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INTRODUCTION Increased levels of Wilms' tumor (WT1) mRNA have been used to establish risk categories in patients with acute myeloid leukemia (AML). Raised values of WT1 have been associated with progression in myelodysplastic syndrome (MDS). METHODS We retrospectively analyzed the available bone marrow (BM) samples from 115 patients with myeloid neoplasms obtained before and during treatment with 5-azacytidine. A threshold of 100 copies in BM was used to define risk groups: group 1: patients with WT1 levels always below

Vascular endothelial growth factor C (VEGFC) stimulates leukemia cell proliferation and survival, and promotes angiogenesis. We studied VEGFC expression in bone marrow samples from 353 adult acute myeloid leukemia (AML) patients and its relationship with several clinical, cytogenetic, and molecular variables. We also studied the expression of 84 genes involved in VEGF signaling in 24 patients. We found that VEGFC expression was higher in AML patients with myelodysplasia-related changes (AML-MRC) than in patients with non-AML-MRC. We also found an association between VEGFC expression and the patient cytogenetic risk group, with those with a worse prognosis having higher VEGFC expression levels. No correlation was observed between VEGFC expression and survival or complete remission. VEGFC expression strongly correlated with expression of the VEGF receptors FLT1, KDR, and NRP1. Thus, in this series, VEGFC expression was increased in AML-MRC and in subgroups with a poorer prognosis, but has no impact on survival.

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:Acute myeloid leukemia (AML) real-world incidence has been investigated in a limited number of European countries such as Sweden, Denmark and a few others. These prospective population-based analyses give a more precise idea of AML as a health problem than registry data from cooperative groups or other sources, that usually include selected cases as part of research studies and/or therapy trials. In October 2016, the Autonomous Government of Catalonia funded a project (PERIS SLT002/16/00433) to prospectively collect all AML cases from our territory. Objective:To investigate the incidence, characteristics and treatment decisions in all consecutive AML patients diagnosed in Catalonia between January 2017 and December 2019. Methods:Inclusion criteria were diagnosis of AML according the WHO 2016 criteria, both primary and secondary (APL excluded) with an age >18 years. The project was disseminated to all hospitals from Catalonia regardless of their size, having at least one hematologist. A specific informatics tool was implemented for remote reporting of the cases. All data were anonymized. In parallel, a circuit for centralized bio banking of patients' samples was designed. The database included the main clinical, laboratory data as well as the initial therapeutic approach. Cases included in our CETLAM group cooperative studies were automatically linked to the trial database for collecting detailed information. Statistical analyses were performed with R packages. Results:Assuming an incidence of AML of 4 cases per 100,000 inhabitants (based on previous reported data from others), we expected 912 cases during three years in the 7,6 million population of Catalonia. Our prospective registry included 750 consecutive AML patients, 82% of the expected cases. The remaining 18% could be explained by the exclusion of APL, age below 18 years, or underreporting. Seventy percent of patients (n=527) were diagnosed and treated in the 5 large University Hospitals from Barcelona and the two adjacent cities (Badalona and Hospitalet). Table 1 shows the main characteristics of the patients. Among the 390 patients up to 70 years, 272 (70%) were enrolled in the CETLAM AML-12 protocol that included intensive chemotherapy (ICT) and risk adapted hematopoietic cell transplantation (HCT). Forty-one additional patients (11%) in this age group received other ICT in different clinical trials. A remaining 73 patients (20%) were treated with other intensive or non-intensive approaches outside trials. In the group of 360 patients older than 70 years only a 33% (n= 119) were treated under the risk-adapted CETLAM AML-16 protocol for elderly AML patients. This trial included ICT as in the CETLAM-12 in case of favorable genetic features; this was received by 13 of the 119 patients (11%) enrolled. The remaining patients of CETLAM-16 were treated with low-intensity chemotherapy (oral fludarabine, subcutaneous (SC) cytarabine and G-CSF or azacytidine) and 97 additional elderly patients were included in other clinical trials mostly with targeted and hypomethylating agents (27%). Other active therapies outside trials (usually low-intensity) were administered in 50 additional patients (14%) whereas the remaining 94 patients (26%) only received supportive measures (transfusions, hydroxyurea, antibiotics, palliation, or no treatment), because of one or more of the following: advanced age, poor AML features or severe clinical condition. Overall survival (OS) of the whole series at 2 years was 31±2% (CI: 27-35). Patients younger than 70 years had a 2-year OS of 47±3% (CI: 41-53) compared to 11±3% (CI: 7-17) for those above 70 years (p Conclusions:This prospective study is highly representative of the diagnosis and treatment of AML in Catalonia. The median age at diagnosis was 70 years. Of note, 81% of patients up to 70 years were enrolled in ICT trials. The proportion of patients in trials in the elderly group was lower although still remarkable (60%). In this advanced age group, a 26% of patients were treated with supportive measures only. Despite the high inclusion rate in clinical trials, only one third of newly diagnosed AML patients have the probability to survive at 2 years, with a dismal outcome in those above 70 years. Therefore, the investigation of novel and more effective treatments remains mandatory. This series will be detailed and updated during the meeting. Disclosures Salamero: Pfizer:Consultancy;Jazz Pharmaceuticals:Consultancy, Honoraria;Daichii Sankyo:Honoraria;Novartis:Consultancy, Honoraria;Celgene:Consultancy, Honoraria.Olivera:BAYER:Consultancy;Pfizer:Consultancy, Speakers Bureau;Daiichi Sankyo:Consultancy, Speakers Bureau;Boehringer Ingelheim:Consultancy, Speakers Bureau.Sureda Balari:Celgene:Consultancy, Honoraria;Merck Sharpe and Dohme:Consultancy, Honoraria, Speakers Bureau;Sanofi:Consultancy, Honoraria;Novartis:Consultancy, Honoraria;Gilead/Kite:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;Incyte:Consultancy;Roche:Honoraria;BMS:Speakers Bureau;Celgene/Bristol-Myers Squibb:Consultancy, Honoraria;Takeda:Consultancy, Honoraria, Speakers Bureau.Ribera:Pfizer, Amgen:Research Funding;Pfizer, Amgen, Ariad, Novartis:Consultancy, Speakers Bureau.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.

