-135a, let-7a* or miR-23a* showed independent prognostic value, and contribute to predict the outcome within specific molecular subgroups. Nonetheless, confirmation of the prognostic impact of these miRNAs and investigation of possible underlying mechanisms account for this effect require future studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2434 Introduction Acute myeloid leukemia (AML) with t(8;16)(p11;p13) and MYST3-CREBBP rearrangement [t(8;16) AML] is an infrequent leukemia subtype with specific clinical and biological characteristics including a specific gene expression profile with overexpression of RET protooncogene. Our group has previously described a specific signature of microRNAs (miRNAs), most of which were downregulated, and found an inverse correlation between RET mRNA expression and the expression of several of these miRNAs. Since MYST3 and CREBBP are chromatin-modifying proteins, we have now investigated the role of epigenetic mechanisms in the downregulation of the miRNAs in our signature and examined RET as a potential target of some of these miRNAs. Methods To analyze the possible epigenetic regulation of microRNA signature of t(8;16) AML, we first selected those miRNA genes with CpG islands in the promoter regions according to UCSC Genome Browser (http://genome.ucsc.edu). Changes in the expression of selected miRNA levels were studied in cryopreserved cells from a t(8;16) AML patient and in the K562 cell line after treatment for 72 hours with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC, decitabine) and histone deacetylase inhibitor trichostatin A (TSA). Expression of selected miRNAs after treatment with 5-aza-dC and TSA was analyzed using TaqMan MicroRNA Assays (Applied Biosystems). In addition, the degree of quantitative methylation in the promoter region of these miRNA genes was measured with Human Cancer miRNA Genes, Signature Panel Methyl-Profiler DNA Methylation PCR Array (Qiagen). For the validation of RET as the target of specific miRNAs, we performed a Renilla/Luciferase assay with a modified expression vector psiCHECK-2 containing the 3'UTR region of RET cloned in the 3'UTR region of Renilla gene. Modulation of RET expression by selected miRNAs was analyzed after transfection of the corresponding pre-miRNAs in the K-562 cell line, and luminescence was read at 24 hours. Results Since methylation is a common mechanism of the regulation of miRNA expression, we performed a bioinformatic analysis which showed that 40% of the miRNAs in our t(8;16) AML signature harbored CpG islands in their promoter gene region, making them susceptible to silencing by methylation. After treatment with 5-AZA-dc, 60% of these miRNAs were re-expressed more than two fold-change. Interestingly, TSA induced a greater re-expression in 75% of these miRNAs. The degree of methylation status was analyzed in 8 of these miRNAs using the DNA methylation PCR Array. CpG hypermethylation was present to a certain degree in three miRNAs (22% in let-7g, 29% in miR-1, and 56% in miR-126) and to a greater extent (99%) in the miR-23b cluster, which comprises miR-23b, miR-24 and miR-27b. Due to the strong inverse correlation of several miRNAs of the t(8;16) AML signature and RET mRNA levels, we then focused on miRNA target validation of RET. The analysis of the 3'UTR region of RET with TargetScan v5.1 revealed the presence of target sites for 9 miRNAs of the signature (miR-15b, miR-195, miR-218, miR-15a, miR-34a, miR-424, miR-128, and miR-27a/b), including 2 putative binding sites for miR-218. The Renilla/Luciferase assay, performed with the modified psicheck2 vector containing the 3'UTR of the RET gene, showed that 6 of these 9 miRNAs (miR-218, miR-128, miR-27b, miR-15a, miR-195, and miR-424) produced a significant downregulation of Renilla translation (41–49%), confirming our hypothesis that RET is a target of some of the miRNAs of our t(8;16) AML signature. Interestingly, 4 of the miRNAs targeting RET (miR-218, miR-27b, miR-15a and miR-424) were re-expressed after treatment with 5-AZA-dc and TSA. Conclusions Our findings provide greater insight into the genetic and epigenetic abnormalities in t(8;16) AML. Here we have shown that our t(8,16) AML-specific miRNA signature could be a functional consequence of impaired epigenetic mechanisms, including hypermethylation and abnormal histone acetylation. In addition, we provide the first validation of RET as a target of several miRNAs in this t(8;16) AML miRNA signature. Disclosures: No relevant conflicts of interest to declare.

