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Pethema Group., [Simple Summary] The use of azacitidine (AZA) and decitabine (DEC) have allowed more elderly acute myeloid leukemia (AML) patients to be treated. However, scarcely any direct comparative data exist between both drugs. This study shows no significant differences in response rates or overall survival (OS) between upfront AZA and DEC treatment in a large retrospective with long-term follow-up cohort of AML patients. However, we identified for the first time the baseline characteristics of patients benefitting from AZA vs. DEC in terms of responses, 120-day mortality and OS. We also show differences in salvage treatment patterns and outcomes after failure to both hypomethylating agents in a real-life setting. Taken together, these findings could help to select the most appropriate hypomethylating agent in monotherapy., The hypomethylating agents, decitabine (DEC) and azacitidine (AZA), allowed more elderly acute myeloid leukemia (AML) patients to be treated. However, there are little direct comparative data on AZA and DEC. This multicenter retrospective study compared the outcomes of AZA and DEC in terms of response and overall survival (OS). Potential predictors associated with response and OS were also evaluated. A total of 626 AML patients were included (487 treated with AZA and 139 with DEC). Response rates were similar in both groups: CR was 18% with AZA vs. 23% with DEC (p = 0.20), CR/CRi was 20.5% vs. 25% (p = 0.27) and ORR was 32% vs. 39.5% (p = 0.12), respectively. Patients with leukocytes < 10 × 109/L, bone marrow blasts < 50% and ECOG ≥ 2 had higher ORR with DEC than with AZA. OS was similar in both groups: 10.4 months (95% CI: 9.2–11.7) vs. 8.8 months (95% CI: 6.7–11.0, p = 0.455), for AZA and DEC, respectively. Age (≥80 years), leukocytes (≥ 10 × 109/L), platelet count (

We have analyzed treatment patterns and outcomes of relapsed/refractory(R/R) FLT3mut AML adult patients registered in our institutional data base between 1998 and 2018. Overall, 147 patients were evaluable: 34 from 1998 to 2009, 113 from 2010 to 2018. Salvage treatments were intensive chemotherapy ( n = 25, 74%), and supportive care ( n = 9, 26%) in the 1998-2009 period, and intensive chemotherapy ( n = 63, 56%), hypomethylating agent ( n = 7, 6%), low-dose cytarabine-based ( n = 8, 7%), clinical trial ( n = 16, 14%) and supportive care ( n = 19, 17%) in the 2010-2018 period. Complete remission (CR) or with incomplete recovery (CRi) rate was 44%, 49% among patients treated intensively (vs 30% with non-intensive p = 0.005). Median overall survival since first R/R was 5.8 months, and 16.3 months in subjects receiving an allo-HSCT in CR/CRi after first salvage (vs 3.8 in the remaining patients p < 0.0001). Clinical outcomes of R/R FLT3mut AML remain unsatisfactory. Inclusion in clinical trials and expanding options could lead to improved outcomes.

Este artículo se encuentra disponible en la siguiente URL: https://www.nature.com/articles/s41598-020-61589-9.pdf En este artículo también participan: David Hervás-Marín, Eva Barragán, Rosa Ayala, Marta LLop, María López-Pavía, Inmaculada Rapado, Alex Neef, Alejandra Sanjuan-Pla, Claudia Sargas, Elisa Gonzalez-Romero, Mireia Boluda-Navarro, Rafa Andreu, Leonor Senent, Pau Montesinos, Joaquín Martínez-López, Miguel Angel Sanz, Guillermo Sanz y José Cervera. Nearly 50% of patients with de novo acute myeloid leukemia (AML) harbor an apparently normal karyotype (NK) by conventional cytogenetic techniques showing a very heterogeneous prognosis. This could be related to the presence of cryptic cytogenetic abnormalities (CC A) not detectable by conventional methods. The study of copy number alterations (CNA) and loss of heterozygozity (LOH) in hematological malignancies is possible using a high resolution SNP-array. Recently, in clinical practice the karyotype study has been complemented with the identification of point mutations in an increasing number of genes. We analyzed 252 de novo NK-AML patients from Hospital La Fe (n = 44) and from previously reported cohorts (n = 208) to identify CCA by SNP-array, and to integrate the analysis of CCA with molecular alterations detected by Next-Generation-sequencing. CCA were detected in 58% of patients. In addition, 49% of them harbored CNA or LOH and point mutations, simultaneously. Patients were grouped in 3 sets by their abnormalities: patients carrying several CCA simultaneously, patients with mutations in FLT3, NPM1 and/or DNMT3A and patients with an amalgam of mutations. We found a negative correlation between the number of CCA and the outcome of the patients. This study outlines that CCA are present in up to 50% of NK-AML patients and have a negative impact on the outcome. CCA may contribute to the heterogeneous prognosis.