BACKGROUND: The progress in the understanding of pathophysiology of AML has allowed the identification of genetic and immune abnormalities with prognostic impact on outcome and suitable as therapeutic targets. The genetic abnormalities are essential for risk allocation and risk-adapted treatment included the indication of hematopoietic cell transplantation. In the last decade, several studies have shown that persistence of measurable residual disease (MRD) after chemotherapy increases relapse incidence and the probability of leukemia recurrence and survival. Therefore, MRD has been progressively incorporated in prognosis estimation. In the last 25 years the CETLAM cooperative group has promoted 4 consecutive trials for AML patients fit for intensive chemotherapy and eventually HCT. Post-remission treatment was based on genetics of the disease and more recently on MRD. The aim of this study has been to investigate if the survival of patients has improved and, if so, to identify the factors that have influenced on the better outcome. METHODS: We included all patients with primary AML up to the age of 60 years enrolled in 4 consecutive Spanish CETLAM group trials. In brief, induction chemotherapy included idarubicin, cytarabine and etoposide in AML-94, AML-99 and AML-03 protocols and without etoposide in the AML-12. G-CSF priming was allowed in the two more recent trials. Post remission therapy included 1 to 3 consolidations including intermediate or high dose cytarabine. Hematopoietic transplantation indication was based on availability of an HLA-compatible donor, genetic findings and more recently MRD. Follow-up was extended to June 2020. The survival and relapse incidence analyses were censored at 5 years. Informed consent was obtained in all cases and the institutional review boards approved the protocols. RESULTS: Between 1994 and 2019, 1755 primary AML patients between 18 and 60 years-old fulfilled the inclusion criteria. The main characteristics of patients appear in table 1. Median age of the whole group was 46 years old. Overall survival (OS) in the whole group was 45% at 5 years, being significantly better in AML-03 and AML-12 than in AML-94 and AML-99 (image 1). Event free survival (EFS) in the whole group was 37% at 5 years, with also significant differences between trials. Also, the cumulative incidence of relapse (CIR) was 39% in the whole group with less relapses in the two more recent trials (image 2). To understand these findings, we analyzed first the CR rate over time that was higher in the AML-03 and AML-12 protocols (table 2). The results were different depending on genetics of AML with highest CR rate in patients with CBF AML and in those with intermediate-risk cytogenetics and favorable molecular findings; in contrast, patients with adverse cytogenetics had the lowest CR rate mainly because frequent refractoriness to therapy. According to outcomes in each MRC cytogenetic group 5y-OS was: 69±3% (63-76) in favorable group, 46±2% (43-49) in intermediate and 21±3% (16-27) in adverse group (p Referent to feasibility of allogeneic HCT, there was an increased access to the procedure over the years. A higher proportion of patients allografted in AML-03 and AML-12, 32% and 41% of patients in CR, respectively, compared to 16% in AML-94 and 19% in AML-99. A shortening of the interval between CR and transplantation has been observed in recent years; 3.9 months (mo) in AML-94, 2.7 mo in AML-99, 2.9 mo in AML-03 and 2.2 mo in AML-12. CONCLUSIONS: In adults with primary AML and age up to 60 years-old have improved over the last 25 years. During this period, the CETLAM group has refined the biological characterization of AML patients and tailored the post-remission therapy based on genetic markers with prognostic impact. The increased feasibility of allogeneic HCT may also justify the better results in more recent trials. Even though, there is substantial room for improvement, particularly in patients with AML and adverse genetic features. Disclosures Salamero: Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria; Daichii Sankyo: Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Moraleda:Takeda: Consultancy, Other: Travel Expenses; Sandoz: Consultancy, Other: Travel Expenses; Novartis: Consultancy, Other: Travel Expenses; Gilead: Consultancy, Other: Travel Expenses; Jazz Pharmaceuticals: Consultancy, Research Funding. Tormo: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; Janssen: Honoraria; MSD: Honoraria; 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. Ribera:Pfizer, Amgen: Research Funding; Pfizer, Amgen, Ariad, Novartis: Consultancy, Speakers Bureau. Sureda Balari:Roche: Honoraria; Incyte: Consultancy; Janssen: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Merck Sharpe and Dohme: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; BMS: Speakers Bureau; 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.