Abstract 230 Acute myeloid leukemia (AML) with t(8;16)(p11;p13) and MYST3-CREBBP rearrangement [t(8;16) AML] is an infrequent leukemia subtype with specific clinical and biological characteristics. Although a characteristic gene expression pattern has been identified with t(8;16) AML, to date, the microRNA (miRNA) expression pattern of this subtype has not been described. The main objective of this study was to analyze the expression pattern of mature miRNAs in patients with t(8;16) AML and compare it with other well defined AML subtypes. We have analyzed samples from 117 AML patients and three CD34+ bone marrow specimens from healthy donors. In addition to five cases of t(8;16) AML, samples from nine other AML subtypes were included.The expression of 670 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2−ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software. In order to identify miRNA targets, we used the RmiR package (Bioconductor) to cross-correlate the miRNA expression data from the present study with our previous findings on gene expression in the same patient samples (Camos M et al, Cancer Res 2006), based on the predicted targets from TargetScan and Pictar databases. The unsupervised hierarchical cluster analysis showed well-differentiated groups correlating with cytogenetic and molecular categories. Interestingly, all t(8;16) AML patients were grouped in an independent cluster. Supervised analysis by means of t-test and SAM analysis revealed a distinctive 108-miRNA signature in t(8;16) AML patients. All 108 miRNAs were downregulated in t(8;16) AML in comparison to the other subtypes, including all the miRNAs of cluster miR-17-92 and its paralogs miR-106b-25 and 106a–363, as well as miR-29b.Since 14% of the 108 miRNAs in the signature, including miR17-92 and its paralogs, are transcriptionally activated by c-Myc, we then examined c-Myc mRNA analysis by RT-QPCR. Expression of c-Myc in our t(8;16) AML patients was significantly downregulated (p=0.02).miR-29b, also downregulated in our t(8;16) AML patients, is known to target DNA methyltransferase (DNMT) genes, which play a key role in the regulation of DNA methylation through CpG methylation. Since 8% of the 108 miRNAs in our signature are located in CpG islands and 29% are intronic miRNAs contained in genes with a CpG island in their promoter region, we speculated that DNMT activity could also be a factor in the overall downregulation of the miRNAs in the signature.When miRNA data from the present study were cross-correlated with our previous findings on mRNA expression in the same patients, we found an inverse correlation of miR-130a and miR-130b with HOXA3 (r2= -0.5; -0.8; respectively), of miR-1 and miR-23b with MEIS1 (r2= -0.62; -0.62; respectively) and of miR-15b, miR-195 and miR-218 with RET (r2= -0.92;-0.58;-0.87; respectively). In summary, t(8;16) AML exhibits a distinctive 108-miRNA signature, with an overall downregulated profile, which may be partially explained by c-Myc downregulation. Moreover, we have identified potential target genes of these miRNAs which had previously been shown to be characteristic of this AML subtype. Our findings thus contribute some insight into the biological profile of t(8;16) AML and can provide a greater understanding of genetic and epigenetic abnormalities in t(8;16) AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1712 The study of chromosome alterations helps to classify acute myeloid leukemia (AML) into three prognostic groups, favorable, intermediate and adverse. The prognostic value of favorable risk and adverse risk abnormalities is well defined; in contrast, the outcome of intermediate-risk group is heterogeneous. Molecular characterization of patients with intermediate-risk AML has identified subcategories with diverse prognosis. All this knowledge has translated into a recent ELN proposal for the genetic classification of AML. Additionally, the intermediate and particularly the cytogenetically adverse groups have been refined by the HOVON group by introducing the concept of “monosomal karyotype” (MK), consisting of at least two autosomal monosomies or one monosomy plus a structural abnormality. Objective: To validate the recent ELN classification in a large series of AML patients and to investigate if the inclusion of MK improved the prognostic stratification. Patients and methods: 804 consecutive patients treated under the CETLAM AML-99 (n=353) and CETLAM-03 (n=451) trials were analyzed. The two protocols included idarubicin, intermediate-dose cytarabine and etoposide as induction and mitoxantrone and intermediate-dose cytarabine as