INTRODUCTION The hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), have made it possible to treat more elderly patients with acute myeloid leukemia (AML). Both HMAs have demonstrated efficacy in monotherapy and in combination with targeted therapies. However, there is little direct comparative data on AZA and DEC in first-line treatment, and we do not know which group of patients might benefit from each drug. Results of the full analysis set (FAS) were presented previously (Labrador J, et al. ASH 2020). Here, we report long-term clinical efficacy from prespecified patient subgroup analyses. METHODS We conducted a retrospective study to compare real-life clinical outcomes between AZA and DEC in patients with AML ineligible for intensive chemotherapy included in the PETHEMA registry, and analyzed clinical variables associated with response and overall survival (OS) between AZA and DEC. RESULTS A total of 626 patients were included for the FAS between 2006 and 2019. 487 (78%) received AZA and 139 (22%) received DEC. Baseline characteristics were comparable in both groups, except for the percentage of bone marrow blasts (44% vs. 34% in the DEC group compared to AZA, p=0.010). In the FAS, there was no difference in the CR, CR/CRi or ORR (CR/RCi + PR) rate: 18%, 20.5% and 32% with AZA vs. 23%, 25% and 39.5% with DEC (p=0.20, p=0.27 and p=0.12). In the subgroup analysis, DEC was associated with higher CR/CRi rate than AZA in patients with ECOG ≥ 2 (95% CI: 0.088 - 0.801), bone marrow blast count < 50% (95% CI: 0.293 - 0.965), secondary AML (95% CI: 0.223 - 0.918) and adverse cytogenetics (95% CI: 0.171 - 0.857) (Figure 1A). DEC was associated with higher ORR rate than AZA in patients with ECOG ≥ 2 (95% CI: 0.116 - 0.782), leukocytes < 10 x10 9/L (95% CI: 0.321 - 0.920) and bone marrow blasts < 50% (95% CI: 0.321 - 0.920) (Figure 1B) 120 days-mortality was 25.4% after AZA and 27.1% after DEC, p=0.70. Patients who did not achieve at least a PR had significantly higher 120-day mortality with both HMAs (OR 8.85 and 8.22 for AZA and DEC, respectively). In the subgroup analysis, patients with leukocytes ≥ 10 x10 9/L (95% CI: 1.069 - 4.157) and those with estimated glomerular filtration rate (eGFR) ≥ 45 mL/min/1.73m 2 (95% CI: 1.249 - 4.664) had higher 120-day mortality with DEC than with AZA (Figure 1C) With a median follow-up of 12 months, median OS was 10.4 months (95% CI: 9.2 - 11.7) for AZA vs. 8.8 months (95% CI: 6.7 - 11.0) for DEC (p = 0.455). The subgroup analysis revealed that patients ≥ 80 years (95%: CI 1.005 - 2.341), with leukocytes ≥ 10 x10 9/L (95% CI 1.039 - 2.062), platelet count CONCLUSIONS Our study provides real-life data on the outcomes of AML patients treated with AZA compared to DEC in a large retrospective cohort with long-term follow-up. In addition, we identify for the first time some baseline characteristics that could benefit from AZA or DEC in terms of responses, 120-day mortality and OS. These findings could help us to choose the most appropriate HMA in monotherapy or for the development of new combinations. Figure 1 Figure 1. Disclosures de la Fuente: Novartis: Research Funding; Abbie: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Tormo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Montesinos: Forma Therapeutics: Consultancy; Tolero Pharmaceutical: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Glycomimetics: Consultancy; Agios: Consultancy; Stemline/Menarini: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau.

INTRODUCTION The hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), are a common choice for initial treatment in elderly patients with acute myeloid leukemia (AML). However, about 60% of patients are resistant and most will relapse after a complete response (including CR/CRi). Although relapse or refractoriness to HMAs is very common, very little is known about the therapeutic management of these relapsed or refractory AML (RR-AML) patients after HMAs. METHODS We conducted a retrospective study to describe and compare salvage treatment patterns and real-life clinical outcomes of those AML patients treated upfront with AZA or DEC included in the PETHEMA-AML Registry. RESULTS Between 2006 and 2019, we included 626 AML patients treated in first-line with HMAs, 487 (78%) received AZA and 139 (22%) received DEC. Overall response rate (CR/CRi+PR) was 33.7%, 32% in the AZA vs 39.5% in the DEC group (p=0.120). Patients treated with DEC had a higher median relapse free survival than those treated with AZA (25.6 vs. 17.5 months, p=0.027). No differences were observed in the overall survival between AZA and DEC. We observed 59.7% resistance after HMAs, 60% after AZA and 58.8% after DEC; and 6.5% had other type of response (< PR), 7.9% with AZA and 1.6% with DEC. In addition, 76/121 patients who achieved CR/CRi (62.8%) relapsed, 66/90 in the AZA group (73.3%) vs 10/31 in the DEC group (32.3%) (p=0.000). After relapse or resistance, 74.5% of patients received supportive care only (BSC), which included patients receiving transfusions and other supportive measures, including oral agents to control the white blood cell counts; 71.5% of patients in the AZA group and 84.3% of patients in DEC group (p= 0.004). No differences were observed in baseline characteristics at diagnosis of patients treated with BSC only, except for a higher proportion of patients with adverse cytogenetic risk in the AZA group (46.6% vs. 33.7%, p=0.039). Only 135 patients received a salvage therapy, 116 in the AZA group and 19 in the DEC group. Thirty-five out of 135 RR-AML treated patients (26%) continued receiving HMAs: 31 (26%) in the AZA group and 4 (22%) in the DEC group. In the AZA group, 19 (16%) patients continued with AZA and 12 (10%) switched to DEC, while in the DEC group 2 patients (11%) continued with DEC and 2 (11%) switched to AZA. Fifty-one patients (37.8%) received FLUGA (fludarabine and Ara-C), FLAG-IDA-Lite (fludarabine, Ara-C and idarubicin), Low-dose Ara-C or other non-intensive regimens, 43 patients (37%) in the AZA group and 8 (42%) in the DEC group. Other salvage therapies were administered in 34% of patients (33% in the AZA group and 37% in the DEC group). Salvage therapy was not available in 1% of patients. Response assessment was available in 113/135 of RR-AML treated patients, 98 in the AZA group and 15 in the DEC group. In the AZA group, 13.2% of the patients achieved a CR (n=13), 5.1% achieved CRi (n=5), 6.1% achieved a PR (n=6), 65.3% resistance (n=64) and 8.1% died (n=8). However, no patients in the decitabine group responded to salvage therapy, 86.7% resistance (n=13) and 13.3% died (n=2). If we exclude those patients who died before response was evaluated, the ORR (CR/CRi+PR) after salvage therapy was statistically significant between AZA (n=24/90, 26.7%) and DEC group (n=0/13, 0%), p=0.035. CONCLUSIONS This study shows and compares, for the first time to our knowledge, the patterns of salvage therapy in patients with RR-AML treated upfront with HMAs. Despite the similar response rate with both HMAs, the relapse free survival was lower after AZA. The absence of responses in patients with RR-AML initially treated with DEC could justify the similar OS between AZA and DEC observed. However, we should be very cautious due to the low number of RR-AML patients treated. Disclosures de la Fuente: BMS: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Abbie: Consultancy, Speakers Bureau; Novartis: Research Funding. Tormo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Montesinos: Forma Therapeutics: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline/Menarini: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Glycomimetics: Consultancy; Tolero Pharmaceutical: Consultancy; Agios: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau.

