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Background The resulting clinical impact of the combined use of G-CSF with chemotherapy as a chemosensitizing strategy for treatment of acute myeloid leukemia (AML) patients is still controversial. In this study, the effect of ex vivo treatment with G-CSF on AML primary blasts was studied. Methods Peripheral blood mononuclear cells from AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stromal cells. Cell viability and surface phenotype was determined by flow cytometry 72 h after treatment. For clonogenicity assays, AML primary samples were treated for 18 h with G-CSF at increasing concentrations and cultured in methyl-cellulose for 14 days. Colonies were counted based on cellularity and morphology criteria. Results The presence of G-CSF reduced the overall viability of AML cells co-cultured with bone marrow stroma; whereas, in absence of stroma, a negligible effect was observed. Moreover, clonogenic capacity of AML cells was significantly reduced upon treatment with G-CSF. Interestingly, reduction in the AML clonogenic capacity correlated with the sensitivity to chemotherapy observed in vivo. Conclusions These ex vivo results would provide a biological basis to data available from studies showing a clinical benefit with the use of G-CSF as a priming agent in patients with a chemosensitive AML and would support implementation of further studies exploring new strategies of chemotherapy priming in AML. Electronic supplementary material The online version of this article (doi:10.1186/s12935-015-0272-3) contains supplementary material, which is available to authorized users.

epartment of Hematology, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, SpainHematopathology Unit, Pathology Department, Hospital Clinic de Barcelona, Villarroel 70, 08036 Barcelona, SpainDepartment of Hematology, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, SpainHematopathology Unit, Pathology Department, Hospital linic de Barcelona, Villarroel 170, 08036 Barcelona, SpainDepartment of Hematology, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain

Review on MIR135A1, with data on DNA/RNA and where the gene is implicated.

Acute myeloid leukemia (AML) is a heterogeneous disease, and optimal treatment varies according to cytogenetic risk factors and molecular markers. Several studies have demonstrated the prognostic importance of microRNAs (miRNAs) in AML. Here we report a potential association between miRNA expression and clinical outcome in 238 intermediate-risk cytogenetic AML (IR-AML) patients from 16 institutions in the CETLAM cooperative group. We first profiled 670 miRNAs in a subset of 85 IR-AML patients from a single institution and identified 10 outcome-related miRNAs. We then validated these 10 miRNAs by individual assays in the total cohort and confirmed the prognostic impact of 4 miRNAs. High levels of miR-196b and miR-644 were independently associated with shorter overall survival, and low levels of miR-135a and miR-409-3p with a higher risk of relapse. Interestingly, miR-135a and miR-409-3p maintained their independent prognostic value within the unfavorable molecular subcategory (wild-type NPM1 and CEBPA and/or FLT3-ITD), and miR-644 retained its value within the favorable molecular subcategory. miR-409-3p, miR-135a, miR-196b and mir-644 arose as prognostic markers for IR-AML, both overall and within specific molecular subgroups.

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)

Acute myeloid leukemia (AML) with t(8;6)(p11;p13) (t(81 6) AML) has unique clinico-biological characteristics, but its microRNA pattern is unknown. We analyzed 670 microRNAs in seven patients with t(8;16) AML and 113 with other AML subtypes. Hierarchical cluster analysis showed that all t(8;16) AML patients grouped in an independent cluster. Supervised analysis revealed a distinctive signature of 94-microRNAs, most of which were downregulated, including miR-21 and cluster miR-17-92. The mRNA expression analysis of two known transcription factors of these microRNAs (STAT3 and c-Myc, respectively) showed significant downregulation of STAT3 (P=0.04). A bioinformatic analysis showed that 29 of the downregulated microRNAs might be regulated by methylation; we treated a t(81 6) AML sample with 5-aza-2'-deoxycytidine (5-AZA-dC) and trichostatin A and found that 27 microRNAs were re-expressed after treatment. However, there was no difference in methylation status between t(8;16) and other AML subtypes, either overall or in the microRNA promoter. Cross-correlation of mRNA and microRNA expression identified RET as a potential target of several microRNAs. A Renilla-luciferase assay and flow cytometry after transfection with pre-microRNAs confirmed that RET is regulated by miR-218, nniR-128, miR-27b, miR-15a and miR-195. In conclusion, t(8;16) AML harbors a specific microRNA signature that is partially epigenetically regulated and targets RET proto-oncogene. Leukemia (2013) 27, 595-603; doi:10.1038/leu.2012.278