consolidation. G-CSF priming was given in the CETLAM-03 trial. Following, risk-adapted treatment with chemotherapy or hematopoietic transplantation was administered. Parameters analyzed were relapse rate (REL), leukemia-free survival (LFS) and overall survival (OS). Results: Median age of the series was 46 years (range 16–60). Median follow-up of patients alive was 15 months. The ELN classification resulted in different prognostic risk groups. At 5 years, ELN favorable risk category had an OS of 60±4%, intermediate-I of 32±%, intermediate-II of 46±% and adverse of 17±3% (p Conclusion: In the present study, the ELN genetic classification did not discriminate the prognosis of patients in the intermediate-I and intermediate-II categories. In contrast, genetic grouping that considered CBF AML, favorable mutations in the intermediate cytogenetics category and that separated adverse karyotype with or without MK translated into significantly different OS. This classification, together with the study of mutations recently described and the expression of certain genes may contribute to a more precise prognostic stratification and risk-adapted therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 2611 Poster Board II-587 The prognostic heterogeneity of patients with intermediate-risk cytogenetics AML (AML-IR) is mostly clarified by determination of mutations of NPM1 gene (NPMmut) and internal-tandem duplication of FLT3 gene (FLT3-ITD). Nonetheless, other genetic lesions described in this population might contribute to a better prognostic categorization. In this context, we analyzed the presence of CEBPA mutations and associated features in patients with AML-IR lacking both NPMmut and FLT3-ITD. Overall, 136 patients (51% female; median age: 53, range: 17=74) diagnosed with de novo AML-IR (MRC definition) in our institution between 1994 and 2008 who received standard AML chemotherapy were included in the analysis. CEBPA mutations (CEBPAmut) were investigated by whole gene sequencing using 4 primer pairs according to previously reported methods (Fröhling et al, 2004). Sixty-five patients (48%) harbored NPM1 mutations, 30 of them having concomitant FLT3-ITD. Among NPM1 wild-type patients (NPMwt), FLT3-ITD, CEBPA and MLL mutations were detected in 18, 11, and 5 patients, respectively. Regarding cases with CEBPAmut, biallelic mutations were found in 8 patients, including 6 cases with combined mutations of N-terminal and bZIP domains and two homozygous mutations, whereas a single mutation in bZIP domain was found in the three remaining patients. As compared to patients with wild-type CEBPA (CEBPAwt), those with CEBPAmut were younger (29 vs. 53, p=0.027), and showed a trend to male predominance (73 vs. 46%, p=0.09) and lower WBC count at presentation (16 vs. 33.5 × 109/L, p=.095). Of note, CD7 antigen was aberrantly expressed in virtually all CEBPAmut cases (10/11), compared to only 21% of CEBPAwt patients (p Disclosures: No relevant conflicts of interest to declare.

The heterogeneous prognosis of patients with intermediate-risk cytogenetics AML (AML-IR) can be partially clarified by screening of NPM1 mutations (NPMmut) and internal tandem duplication of FLT3 (FLT3-ITD). Nonetheless, additional factors might influence the prognostic effect of these molecular lesions, such as the FLT3-ITD mutant level. Moreover, the optimal post-remission strategy might differ depending on the underlying molecular lesion. In this regard, we analyzed the outcome, according to NPM1 and FLT3 mutations and post-remission therapy given, of a series of patients diagnosed with de novo AML-IR in a single institution who received intensive chemotherapy. Patients were treated following 4 sequential protocols of CETLAM group during the period 1994–2006, consisting of 1 or 2 cycles of standard induction chemotherapy and 1 course of high-dose cytarabine-based consolidation therapy. Thereafter, patients underwent hematopoietic stem cell transplantation (HSCT) according to donor availability and presumed risk (protocols LAM 99 & 2003). NPM1 mutations and FLT3-ITD were screened in diagnostic DNA by PCR amplification followed by Genescan analysis. The ratio between FLT3-ITD and wildtype FLT3 alleles (ITD/wt ratio) was calculated using the area under the peak of corresponding alleles. Overall, 134 patients (51% male; median age, 53; range: 17–70) with AML-IR (normal karyotype, 66%) were studied. NPM1mut and FLT3-ITD were found in 45% and 37% of patients, respectively, with a median ITD/wt ratio of 0.59 (0.045–5.5). After induction regimen, 109 patients (81%) achieved complete response (CR). The only variables