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.

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

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (> 75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45 + CD123 + cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages. (C) 2010 Elsevier Ltd. All rights reserved.

CTLA-4 is an inhibitory molecule that down-regulates T-cell activation. Although polymorphisms at CTLA-4 have been correlated with autoimmune diseases their association with clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has yet to be explored. A total of 5 CTLA-4 single-nucleotide polymorphisms were genotyped on 536 HLA-identical sibling donors of allo-HSC transplants. Genotypes were tested for an association with patients' posttransplantation outcomes. The effect of the polymorphisms on cytotoxic T-lymphocyte antigen 4 (CTLA-4) mRNA and protein production were determined in 60 healthy control participants. We observed a reduction in the mRNA expression of the soluble CTLA-4 isoform in the presence of a G allele at CT60 and +49. Patients receiving stem cells from a donor with at least 1 G allele in position CT60 had worse overall survival (56.2% vs 69.8% at 5 years; P = .001; hazard ratio [HR], 3.80; 95% confidence interval [CI], 1.75-8.22), due to a higher risk of relapse (P = .049; HR, 1.71; 95% CI, 1.00-2.93). Acute graft-versus-host disease (aGVHD) was more frequent in patients receiving CT60 AA stem cells (P = .033; HR, 1.54; 95% CI, 1.03-2.29). This is the first study to report an association between polymorphisms at CTLA-4 and clinical outcome after allo-HSCT. The CT60 genotype influences relapse and aGVHD, probably due to its action on CTLA-4 alternative splicing.

Acute myeloid leukemia (AML) comprises a biologically and clinically heterogeneous group of aggressive disorders that occur as a consequence of a wide variety of genetic and epigenetic abnormalities in hematopoietic progenitors. Despite significant advances in the understanding of AML biology

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.