The incidence of SF3B1 mutations in patients with RARS is high. Recently, it has been shown that SF3B1 and DNMT3A mutations overlap more often than expected, although it is not clear how this could affect the disease. We studied SF3B1 and DNMT3A in 123 RARS patients: 101 out of 123 samples (82%) had somatic mutations in SF3B1, and 13 of them (13%) showed a co-mutation (SF3B1mutDNMT3Amut). All co-mutated patients had a normal karyotype, and 12 of them (92%) were lower-risk patients (IPSS and IPSS-R). Despite their favorable profile, SF3B1mutDNMT3Amut patients showed a higher RBC transfusion dependency (92% versus 48%, p = .007), a shorter overall survival (OS) (median, 30 versus 97 months, p = .034), and a higher risk of progression to acute myeloid leukemia (AML) at 5 years (25% versus 2%, p = .023) than SF3B1mutDNMT3Awt patients. In conclusion, DNMT3A mutations are present in a significant proportion of SF3B1mut patients with a negative clinical impact.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials. © 2016 by The American Society of Hematology.

Keywords: mantle cell lymphoma; MCL International Prognostic Index; prognostic factors

INTRODUCTION: A sizable proportion of elderly acute myeloid leukemia (AML) patients receive frontline hypomethylating agents (HMAs), namely azacitidine (AZA) and decitabine (DAC), as they are deemed unfit for intensive chemotherapy (ICT) by their treating physicians. A foreseeable high early death (ED) rate and lack of overall survival (OS) benefit under ICT are the main drivers for this decision. Several groups have published different predictive tools for ED or OS in elderly patients receiving ICT but, since ED in patients treated by HMAs is lower, the research activity has been restricted to OS in this population. METHODS: 415 elderly AML patients (264 M, 152 F) aged 61-90 receiving frontline HMAs (AZA 297, DAC 118), either in daily practice or within clinical trials (AZA 27, DAC 17), with complete relevant clinical information (see Table I) were available from the PETHEMA epidemiologic AML registry (NCT02006004). We analyzed the predictive value for ED (8wk) of the prognostic factors for OS/ED in AML included in the Walter, MRC/LRF, ALFA and ALMA scoring systems, namely age, WBC count, performance status (PS), MRC 2010 cytogenetics, platelet count and secondary disease, as well as the type of HMA. The potential predictors were categorized following previous published models (Walter, MRC/LRF, ALFA, ALMA). Cumulative early death rate at 8 weeks was calculated by the life-time method and the relevant strata were tested for univariate significance by the Wilcoxon test. All significant covariates were included in a Cox multivariate regression model and those significant for death at 8wk were included in a new predictive tool (HMA-EDS). Patients were assigned randomly in a 1:1 ratio to a training cohort (TC) and a validation cohort (VC). The different scoring systems (Walter, MRC/LRF, ALFA, ALMA, HMA-EDS) were checked for their prognostic impact on ED. Finally the 95% CI for the expected death rate at 8wk for the different strata of the new model was calculated for the full patient series. RESULTS: 51 patients out of 415 died and 13 were lost to follow-up before day 56 (cumulative ED rate at 8wk 13%, 95%CI 9-17%). Age, cytogenetics, secondary AML, platelet count and type of HMA were not significantly associated to ED. PS and WBC count strata confirmed their prognostic utility both in univariate and multivariate analysis (Table II). We developed the HMA-EDS by adding WBC (cutoffs 10 and 50, scores 1/2/3) and PS (0-1/2-4, scores 0/1) that classified patients in low-risk (score 1-2/ 84.6% of patients) and high risk (3-4/ 15.4% of patients) strata. When the prognostic utility for ED in the TC and the VC for the different scoring AML systems were checked, only HMA-EDS predicted ED in both cohorts (see Table III). The new EDS discriminates 2 different strata for ED at 8wk in unfit AML patients treated by HMA (see Figure 1 & Table III), namely a lower-risk group (ED rate 10%, 95% CI 6-14) and a high-risk group (ED rate 26%, 95% CI 14-38). CONCLUSIONS: WBC count and PS are the main predictors for ED in unfit AML patients treated by HMAs. A new tool (HMA-EDS) discriminates two different risk groups and supersedes other previously published prognostic systems (Walter's, Wheatley's MRC/LRF, ALFA and ALMA) for this purpose. This score could be useful to select patients for front-line HMA or even HMAs-based combination therapies, given that several cycles are usually needed to achieve a clinical response. We suggest that other patient-related covariates such as geriatric assessment be checked in future studies. Disclosures Ramos: Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Honoraria. Fernandez:Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations.

The prognostic value of cytogenetics in adult acute lymphoblastic leukemia (ALL) is not as established as in childhood ALL. We have analyzed the outcome and prognostic value of karyotype in 84 adults diagnosed with Philadelphia-negative ALL from a single institution that received induction chemotherapy and had successful karyotype performed. The most frequent finding was normal karyotype in 35 (42%) cases, followed by aneuploidies in 20 cases (24%) and t(4;11)(q21;q23)/MLL/AF4 in 5 (6%), and the remaining 24(27%) cases carried miscellaneous clonal abnormalities. The group of patients with t(4;11)(q21;q23)/MLL/AF4, hypodiploidy and low hyperdiploidy (less than 50 chromosomes) showed a worse outcome than those with normal karyotype and miscellaneous abnormalities in terms of overall survival (OS) (3 years OS; 47% vs. 13%, p = 0.014) and relapse-free survival (RFS) (3 years RFS; 44% vs. 27%, p = 0.005). Other cytogenetic prognostic classifications reported to date were tested in our series, but any was fully reproducible. In conclusion, karyotype is a useful tool for risk assessment in adult ALL. We have confirmed the bad prognosis of t(4;11)(q21;q23)/MLL/AF4 and hypodiploidy. Besides, low hyperdiploidy could also define a high-risk group of patients who might be candidates for more intensive treatment.