predictive of a favorable response were NPMmut (90% vs. 75%; p=0.01) and age 50 × 109/L), NPMwt, and FLT3-ITD. Interestingly, the prognostic value of FLT3-ITD depended on the relative mutant level, and detection of FLT3-ITD with a low ITD/wt ratio (i.e., Moreover, the effect of post-remission therapy varied in both molecular-defined subgroups. Thus, among patients with an age ≤60, 5-year survival in LOWmol patients with a planned autologous HSCT (autoHSCT) was 83%±9%, not differing significantly from that of patients undergoing allogeneic HSCT (intention-to-treat analysis; figure On the other hand, 5-year OS of HIGHmol patients who underwent allogeneic HSCT in first CR was 73%±13, which compared favorably with other post-remission strategies (5-yr OS: 27%±7%; p=0.019). In conclusion, in patients with intermediate-risk AML, the combined assessment of NPM1 mutations and quantitative estimation of FLT3-ITD allows the distinction of 2 categories of patients with different prognosis. Thus, whereas autoHSCT arises as an effective postremission therapy in patients harboring low-risk molecular features, allogeneic HSCT in first CR seems to overcome adverse prognosis of patients with high-risk disease. Nonetheless, the validity of this molecularly-based therapeutic algorithm warrants confirmation in other studies. Figure Figure

Post-remission therapy in patients with acute myeloid leukemia (AML) is assigned according to the predictable biological risk of the disease, mainly based on cytogenetics. Nonetheless, optimal post-remission strategy for the intermediate-risk subtype, given the prognostic heterogeneity of this category, is currently undefined. Analysis of potentially relevant molecular features within this subgroup might contribute to clarify the role of autologous stem cell transplantation (autoHSCT) in these patients. Thirty seven patients (age: 53, 15–66; 51% female) diagnosed with intermediate-risk de novo AML during the period 1994–2006 who received an autoHSCT in first complete response were included in the study. Pre-transplant therapy was similar in all patients, consisting of standard induction chemotherapy (ICE, n=8, IDICE, n=29) and one cycle of high-dose ara-C-based consolidation chemotherapy. Internal tandem duplication of flt-3 (flt-3 ITD) and exon 12 NPM1 mutations were studied by either PCR or RT-PCR following standard methods. Gene expression profiling was examined in 28 patients with oligonucleotide HGU133 Plus 2.0 arrays (Affymetrix). Gene expression measures were normalized using RMA methodology (Affy package), and dChip v1.3 and Limma software (Bioconductor) were used for unsupervised and supervised analyses. In order to identify genes with prognostic value, a supervised analysis based on patients' outcome (relapsed patients vs. long-term responders, i.e. >2-year duration) was performed. The combined results of NPM mutation and flt-3 ITD defined three subgroups of patients with different outcome: group 1 (n=12), constituted by patients with mutated NPM1 without flt-3 ITD; group 2 (n=20), which included patients with neither NPM1 mutation or flt-3 ITD; and group 3 (n=5), defined by flt-3 ITD regardless NPM1 mutational status. Thus, 5-year survival of these 3 subgroups of patients was 91%±9%, 52%±12%, and 20%±18%, respectively (p=0.02; see figure). Preliminary results of multiple gene profile comparisons between subgroups of patients with different outcome disclosed a cluster of genes with differential expression. Thus, in the most significant balanced comparison, 1238 genes were found to vary significantly in the unsupervised analysis, and 109 differentially expressed genes were identified in the supervised analysis. Interestingly, overexpression of genes such as TNF, RETN, CFLAR, SLC16A7, ENG, CD48, PLCR1, and SULTB1 correlated with a high relapse risk, whereas increased expression of YY1, FBXL12 and EXOSC6 were associated with a favorable outcome. In conclusion, presence of NPM1 mutation and flt-3 ITD are strong predictors of the outcome after autoHSCT in patients with intermediate-risk AML. Furthermore, genome-wide analysis may contribute to further define gene clusters with prognostic significance in patients with cytogenetically intermediate-risk AML receiving autoHSCT as consolidation therapy. Download : Download high-res image (88KB) Download : Download full-size image Figure