Purpose Most patients with acute myeloid leukemia (AML) and genetic rearrangements involving the core binding factor (CBF) have favorable prognosis. In contrast, a minority of them still have a high risk of leukemia recurrence. This study investigated the adverse features of CBF AML that could justify investigational therapeutic approaches. Patients and methods One hundred and fifty patients (median age 42 yr, range 16–69) with CBF AML (RUNX1-RUNX1T1 n = 74; CBFB-MYH11 n = 76) were prospectively enrolled into two consecutive CETLAM protocols at 19 Spanish institutions. Main clinic and biologic parameters were analyzed in the whole series. In non-selected cases with available DNA samples, the impact of molecular characterization and minimal residual disease (MRD) was also studied. Results Overall, complete remission (CR) rate was 89% (94% in ≤50 yr old and 72% in >50 yr, P = 0.002). At 5 yr, cumulative incidence of relapse (CIR) was 26 ± 1%, disease-free survival (DFS) 62 ± 6%, and overall survival (OS) 66 ± 4%. In multivariate analyses, leukocyte count above 20 × 109/L, BAALC over-expression, and high copy numbers of RUNX1-RUNXT1 or CBFB-MYH11 after induction chemotherapy (CT) led to increased relapse rate. Regarding OS, age >50 yr, leukocyte count above 20 × 109/L, and increased MN1 expression were adverse features. Conclusion Age, leukocyte counts, BAALC, and MN1 gene expressions as well as high copy numbers of RUNX1-RUNXT1 or CBFB-MYH11 after induction chemotherapy are useful tools to predict the outcome and should be considered for risk-adapted therapy.

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)

We retrospectively assessed whether normalized bone marrow WT1 levels could be used for risk stratification in a consecutive series of 584 acute myeloid leukemia (AML) patients. A cutoff value of 5065 copies at diagnosis identified two prognostic groups (overall survival (OS): 44 ± 3 vs 36 ± 3%, P=0.023; leukemia-free survival (LFS): 47 ± 3 vs 36 ± 4%, P=0.038; and cumulative incidence of relapse (CIR): 37 ± 3 vs 47 ± 4%, P=:0.043). Three groups were identified on the basis of WT1 levels post-induction: Group 0 (WT1 between 0 and 17.5 copies, 134 patients, OS: 59 ± 4%, LFS:59 ± 4% and CIR: 26 ± 4%); Group 1 (WT1 between 17.6 and 170.5 copies, 160 patients, OS: 48 ± 5%, LFS:41 ± 4% and CIR: 45 ± 4%); and Group 2 (WT1170.5 copies, 71 patients, OS: 23 ± 6%, LFS: 19 ± 7% and CIR: 68 ± 8%) (P0.001). Post-intensification samples distinguished three groups: patients with WT1100 copies (47 patients, 16%); an intermediate group of patients with WT1 between 10 and 100 copies (148 patients, 52%); and a third group with WT110 copies (92 patients, 32%). Outcomes differed significantly in terms of OS (30 ± 7%, 59 ± 4%, 72 ± 5%), LFS (24 ± 7%, 46 ± 4%, 65 ± 5%) and relapse probability (CIR 72 ± 7%, 45 ± 4%, 25 ± 5%), all P0.001. WT1 levels in bone marrow assayed using the standardized ELN method provide relevant prognostic information in de novo AML.

The study of genetic lesions in AML cells is helpful to define the prognosis of patients with this disease. This study analyzed the frequency and clinical impact of recently described gene alterations, isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations, in a series of homogeneously treated patients with primary (de novo) AML. Two-hundred and seventy-five patients enrolled in the CETLAM 2003 protocol were analyzed. IDH1 and IDH2 mutations were investigated by well-established melting curve-analysis and direct sequencing (R140 IDH2 mutations). To establish the percentage of the mutated allele a pyrosequencing method was used. Patients were also studied for NPM, FLT3, MLL, CEBPA, TET2 and WT1 mutations. IDH1 or IDH2 mutations were identified in 23.3% AML cases and in 22.5% of those with a normal karyotype. In this latter group, mutations were associated with short overall survival. This adverse effect was even more evident in patients with the NPM or CEBPA mutated/FLT3 wt genotype. In all the cases analyzed, the normal allele was detected, suggesting that both mutations act as dominant oncogenes. No adverse clinical impact was observed in cases with TET2 mutations. IDH1 and IDH2 mutations are common genetic alterations in normal karyotype AML. Favourable genotype NPM or CEBPA mutated/FLT3 wt can be further categorized according to the IDH1 and IDH2 mutational status. (C) 2012 Elsevier Ltd. All rights reserved.

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 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.