The current classification system for Myelodysplastic Syndromes lumps all therapy-related (tMDS) into one subgroup assuming all tMDS had the same poor prognosis. We have put together a database including 2032 patients with a diagnosis of tMDS from several different IWG centers and the MDS clinical research consortium. With the idea of developing an individual scoring system for tMDS, we decided to start by optimizing the cytogenetic part of the IPSSR. First, we did an extensive review of karyotypes. Finally, 1245 patients had complete data and correct ISCN formula to be used for score development. We could show regarding karyotypes there are very limited differences between primary and tMDS. Mainly the distribution of risk groups differs with complex occurring more (37%) and normal karyotypes occurring less frequent, although still accounting for 30%. There are few exceptions that are relatively special for tMDS, like translocations including 11q23. A few karyotypes are less frequent; therefore, we could not evaluate the value of IPSS-R cytogenetics for all karyotypes. However, if we apply IPSS-R cytogenetics to our patient cohort, we can separate 5 different risk groups as in pMDS. We tested the performance of the score by using the Dxy. As main endpoint we chose transformation-free survival giving better information about the severity of the disease compared to the single endpoints survival and AML transformation that where calculated for completeness as well. The Dxy for the IPSS-R cytogenetic part is 0.31 for transformation-free survival. This indicates an effective prognostic performance although not as good as in pMDS. Several attempts were done to develop a tMDS specific cytogenetic score. The best draft scoring component achieves a Dxy of 0.33. Counting the number of aberrations achieves a score of 0.30. If normal clone present or not is added, the performance of this very simple model is improved with a Dxy of 0.32. As we could show, all these different approaches lead to a comparable performance. One can argue that still regarding a few karyotypes the prognostic impact is slightly different between p and tMDS (e.g. +8). On the other hand, the most practical approach seems to be to adopt the original cytogenetic part of the IPSS-R for further score development since clinicians do not need to use different scoring systems for different MDS subtypes. While the final analyses for the development of a tMDS specific risk score are currently under way, extensive calculations regarding the performance of different scores like WHO- (Dxy 0.24), FAB-classification (Dxy 0.19), WPSS-R (Dxy 0.35), IPSS-R (Dxy 0.37), and IPSS-R+age (Dxy 0.36), show all these systems can separate different risk groups within our cohort. However, these results also show an inferior performance of the scoring systems in t compared to pMDS. There are multiple possible reasons for this. The most important seem to be tMDS patients are often not cured from the primary disease and its disease specific risk of death should ideally be considered. Unfortunately, we don't have that data. And second, we included treated as well as untreated patients. It seems not to be feasible otherwise since the selection bias for old unfit patients would be unacceptable. We could show already in pMDS that the score performances are considerably worse if we analyze treated patients and the score performance in our cohort is better if limited to untreated patients. To conclude, we can say existing classification and scoring systems work in tMDS and can separate groups with clearly different risk for death and transformation. Although we could not develop a tMDS specific cytogenetic score this could be seen positively since it underlines tMDS do not seem to be much different regarding disease specific risk. This should initiate a discussion of a revision of the WHO-classification and encourage clinicians to use the existing tools for risk assessment and treatment decisions. A simple solution could be to use the WHO classification for pMDS and precede each subgroup with a t, like tMDS-SLD, and so on. Such an approach would be of importance for patients falsely classified as tMDS. After all this classification is done according to anamnestic information only and sporadic cases cannot be excluded. Until now, in the first analyzes performed with the final tMDS-database, we did not find any indication that risk factors established in pMDS would lose or change their meaning in tMDS. Figure. Figure. Disclosures Komrokji: Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. List:Celgene: Research Funding. Roboz:Orsenix: Consultancy; Eisai: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Janssen Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Argenx: Consultancy; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Cellectis: Research Funding; Daiichi Sankyo: Consultancy; Sandoz: Consultancy; Otsuka: Consultancy; Daiichi Sankyo: Consultancy; Eisai: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Celgene Corporation: Consultancy; Cellectis: Research Funding; Orsenix: Consultancy; Aphivena Therapeutics: Consultancy; Otsuka: Consultancy; Jazz Pharmaceuticals: Consultancy; Sandoz: Consultancy; Roche/Genentech: Consultancy; Aphivena Therapeutics: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Bayer: Consultancy; Astex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; AbbVie: Consultancy. Döhner:Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Platzbecker:Celgene: Research Funding. Lübbert:TEVA: Other: Study drug; Celgene: Other: Travel Support; Cheplapharm: Other: Study drug; Janssen: Honoraria, Research Funding. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stauder:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva: Research Funding. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression.

BRAF V600E is a point mutation located in exon 15 of the serine/threonine protein kinase B-Raf (BRAF) gene, and was originally discovered to recurrently occur in melanoma [1]. Because of this acqui...

Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1(R132) and 6% with IDH2(R140)), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, [corrected] or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

An abnormal increase of nonleukemic blastic-appearing lymphocytes in bone marrow (BM) specimens has been reported after unrelated cord blood transplantation (UCBT). This study analyzed the incidence, chronology, biological features, and clinical significance of elevated numbers of these cells in a series of 165 consecutive adult patients demonstrating myeloid engraftment after myeloablative UCBT in a single institution. The patients' BM samples were routinely evaluated by cytomorphology at different time points after UCBT. When ≥5% of blastic-appearing cells were detected by cytomorphology in the BM, samples were also evaluated by multiparametric flow cytometry to characterize these cells. Systematic chimerism analyses of BM samples using PCR amplification of short tandem repeat markers were performed. Forty-three patients (cumulative incidence, 26.1%) demonstrated ≥5% of nonmalignant blastic-appearing cells in BM after a median of 101 days after UCBT (range, 28-377 days). All of these patients had full-donor chimerism and a clinical course without leukemic relapse. Multiparametric flow cytometry analyses performed in 36 of the 43 patients showed a polyclonal expansion of B lymphocytes with a broad spectrum of maturation stages. An increased number of nonmalignant blastic-appearing cells was significantly associated with a high number of lymphocytes infused at the time of UCBT and with low rates of acute and chronic extensive graft-versus-host disease, suggesting a potential immunoregulatory role of these cells. The observation of ≥5% nonmalignant blastic-appearing cells in BM samples after myeloablative UCBT is frequent, and these should be distinguished from malignant blasts.

Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

INTRODUCTION Anemia is the most frequent cytopenia in lower-risk MDS. Erythropoietic-stimulating agents (ESAs) are commonly used in these patients. The use of ÒclassicalÓ parameters (EPO and ferritin levels) and the revised IPSS (IPSS-R) has been proposed1 (SantiniÕs score) to predict response to ESAs and overall survival (OS) among patients with lower risk MDS by IPSS and a favorable Nordic group score2. OBJECTIVES The main objective of the study was to evaluate overall response rate (ORR) to ESAs and OS according to the proposed SantiniÕs score in an independent and large cohort of anemic lower risk MDS patients receiving treatment with ESAs. METHODS Data from 530 anemic patients with low/int1 risk IPSS de novo MDS (according to FAB and WHO criteria) and sufficient follow-up data available were recorded in Spresas3 (SPanish Registry of Erythropoietic Stimulating Agents Study from GESMD). Two hundred and twenty six patients (42.6% of the patients) were selected according to specific criteria regarding the published SantiniÕs score1: Hb level 200mU/mL(=1), serum ferritin (SF) >350 ng/mL(=1) and IPSS-R very low=0, low=1, intermediate=2 and high=3) yielded a score ranging from 0 to 5. ESAs response rate and overall survival were analysed according to these score. Response to treatment was evaluated according to IWG 2006 response criteria and a multivariate logistic regression analysis was used to identify independent predictors of erythroid response (ER). OS were defined as the time between diagnosis and the corresponding event or last follow up (Feb 2015) and were analyzed using univariable and multivariable Cox proportional hazards regression methods. RESULTS Median age was 77 years (interquartile range [IQR] 25%-75%: 71-83 y), median Hb level at start of treatment was 10 g/dL (IQR25-75: 9-10), median EPO level was 90 (IQR25-75: 27,25-108) and median ferritin level was 338,5 (IQR25-75: 146,5-568,75). Among 139 patients with this data available, 85 patients (61,1%) were RBC transfusion dependent before ESAs treatment. Median time from diagnosis to ESAs treatment was 82 (IQR25-75: 27-353) days. According to the IPSS, 68.6% (N=155) and 31.4% (N=71) were in low and Int-1 risk groups, respectively. Regarding IPSS-R, 23% (N=52), 66.8% (N=151), 9.7% (N=22) and 0.4% (N=1) were in very low, low, intermediate and high risk, respectively. ORR to ESA treatment was 71.2% (N=161), with a median duration of response of 2.06 years. Prognosis factors of ER showed a trend toward to a higher ER among patients in the lower IPSS-R (P>0.05), low IPSS (p=0.039) and lower EPO levels (p According to SantiniÕs score, 11.5%(N=26), 42.9%(N=97), 25.8%(N=81), 8%(N=18) and 1.8%(N=4) of the patients were in the 0, 1, 2 3 and 4 score. Erythroid response was better for patients in the lower scores, with response rates of 73.1%, 82.5%, 65.4%, 50% and 0%, for patients in 0, 1, 2, 3 and 4 score, respectively (p CONCLUSIONS The present study confirms that SantiniÕs score is useful to identify patients with a higher probability of response to ESAs and better OS among lower risk MDS patients with an expected favorable response to ESA according to Nordic group score. Spresas study was partly supported by Janssen 1.-Santini et al, Blood 122(13), 2013. 2.-Hellstršm-Lindberg, Br J Haematol 120(6), 2003. 3.-D'ez Campelo, EHA 2015 meeting, P244. Disclosures Díez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Off Label Use: Use of erythropoietic stimulating agents for anemia in patients with myelodysplastic syndromes. Ramos:JANSSEN: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Consultancy, Honoraria; NOVARTIS: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Falantes:Celgene: Honoraria. Garcia:Celgene: Research Funding. Sanz:JANSSEN CILAG: Honoraria, Research Funding, Speakers Bureau.

Recent genome-wide sequencing eff orts have led to the identifi cation of a number of new candidate genes involved in the pathogenesis of myeloid neoplasms. Among them, variations in isocitrate dehydrogenase genes ( IDH1 and IDH2 ) have been identifi ed in several hematologic malignancies. IDH1 and IDH2 mutations have been found in 8 – 10% and 15% of patients with acute myeloid leukemia, res pectively, especially in those having normal karyotype (NK-AML). Both gene mutations were recently also reported in myelodysplastic syndromes and chronic myeloproliferative disorders, and were associated with a high rate of leukemic transformation and poor prognosis [1 – 3]. A few studies have been published on somatic mutations of IDH1 and IDH2 in chronic myelomonocytic leukemia (CMML). Th e reported incidence of these mutations in CMML ranged from 0 to 9% [3 – 7], although their prognostic relevance remains uncertain. All reported IDH1 mutations involve the R132 residue, while IDH2 mutations involve either the R140 or the R172 residue. Mutations in IDH decreased affi nity to isocitrate, displaying a neomorphic catalytic activity toward α -ketoglutarate, producing an accumulation of 2-hydroxyglutarate [2,8 – 10]. It is currently believed that these intracellular changes facilitate oncogenic pathways, including activation of hypoxia-inducible factor-1 α (HIF-1 α ). Nevertheless, their relative contribution is still unclear [2].