Abstract 1066 Different approaches have been investigated to improve the prognosis of adult patients with primary acute myeloid leukemia. In two consecutive phase II trials our group has explored the use of intermediate-dose cytarabine in induction associated with idarubicin and etoposide, the addition of G-CSF priming to the previous combination, and risk-adapted postremission therapy. Objective: To compare the results of two consecutive trials for primary AML and to analyze the factors influencing the outcome. Patients and methods: Adult patients between 17 and 60 years of age with de novo AML, diagnosed between 1999 and 2009, were included in the CETLAM AML-99 and AML-03 trials. Induction chemotherapy (CT) included one or two courses of idarubicin 12 mg/m2 IV days 1,3,5, cytarabine 500 mg/m2/12h over 2h IV days 1,3,5,7 and etoposide 100 mg/m2 IV days 1,2,3. This was followed by one consolidation with mitoxantrone 12 mg/m2 IV from day 4 to 6, and cytarabine 500 mg/m2/12h IV from day 1–6. In the AML 03 trial, patients also received G-CSF priming, 150 mg/m2 subcutaneously (SC) from day 0 to the last day of induction and consolidation CT. Postremission therapy consisted of high-dose cytarabine (CBF AML), autologous or allogeneic hematopoietic transplantation depending on cytogenetics, courses to complete remission (CR), and in the AML-03 protocol also based on molecular abnormalities involving FLT3 or MLL genes and/or the persistence of minimal residual disease after consolidation. Results: Overall, 788 patients were included, 353 in the AML-99 trial and 435 in the AML-03. Median age of the patients was 46 years (range 17–60). There were no differences between patients included in the two protocols regarding age, gender, leukocyte counts, cytogenetics and proportion of favourable and unfavourable molecular cases in the group with intermediate-risk karyotype. The main results achieved appear in the table. Multivariate analysis confirmed the favourable impact of AML-03 protocol on outcome. Other significant factors influencing survival were age, leukocyte counts and cytogenetics. Conclusion: G-CSF priming improved the CR rate of adult patients with primary AML and favourable or unfavourable cytogenetics. This fact and a more precise risk-adapted therapy taking into account genetic data and MRD studies translated into improved overall survival. Disclosures: No relevant conflicts of interest to declare.

Abstract 2530 Macrophage migration inhibitory factor (MIF) is a key mediator of the innate immune system by promoting pro-inflammatory functions, being involved in the pathogenesis of the sepsis, in inflammatory and autoimmune diseases, growth and cell differentiation, and in carcinogenesis. The recessive CC gene variant in -173G/C in the promoter region causes higher levels of MIF and it has been associated with sepsis, gastric cancer, and autoimmune diseases. We aimed to see whether this genetic variant influenced the clinical outcome of acute myeloid leukemia (AML) patients. Population study consisted of 277 AML patients from the Spanish cooperative CETLAM group. Median age of the patients was 45 (range, 15–74). According to MRC classification, 11% were of good risk, 70% of intermediate risk, and 11% of poor risk. For 8% of the patients MRC cytogenetical data was not available. Multivariate analysis for outcome was performed including age, MRC classification and leukocyte count. Results showed that recessive CC gene variant was associated with worse outcome than the other two genotypes (GG+GC). Thus, this gene variant was associated with lower overall survival (OS): 0% vs 28%, p Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a neoplasia characterized by the rapid expansion of immature myeloid blasts in the bone marrow, and marked by poor prognosis and frequent relapse. As such, new therapeutic approaches are required for remission induction and prevention of relapse. Due to the higher chemotherapy sensitivity and limited life span of more differentiated AML blasts, differentiation-based therapies are a promising therapeutic approach. Based on public available gene expression profiles, a myeloid-specific differentiation-associated gene expression pattern was defined as the therapeutic target. A XIAP inhibitor (Dequalinium chloride, DQA) was identified in an in silico screening searching for small molecules that induce similar gene expression regulation. Treatment with DQA, similarly to Embelin (another XIAP inhibitor), induced cytotoxicity and differentiation in AML. XIAP inhibition differentially impaired cell viability of the most primitive AML blasts and reduced clonogenic capacity of AML cells, sparing healthy mature blood and hematopoietic stem cells. Taken together, these results suggest that XIAP constitutes a potential target for AML treatment and support the evaluation of XIAP inhibitors in clinical trials.