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 Quantitation of WT1 mRNA levels may be used in minimal residual disease studies in AML cases lacking a suitable molecular target. The normal thresholds of bone marrow WT1 levels at different time–points have not been clearly stated. Difficulties arise as a consequence of the relatively high WT1 expression in normal bone marrow and the unknown influence of chemotherapy. Objective To establish the reference range value of bone marrow WT1 levels in treated CBF-AML and APL in complete remission. Patients and Methods Patients with bone marrow samples obtained from ≥2 years (2-11 y) after initial diagnosis of CBF-AML (AML1-ETO n:21 patients CBFb-MYH11 n:21 patients) or APL(n:22 patients) were included in the study (120 samples). Ages at diagnosis ranged from 1 to 76 years. Mean age was: AML1-ETO 36.8(2-76y),CBFb-MYH11 32.4(1-55y), PML-RARa 47.3(21-76y). Peripheral blood, reticulocyte count and a bone marrow aspirate were also available in 86 cases. WT1 was analyzed following the primers and conditions of the ELN group. Specific chimeric real time PCR was simultaneously performed in each sample in accordance with the BIOMED protocol. Each WT1 or chimeric PCR was analyzed in triplicate whereas the control gene (abl) was analyzed in duplicate. Results All patients with an available BM study were in morphologic complete remission. Two samples corresponded to an anemic patient (2/86). Six cases had a reticulocyte count above 2%. Platelet counts were below 120x109/l in 3 cases (3/86). Leukocyte counts below 3.5x109/l were observed in 6 samples (6/86). The calculated mean WT1 copy number from the 120 bone marrow samples was 47.23 (AML1-ETO:61.33, CBFb-MYH11:43.51, PML-RARa:36.4 p:ns). The mean chimeric copy number was 0.75 (AML1-ETO:0.82, CBFb-MYH11:0.72, PML-RARa:0.69 p:ns). Only 5 samples had a specific copy number of chimeric transcripts above 5 (3 AML1-ETO,1 CBFb-MYH11 and one PMAL-RARa patient who had a molecular relapse one month later and was succesfully treated). In 6 samples, the specific chimeric copy number was between 1 and 5 copies ( 4 AML1-ETO and 2 CBFb-MYH11). In the remaining 109 BM samples, the specific copy number was Conclusions Bone marrow WT1 copy number in treated CBF-AML and APL was 47.23. In ten percent of cases, the WT1 copy number was greater than 100 copies. Occasionally, WT1 peaks (≥300 ) were detected. The meaning of the differential bone marrow WT1 transcriptional activity in long-term survivors of AML remains to be investigated. 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 2524 WT1 monitoring is an almost universal target to follow de novo AML. Its exppression in myeloid malignancies is upregulated in parallel to the blast percentage. Recently, WT1 determination has been standardized as result of an European Leukemia Net initiative. Early reports have demonstrated that the best results are obtained when peripheral blood is used to establish clinical predictions. Pediatric studies in AML have shown that raised WT1 levels after induction associate with unfavourable outcome. Despite all the mentioned, WT1 quantitation has not yet gained widespread use, in part because some AML show normal WT1 levels at diagnosis. To investigate the prognostic impact of the normalized bone marrow WT1 levels at diagnosis and post-induction in a consecutive series of de novo AML patients enrolled in the CETLAM group trials. Available bone marrow samples at diagnosis (586 cases) and post induction (367 cases) were obtained in each participating center and sent to the CETLAM repository center at the Hospital de la Santa Creu i Sant Pau for complete immunophenotype and molecular analyses. One μg of RNA was reverse transcribed to cDNA in a total reaction volume of 20μl containing Cl2Mg 5mM, 10× Buffer, DTT 10mM, dNTP's 10mM each, random hexamers 15μM, RNAsin 20 units (Promega) and 200 units of MMLV enzyme. WT1 expression levels were determined by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI PRISM 7700® Genetic Analyzer (Applied Biosystems, Foster City, CA) using the primers and conditions described by the ELN group (Cilloni et al J. Clin. Oncol 2009;27:5195-201). For WT1 copy number titration, the IPSOGEN® (Marseille, France) plasmid was employed. Results were expressed as copies and four normal bone marrow samples were used as test controls. Patients were treated between 2004 and 2011 according to the CETLAM03 protocol. Adults up to 70 years of age received induction chemotherapy with idarubicin, intermediate-dose cytarabine and etoposide, followed by consolidation with mitoxantrone and intermediate-dose ara-C. Subsequently, patients with favourable cytogenetics at diagnosis received one cycle of high-dose cytarabine.G-CSF priming during induction and consolidation was used. Patients with favorable cytogenetics and high leukocyte counts at diagnosis were treated with autologous transplantation instead of high-dose cytarabine. Furthermore, patients with a normal karyotype but an adverse molecular profile (FLT3 mutations or MLL rearrangements) were allocated to the treatment for unfavorable cases; this included allogeneic transplantation from an HLA-identical