Background: Clinical, morphological and genetic characteristics of chronic myelomonocytic leukemia (CMML) are heterogeneous and vary from a myelodysplastic predominant profile to a myeloproliferative one. CMML has a highly variable course, with a median overall survival (OS) of 20 months and 15-30% of progression to acute myeloid leukemia (AML). Cytogenetic abnormalities are present in only 20-40% of cases. CMML-specific cytogenetic risk classification stratifies karyotypes into three groups: low risk (normal karyotype and isolated loss of Y chromosome, -Y), poor risk (trisomy 8, monosomy 7, 7q deletion and complex karyotype) and intermediate risk (all other chromosomal abnormalities). According to this model, 65-85% of patients fall into the low risk cytogenetic category. The aim of this study was to characterize type, frequency and prognostic impact of cytogenetic alterations detected by SNP arrays (SNP-A) in a series of 128 patients with CMML and low risk cytogenetic features or no metaphases. Methods: A retrospective study was performed on 128 patients with CMML. Cases with normal karyotype (n=120), isolated -Y (n=4) and no metaphases (n=4) were selected. Median age at diagnosis was 73 years (range 39-98), there was a 2.4:1 male predominance and 22.4% (28/125) of cases progressed to AML. Median follow up of patients was 26 months (range 1-115). Morphological WHO subtypes were CMML-1 in 104 (81%) cases and CMML-2 in 24 (19%). According to the FAB criteria 82 (64%) cases were included in the myelodysplastic variant (CMML-MD) and 46 (36%) in the myeloproliferative one (CMML-MP). High density SNP-A (Cytoscan HD, Affymetrix) were performed using DNA extracted from bone marrow (n=124) or peripheral blood (n=4) samples at diagnosis. The statistical analysis was performed with SPSS. Kaplan-Meier method was used for OS and progression-free survival (PFS) analysis and log-rank test was used for comparisons between groups. Results: SNP-A revealed novel chromosomal alterations (copy number alterations, CNA, and loss of heterozygosity, LOH) in 66% (85/128) of cases. Among the abnormal cases (CNA plus LOH), 1 alteration was detected in 65% (55/85) of cases, 2 in 20% (17/85) and ≥3 in 15% (13/85). The median size of the affected genome for CNA and LOH was 759 Kb (range 0-142Mb). CNA were detected in 38% (48/128) of cases, most of them being gains and losses smaller than 10Mb. Only 7 CNA were larger than 10Mb, four of them corresponded to patients with isolated -Y and three were novel alterations that had not been detected by conventional G-banding cytogenetics. Most affected regions (detected in 5 cases) were gains in chromosomes 3q, 8p and 21q as well as losses in chromosomes 10q and 12p. LOH were detected in 39% (50/128) of patients. Interstitial LOH larger than >25Mb were detected in 30% (39/128) of cases. Recurrent interstitial LOH were detected in: 4q24-4q35 region (12 cases), involving TET2 gene, which is mutated in 40-50% of CMML; and in chromosome 11 (9 cases), 7 of which included 11q13.3-11q25 region, involving CBLgene, mutated in 5-20% of CMML. Although the number of total alterations (1, 2 or ≥3) do not have a survival impact, we found a significant correlation between the size of the affected genome (≥30 Mb, including CNA and LOH) and a poorer OS (OS at 3 years 26% vs. 46%, P=0.039). Regarding type of alterations, cases with interstitial LOH had lower OS than cases without LOH (OS at 3 years 19% vs. 45%, P=0.049). Cases with high risk alterations as defined by CMML-specific cytogenetic risk classification (losses in 7q, gains in 8q and ≥3 CNA) had lower PFS than patients with other aberrations (PFS at 1 year 76% vs. 93%, P=0.036), although statistical significance was not reached for OS (OS at 3 years, 27% vs.49%, P=0.096). Conclusions: SNP-A in CMML patients with low risk cytogenetic features or no metaphases has led to the detection of chromosomal alterations in 66% of cases. Using this technique the prognosis can be better defined, and we can detect a group of patients with worse outcome who could be considered for more intensive treatment. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519; PI 11/02010); RTICC, FEDER (RD12/0036/0044; RD12/0036/0014); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras, Obra Social “La Caixa” and Celgene Spain; NHRI-EX103-10003NI, Taiwan. Footnote: Francesc Solé and Lurdes Zamora contributed equally Disclosures Xicoy: Celgene: Honoraria. Sole:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background Therapy-related Myelodysplastic Syndromes (t-MDS) are those MDS occurring after cytotoxic and/or radiation therapy administered for a prior neoplastic or non-neoplastic disorder. Their prognosis is generally very poor. The commonly used risk prognostic models for MDS (IPSS and IPSS-R) are not validated in this entity as they were developed after the exclusion of therapy-related cases (Greenberg et al. Blood 1997; Greenberg et al. Blood 2012). Aims The main aims of this study are: a) to report clinical findings and overall survival on 233 patients with t-MDS, and to compare them with a large series of de novo cases; b) to test if IPSS-R is applicable to t-MDS patients. Patients and methods The study is based on the Spanish Registry for MDS, a retrospective database that includes more than 10000 cases. The investigators were asked to fill in a questionnaire regarding prior disease (PD) and prior therapy in those cases reported to be t-MDS. Herein are described the clinical features and overall survival of the first 233 cases with the required information, and compared with patients with de novo MDS from a single center series (n=725). Log Rank test was applied to asses IPSS-R in t-MDS group. Results The 233 reported patients were diagnosed between January 1993 and February 2014. The series includes 104 women (44,6%) and 129 men (55,4%). One hundred and two patients (43.9%) had a primary hematologic malignancy, 119 (51%) had a solid tumor, and 12 (5.1%) received cytotoxic therapy for autoimmune disorders. Ninety eight patients (42.6%) received only chemotherapy (CT), 45 (19.6%) received only radiotherapy (RT), 44 (19.1%) received combined modality treatment (CMT), and 43 (18.6%) received an autologous stem cell transplantation (ASCT). The median time of latency between PD and diagnosis in t-MDS group was 4.56 years (range: 0.03-29.63) in patients previously treated with CT or CMT, significantly lower than the observed after RT (8.54; range 0.83-23.02) or ASCT (8.64; range 2.87-28.32) groups (p=0.023 and p Conclusions In conclusion, our data supports the notion that t-MDS may contain entities with heterogeneous prognosis due to a diverse biological basis. The capacity of the IPSS-R for separating good prognostic from bad prognostic cases shown in this study could be very useful in the clinical setting in order to offer risk-adapted treatments to patients, although the development of a specific prognostic score for these entities would be needed. Disclosures No relevant conflicts of interest to declare.