donor. Overall survival (OS) was measured from the date of enrolment until the date of death. Leukemia-free survival (LFS) for patients who achieved a CR was calculated from the date of CR to relapse or death. OS and LFS were plotted by the Kaplan-Meier method; differences between curves were analyzed by the log-rank test. The probability of relapse was calculated using cumulative incidence estimates and taking into account the competing risk of death in remission. A WT1 cut-off value of 5065.2 copies at diagnosis was obtained. Two hundred and four samples had WT1 levels greater than this value, whereas 382 samples showed levels below this cut-off. These groups had statistically different OS 55±3 vs 33±5 p As regards the post-induction results, four groups were established: Group 0 (135 patients) with WT1 levels between 0 and 17.5 copies, Group 1 (107 patients) with WT1 values ranging from 17.6 to 76 copies, Group 2 (54 patients) with WT1 between 76.1 and 170.5 copies and Group 3 (71 patients) with WT1 levels after induction greater than>170.6 copies. These groups showed statistically significant differences(p WT1 quantitation at diagnosis and post-induction provide a simple and well standardized measurement of the prognostic risk of adult AML patiens. Larger series need to be analyzed to ascertain whether this determination could be incorporated to initial AML risk stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract 1452 Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods: Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results: Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion: In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3562 5-azacitidine (5-aza) is a widely-used agent for treatment of myelodysplastic syndromes (MDS). In contrast, data on its use in AML are far more limited and, to date, little is known on the clinical or biological markers predicting the response of AML to this drug. We retrospectively analysed a series of patients with AML who received 5-aza due to old age, poor condition or refractoriness to standard treatment, and investigated the potential association of a variety of genetic markers with response and survival. PATIENTS AND METHODS: From 2006 to 2011, 38 patients (23 males, 15 females) diagnosed as AML according to the WHO criteria1 received treatment with 5-aza (75 mg/m2 SQ daily, 5–7 days) at our institution as on-label or compassionate use program. Twenty-four patients (median age 68 years, range 37–85) had primary AML (pAML). Most of them (n=18) had failed to prior standard AML treatment: one line in 12 cases, 2 lines in 5 (including stem cell transplant [SCT] in 2 cases), and 3 lines in one. Fourteen additional patients (median age 68.3; range 32–88) had secondary AML (sAML); among them 8 previously treated: 6 patients had received one line of standard treatment before 5-aza, 1 patient 2 lines, and 1 patient 3 different lines. Twenty-two pAML and 14 sAML cases were screened for the presence of karyotype abnormalities. Screening for gene mutations [FLT3 internal tandem duplication (FLT3/ITD, nucleophosmin-1 gene mutation (NPM1), MLL partial tandem duplication, CCAAT/enhancer binding protein alpha mutation (CEBPA), AML1-ETO and CBFB-MYH11] was performed prior to starting 5-aza whenever possible. The response to treatment was usually assessed between the 4th and 6th courses, and classified according to WHO criteria. RESULTS: Patients received a median 7 courses (range 1–19) of standard-dose 5-aza. Within the pAML group, 13 patients responded [8 complete responses (CR) and 5 partial responses (PR)], and 4 patients with sAML responded (2 CR and 2 PR). Two patients in the pAML group and 1 patient with sAML subsequently received an SCT. After a median 218 days (10–792) follow-up in the pAML group, and 246 (19–995) in the sAML group, 66.7% patients with pAML and 28.6% with sAML were alive. Overall, no significant differences were seen in the rate of patients achieving CR or PR between the pAML and the sAML groups; despite this, the estimate 2-year survival was significantly higher in patients with pAML (p=0.01). Prior exposure to chemotherapy or other forms of treatment had no impact on response or survival. An abnormal karyotype (present in 12 pAML and 8 sAML patients) did not influence the response to treatment. All mutation analysis yielded negative results in sAML cases. In pAML cases, 3/20 were positive for FLT3/ITD, 5/19 for NPM1 and 1/19 for MLL. Most interestingly, NPM1 mutations in the absence of FLT3/ITD, were significantly (p=0.05) associated with improved response rate (5/5) in patients with pAML with no deaths being observed in this group. DISCUSION: Our results show a higher probability of survival after 5-aza treatment in patients with pAML compared to sAML. The presence of NPM1 mutations appeared to identify a subset of pAML patients that, despite having failed first line treatment, may be particularly sensitive to this therapy. If confirmed in larger series, this finding would be of the utmost interest for offering these patients a low toxicity rescue therapy or a maintenance strategy with his agent. Disclosures: No relevant conflicts of interest to declare.