Background Disease karyotype is the parameter with the strongest prognostic impact in the IPSS-R. Around 50% of MDS patients have a normal karyotype. Conventional G-banding cytogenetics has a resolution of 5-10 Mb. SNP microarrays is a genomic array with a higher resolution than cytogenetics and allows the detection of small copy number alterations (CNA) and absence of heterozygosity (AOH). Our hypothesis is that in MDS patients with normal karyotype small cryptic alterations might exist, undetectable by conventional karyotyping, but with an impact on patient's outcome. Our aim is to study a series of MDS patients with normal karyotype belonging to the IWG-PM consortium (MDS Foundation) by SNP microarrays. Herein, we describe the preliminary findings as recruitment is ongoing and complete results will be presented at the meeting. Patients and methods We have recruited microarray data of 125 MDS patients with normal karyotype from centers belonging to the IWG-PM of the MDS Foundation. All cases included were studied with SNP array from Affymetrix®. We collected clinical, biological and microarray data and we studied their impact on overall survival (OS) and acute myeloid leukemia (AML) evolution in 73 patients. The median age was 67.8 years (21.8-92.1), 51.2% were male and the most frequent subtype according 2008 WHO classification was refractory cytopenia with multilineage dysplasia (41.6%), followed by refractory anemia with excess of blasts-1 (RAEB-1) and -2, 18.4% and 16.8%, respectively. Median values at diagnosis were, for hemoglobin: 9.3g/dL, leukocytes count: 3.6x109/L, platelet count: 139x109/L, percentage of polymorphonuclear cells: 47%, and bone marrow blasts: 3.6%. Nineteen patients were analyzed with their matched germ-line control sample. The statistical analysis was performed with SPSS and R software. As level of significance, 5% (two-sided) was chosen. No adjustment for multiple testing was applied. Results Fifty nine cases were found to be altered by microarrays (47.2%), with a median number of alterations of 1 (range: 1-8). CNA were detected in 36.8% of cases, in 8 cases CNA were larger than 5 Mb. AOH were detected in 18.4% of cases. The median size of the affected genome by CNA was 116 Kb, adding up CNA plus AOH was 24745.05Kb. There were significant differences, in distribution of FAB diagnosis and median percentage of bone marrow blasts, in patients altered by SNP arrays in comparison to patients with normal findings by this technique. The impact on the outcome has been studied in 73 patients. The median OS was 35.6 months. The results of SNP array analysis showed that alterations detected by microarrays had no significant impact on outcome (P=0.237 for OS and P=0.807 for AML evolution), but the median OS is higher in cases with a "real" (neither visible by cytogenetics nor submicroscopic alterations) normal karyotype (53.48 vs. 25.90 months). Of note, the presence of AOH has a strong impact on OS (53.48 vs. 16.13 months, P=0.004), less on AML (P=0.086); also its size, greater than 25Mb, has an impact on OS (P=0.012) and AML evolution (P=0.050). To compensate for differing risk distributions, we performed analyses adjusting for IPSS-R. Adjusted for risk, the microarray result adds prognostic power on OS (P=0.010) and AML evolution (P=0.059). As it was observed, the presence of AOH had prognostic impact on OS (P=0.008) and AML evolution (P=0.017). The affected genome size showed a significant impact on OS (P=0.006) and AML (P=0.024), four categories were defined: 0 Kb, >0-500 Kb, >500-20000 Kb and >20000 Kb. Conclusions MDS patients with normal karyotypes and alterations detected by genomic microarrays have lower OS and shorter time to AML compared to those without CNA. While not significant in marginal analysis, it can be demonstrated if adjustment for IPSS-R is performed. Alterations and, particularly, AOH have an independent prognostic impact and add prognostic power to IPSS-R. Acknowledgments Financial support: This work was supported, in part, by a grant from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras, Obra Social “La Caixa”, Celgene Spain and NHRI-EX103-10003NI,Taiwan. Special thanks to Dr. Al-Ali, Dr. Bäsecke, members of Grupo Español de SMD (GESMD) and MDS Foundation. Disclosures No relevant conflicts of interest to declare.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.

Abstract 3539 Background: Microdeletions of genes involved in B lymphopoiesis and cell-cycle regulation, such as CDKN2A/B, PAX5, IKZF1, ETV6, RB1, BTG1 and EBF1 have been reported as a frequent event in pediatric acute lymphoblastic leukemia (ALL). Whether these findings are found in adulthood and the possible differences with childhood ALL, as well as its prognostic implication, are still unknown. Aims: To assess the differences between two cohorts of children and adults diagnosed with ALL on the frequency of deletions in these genes and their relationship with clinical data and prognosis. Methods: We studied 70 children and 83 adults diagnosed with ALL with available DNA sample at diagnosis. In children, median age was 4y. (1 – 14), median leukocytes 10.3×109/L (0.7 – 675) and the cytogenetic risk distribution was 42(39%), 30(27%) and 12(11%) for favourable [t(12;21) and hyperdiploidy], intermediate (normal karyotype and miscellaneous) and high risk [t(9;22), t(4;11), hypodiploid and complex karyotype], respectively. In adults, median age was 38y. (15 – 85), median leukocytes 16.8×109/L (1 – 371) and 29(35%) patients belonged to the high risk cytogenetic group. We performed Multiplex Ligation Probe Amplification (MLPA) using SALSA kit P335-A1 (MRC-Holland). PCR products were separated on an ABIPRISM 310 DNA Analyzer and analyzed using GeneMapper v3.2 (Applied Biosystems). Results: Frequency of deletions in the studied genes was similar in children and adults, except for IKZF1 deletions that were more frequent in adults (P In children, ETV6 deletions occurred more frequently in patients with t(12;21) (67% of patients with deletion vs. 17% without, P In adults, ETV6 and CDKN2A/B deletions occurred more frequently in women (67% vs. 39%, P =.022 and 77% vs. 42%, P =.021, for patients with and without deletions, respectively); PAX5 and IKZF1 deletions appeared more frequently in patients with >30×109/L leukocytes (60% vs. 27%, P =.032 and 52% vs. 21%, P =.007, for patients with and without deletions, respectively); besides, PAX5 deletions occurred in patients who belonged to the standard cytogenetic risk group (55% vs. 6% for patients with and without deletions, P In the pediatric cohort, the leukocytes >30×109/L and the cytogenetic risk group were the variables that reached statistical significance for both overall survival (OS) and relapse free survival (RFS) and also age >10y. for OS, but in the multivariate analyses, just the cytogenetic risk classification remained significant [HR: 4 (CI 95%: 1.6 – 10), P =. 004 for OS and HR: 3.5 (CI 95%: 1.7 – 7.2), P =. 001 for RFS]. In the adult cohort, multivariate analysis for OS including all significant variables in the univariate analysis (age >60y, karyotype, CDKN2A/B and ETV6 deletions) showed as independent variables: age >60y. [HR: 4.3 (CI 95%: 2.1 – 8.6), P Conclusions: This study shows the high incidence of deletions in genes of cell-cycle and B-lymphopoiesis in adult and pediatric ALL. However, the biological and prognostic implications of these deletions seem to differ between both patient groups: while cytogenetics was the strongest variable for risk assessment in children, gene microdeletions in CDKN2A/B and ETV6 added a prognostic value to karyotype in our adult cohort. Fundings: AP-194/10, R06/0020/0031, BES2008–008053, CM10/00321, CM09/00038, and CA08/00141. Disclosures: No relevant conflicts of interest to declare.

Abstract 1450 The clinical relevance and prognostic implications of some recently identified mutations in acute myeloid leukemia (AML) is not yet well established. Among them, we have selected to be analyzed those affecting the following genes: Additional Sex Combs-Like 1 (ASXL1), Isocitrate Dehydrogenase (IDH1 and IDH2), Casitas B-lineage Lymphoma (c-CBL), and Wilms Tumor 1 (WT1). They have been previously reported with a variable incidence: ASXL1 mutations in 10.8% patients with normal karyotype (NK), IDH1 and IDH2 mutations in 8 – 33% of de novo AML, c-CBL mutations in 2% of de novo AML, and WT1 mutations in 5–12% of de novo AML patients. In order to know the incidence and prognostic impact of these mutations and their possible cooperative role in leukemogenesis, we have screened for ASXL1, IDH1, IDH2, c-CBL, WT1, FLT3, NPM1 and CEBPa, mutations in a cohort of de novo AML patients from a single centre. We studied 174 de novo AML patients [98M/76F; median age: 62 yr. (range: 16 – 88); favourable (n= 13), intermediate (n= 86) and high (n= 51) cytogenetic risk classification by the MRC group]. DNA was isolated from bone marrow samples obtained at diagnosis. In order to determine cooperating mutations, we developed a new combination of high-resolution melting (HRM) assays on a LightCycler® 480 and lastly direct sequencing, to detect somatic mutations for ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), WT1 (exons 7, 8 and 9) and c-CBL (exons 8 and 9). All mutations reported in this study were confirmed al least twice. FLT3 (ITD and D835Y), NPM1 (exon 12) and CEBPa were performed as described previously by standard methods. Sequence analysis was checked by its corresponding GeneBank Accession Number. The number of patients found to carry mutations in our series was: 16 patients with ASXL1 mutations (9.2%), 16 patients with IDH mutations (2.9% had a IDH1R132, 12.6% the SNP rs11554137 and 6.3% IDH2R140), 5 patients with WT1 mutations (2.9%), 37 patients with FLT3 mutations (21.3%), 44 patients with NPM1 mutations (25,3%) and 8 patients with CEBPa mutations (4.6%). No mutations where found in c-CBL. We could not found a pattern of cooperating mutations in the studied group of genes. WT1, FLT3 and NPM1 were associated with leukocyte count >30 × 109/L at diagnosis (80% vs. 31% for WT1, P =0,022; 68% vs. 22% for FLT3, P= 0.001; and 50% vs. 24% for NPM1, P= 0.002; in mutated vs. wild-type patients, respectively). WT1 was also associated with a platelet count > 50 × 109/L at diagnosis (100% vs. 57% in mutated vs. wild-type patients, respectively; P =0,048). Besides, FLT3 and NPM1 mutations were more frequent in the intermediate cytogenetic risk group (82% and 74%; P =0.004 and P =0.047; respectively). ASXL1 and IDH mutations were not correlated with any of the clinical and biological features studied. In univariate analysis, only age and cytogenetics had an impact on overall survival (OS, median of 12mo vs. 3mo, for patients < and ≥65 yr., P In multivariate analysis in patients with intermediate cytogenetic risk, the age [HR (95% CI) = 3.3 (1.9 − 5.9) P To sum up, the incidence found for the studied genes was lower than the previously reported: ASXL1, 9.2%; IDH1R132, 2.9%; IDH2R140, 6.3%; WT1, 2.9%; and c-CBL, 0%. We were unable to find a pattern of cooperating mutations in the studied group of genes or any impact of these mutations on the outcome. Disclosures: No relevant conflicts of interest to declare.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm characterized by a highly variable clinical course. Based on clinical, hematologic and cytogenetic parameters, we previously developed a CMML-specific Prognostic Scoring System (CPSS) that stratifies patients into 4 different risk groups [Blood 2013;121:3005-15]. Recently, recurrent somatic mutations have been identified in CMML, and preliminary evidence suggests that selected mutated genes may provide useful prognostic information. In this study, we performed a comprehensive mutation analysis of genes implicated in myeloid malignancies in a large and well characterized cohort of CMML patients with the aim to dissect relationships between genotype and disease phenotype and to integrate somatic mutations into a clinical/molecular prognostic model. Thirty-eight genes were analyzed by high throughput sequencing (Illumina MiSeq, San Diego, CA) in a cohort of 199 CMML patients (pts) diagnosed according to WHO classification, and in 12 pts with monocytosis not fulfilling WHO diagnostic criteria. Myelodysplastic and myeloproliferative subtypes (CMML-MD and CMML-MP, respectively) were defined according to FAB criteria. Least absolute shrinkage and selection operator (Lasso) and Cox proportional hazards methods were adopted to select and weight variables for prognostic scoring. Ninety-three percent of pts showed at least one somatic mutation (median number per patient: 2, range 0-6). The most frequently mutated genes were TET2 (44%), SRSF2 (43%), ASXL1 (34%), KRAS (11%), NRAS (10%), CUX1 (10%), CBL (9%), RUNX1 (7%), SETBP1 (7%), JAK2 (6%), SF3B1 (6%), and U2AF1 (5%). A significant association was found between mutations in TET2 and RNA splicing factors (P=.037), 42 of 199 CMML pts (21%) showing co-occurrence of TET2 and SRSF2 mutations. Mutations in genes involved in signaling were significantly associated with CMML-MP (P=.002), whereas SF3B1 mutations were associated with CMML-MD (P=.024). The number of mutations per patient inversely correlated with overall survival (OS) (HR=1.32, P=.021). In univariate analysis, mutations in ASXL1 (HR=2.31, P=.026) , RUNX1 (HR=3.53, P=.02) and SETBP1 (HR=3.85, P=.005) significantly affected OS. Focusing the analysis on disease subtype, ASXL1 mutations significantly affected survival in CMML-MD (HR=3.45, P=.025), whereas CUX1 and SETBP1 had a significant prognostic value in CMML-MP (HR=4.33, P=.013 and HR=4.4, P=.025, respectively). In order to investigate the additive value of somatic mutations to current prognostic assessment, we first fitted a Lasso Cox regression model for genetic variable selection. The selected variables were then included in an unpenalized Cox regression in order to obtain unbiased coefficients. The statistically significant variables were CPSS-specific cytogenetic risk groups (HR=2.49, P=.001), mutations in ASXL1 (HR=2.77, P=.018), RUNX1 (HR=5.39, P=.009) and SETBP1 (HR=3.96, P=.013). Based on regression coefficients, we defined a CMML-specific genetic risk score that was able to identify three different groups (Low risk: normal karyotype and –Y; Intermediate: other abnormalities, mutations in ASXL1; High: trisomy 8, complex karyotype, mutations in RUNX1 or SETBP1), with significantly different OS (HR=2.24, P In conclusion, this study showed that mutation pattern in CMML significantly correlates with disease phenotype. The integration of somatic mutations in current scoring systems significantly improves prognostic stratification of patients. Disclosures No relevant conflicts of interest to declare.