Abstract 4267 The acceptable toxic profile of 5-aza-citidine (5-aza) allows its use in fragile or elderly patients in whom intensive chemotherapy should be avoided. Whether it is possible to take advantage of this low toxicity in patients awaiting for donor search and/or stem cell transplant (SCT) and in those experiencing leukemia recurrences after the procedure remains unknown. We analysed the clinical results of using 5-aza in these two settings to define the feasibility, safety and results of this approach. Patients and methods: From 2007 to 2011, 15 patients (11 males, 4 females) received 5-aza as last treatment prior to an allogeneic SCT (n=13) or as rescue after an early post-transplant relapse (n=2) at our centre. Diagnosis was MDS in 3 cases (median age 62; range 58–63) and AML in 12 cases (median age 58; range 37–67). Patients with MDS received a median of 6 courses of 5-aza (range 3–8) as the only treatment from diagnosis, except for one patient who had received panobinostat prior to 5-aza. Amongst patients with AML, 12 patients received 5-aza either as treatment for AML (2/12) or after remission (8/12) because of the high relapse risk while awaiting for a suitable donor to be found. Two patients with AML received 5-aza as treatment for early post-SCT relapse. AML patients treated with 5-aza before SCT received a median of 5 courses (range 1–19), whilst patients receiving treatment for relapse received 1 and 3 courses, respectively. Ten patients received a nonmyeloablative conditioning regimen, 1 received a conventional conditioning regimen, 2 patients are still in the process of donor search and the other 2 patients received 5-aza after an autologous stem cell transplantation relapse. RESULTS: All MDS patients engrafted and are in complete remission (CR) after a median of 696 days of follow-up (range 377–1227). One of those patients died because of aGvHD. Nine of 12 AML patients receiving 5-aza prior to SCT are alive after a median 373 days follow-up (133–995). One patient showing refractoriness to 3 different lines of treatment died from disease progression after 211 days. All patients receiving 5-aza as treatment for early relapse are dead, 41 and 401 days after starting treatment. Most interestingly, AML patients receiving 5-aza as maintenance of an already-achieved CR while awaiting transplantation did not experience disease progression despite the median time they remained on this treatment was prolonged (9 months). Graft-versus-host disease ≥ grade II was seen in 3 patients. No graft failures were seen and all patients who received an allogeneic stem cell transplantation remain in complete response. CONCLUSION: The use of 5-aza for maintaining or achieving a response in patients with AML who are awaiting SCT is a safe procedure and adds flexibility to schedule the treatment without the need to administer potentially toxic therapy. The use of 5-aza before transplant did not appear to interfere either with engraftment, incidence of GvHD or short-term relapse after transplant. 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 4711 Aim To analyze the prognostic impact of cytogenetics and main molecular abnormalities on the achievement of complete remission (CR), in patients with primary (de novo) AML (M3 excluded). Patients and Methods Between december-2003 and july-2009, 605 patients up to 70 years-old were included in the CETLAM AML-03 protocol. Induction therapy consisted in one or two courses of idarubicin, intermediate dose ara-C and etoposide, in addition to G-CSF priming from day 0. Cytogenetics classification was as in the MRC studies: Favorable prognosis (FP), intermediate (IP) and adverse (AP). In the IP group, molecular analysis of NPM1 (NPM1+) mutations, CEBPA mutations and internal tandem duplication of FLT3 gene (ITD/FLT3) was performed. In the AP group, the absence (MK-) or presence of monosomal karyotype (MK+) was studied; MK+ was defined as 2 or more autosomal monosomies, or one monosomy and ≥1 structural alteration. Results Median age of the series was 53 years (range, 16-73). In 538 (89%) patients cytogenetic data were available; of them, 64 (10,5%) had FP, 375 (61,8) IP (included 255 with normal cytogenetics) and 99 (16,3%) AP. In the FP group, 33 (5,4%) had the AML1/ETO fusion and 31 (5,1%) the CBF/MYH11. In the IP group, 121 (31%) of 279 patients studied were NPM1+, 100 (26,7%) of 346 were DIT/FLT3+ and 22 (6%) of 235 analyzed were CEBPA+. In patients with AP, 47 were MK- and 33 MK+. Overall, 447 (73,8%) of 588 patients evaluable at the moment of the analysis achieved a CR. CR rates according cytogenetics were: FP 92,2%, IP 76,5% and AP 69,4%, p=0.01. In AML, with AML1-ETO+ and CBF/MYH11+, CR rates were 91% and 93.5%, respectively; of note no chemorefractory cases were observed. In the IP group, CR rates according to mutational status were: NPM1+/FLT3- 91.2%, CEBPA+/FLT3- 94.4%, no mutations 79.1% and DIT/FLT3+ 69.7% (p=0.003). Again, no refractoriness was observed in patients in IP patients belonging to the two groups with favourable molecular profile (p=0.003). In patients with AP, CR rate was 76.1% if MK- and 65.6% if MK+ (p=0.46). The prognostic impact of the herein described cytogenetic and molecular findings was confirmed in multivariate analyses. Comments Genetic characterization is nowadays mandatory in AML. CR rate is high and refractoriness exceptional in the presence of AML1-ETO or CBF/MYH11 rearrangements, or NPM1 or CEBPA mutations without DIT/FLT3. In contrast, CR probability is lower in AP group patients, mainly in those with MK+. These patients require new strategies within clinical trials. Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures: No relevant conflicts of interest to declare.

Abstract 1003 Poster Board I-25 Introduction: Recently, the cooperative group HOVON-SAKK has refined the prognostic impact of cytogenetic abnormalities in acute myeloid leukemia (AML) by introducing the concept of monosomal karyotype (MK). This consists of ≥ 2 autosomal monosomies or one autosomal monosomy in addition to a structural alteration. In their experience, MK would explain the poor prognosis of AML with a complex karyotype. Objective: To investigate the prognostic impact of MK in patients with primary (de novo) AML enrolled in the Spanish CETLAM group protocols (AML 94/99/03). Also, to determine whether considering MK added predictive value to the cytogenetic classification of the Medical Research Council (MRC). Methods: Retrospective analysis of data from 1149 AML patients. Chromosomal formula was centrally reviewed with karyotypes being classified by the presence of MK and allocated into the MRC risk categories. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were calculated. Results: The karyotype was assessable in 904 (79%) of the 1149 cases. In 145 of the 904 cases (16%), abnormalities involving CBF gene were detected and in 437 (48%) the karyotype was normal (NK). In 253 (28%) additional patients the karyotype was not monosomal; of them, 61 (24%) belonged to the unfavorable MRC with 17 cases harboring a complex karyotype ≥ 5 abnormalities, 7 cases with rearrangements 3q, 13 cases with -7, 9 cases with 5q abnormalities and 16 cases with t(6;9)). The remaining 69 (7.7%) patients had a MK; of them, 59 (85.5%) were from the unfavorable MRC category and included 43 cases with complex karyotype ≥ 5 abnormalities, 6 cases with rearrangements 3q, 5 cases with -7, 5 cases with alterations of 5q). The following table summarizes the results in terms of CR rate, DFS and OS: Conclusions: The addition of MK to the MRC cytogenetic classification refines the prognostic prediction. In our series, the dismal outcome of patients with MK is confirmed; these patients had worse prognosis than those with adverse cytogenetics without MK. Alternative treatment strategies are mandatory for MK+ patients. Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures: No relevant conflicts of interest to declare.

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.