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

The evolution of the incidence, morbidity, and mortality of veno-occlusive disease (VOD) was analyzed in 845 allogeneic hematopoietic stem cell transplantations (allo-HSCTs) performed over 24 years. A total of 117 patients and 73 patients developed VOD following the Seattle and the Baltimore diagnostic criteria, respectively (cumulative incidence 13.8% and 8.8%). The cumulative incidence was significantly higher in the period 1985 to 1996 than in 1997 to 2008 (11.5% vs 6.5%; P = .01). This decline was because of the low incidence of VOD among reduced-intensity conditioning-HSCT (RIC-HSCT) (2.1%) and the reduction among those receiving myeloablative-HSCT from unrelated donors (32.7% vs 10.5%, P = .001). A total of 35 patients had severe VOD (26 with multiorgan failure [MOF]), and 20 died by VOD (cumulative mortality rate 17.3%, Seattle, or 22.5%, Baltimore). The mortality declined since 1997 (from 22% to 9%; P = .06, Seattle, and from 36% to 14%; P = .04, Baltimore), with the introduction of defibrotide being the only relevant change in the management of patients. This occurred even though the severity of VOD was similar in both periods. Among those with MOF, only 2 of 8 (25%) receiving defibrotide died versus 14 of 18 (78%) receiving other treatments (P = .007). Myeloablative conditioning, previous liver disease, poor performance status, and alternative donors were the variables with higher impact on VOD development. In summary, although VOD remains a dreaded early complication of HSCT, technical and therapeutic progress in recent decades have notably reduced its incidence and improved the outcome.

Introduction Patients with intermediate-risk cytogenetic AML (IR-AML) have a heterogeneous prognosis, and are currently further stratified based on determined gene mutations. However, the optimal post-remission therapy, especially in younger patients, is unclear. Recently, miR-3151, a novel microRNA (miRNA) located in intron 1 of the BAALC gene, has been identified. High miR-3151 expression –either alone or in combination with high BAALC levels– independently correlates with poor prognosis in patients ≥ 60 years with normal cytogenetics AML (CN-AML) (Eisfeld AK, et al. Blood 2012). However, the prognostic value of miR-3151 in younger patients (≤60 years) with IR-AML has not been examined. We hypothesized that miR-3151 expression could also be a prognostic marker in younger patients. Aim To analyze whether miR-3151 expression – either alone or in combination with BAALC – improved prognostic assessment in younger patients (up to 60 years) with IR-AML and to characterize in this subset of patients the miRNA signature associated with high miR-3151 expression. Methods Samples were available from two separate cohorts of patients with IR-AML who had received intensive therapy: a training set of 76 patients from a single institution and a validation set of 108 patients from several centers who had been treated within the CETLAM AML-2003 protocol. Information on NPM1, FLT3- ITD and CEBPA was available for both patient cohorts. The expression levels of 670 miRNAs had previously been analyzed in the 76 patients in the training set. In the present study, the expression of miR-3151 and BAALC was analyzed using TaqMan® MicroRNA Assay and TaqMan® Gene Expression Assay (Applied Biosystems), respectively. Expression levels of miR-3151 and BAALC –both alone and in combination – were correlated with patient outcome. Statistical analyses were performed with SPSS v.15.0.1, R software v.2.9.0 and TIGR MultiExperiment Viewerv4.0. Results In the training set, higher expression of miR-3151(dichotomized by its median value of normalized expression) correlated with a shorter 5-year overall survival (OS) (32%±17% vs. 61±17%, p=0.029) and 5-year leukemia-free survival (LFS) (29%±17% vs. 58±17%, p=0.036) in patients ≤ 60 yrs. When the analysis was restricted to patients with CN-AML, miR-3151 expression retained its prognostic significance (p= 0.016). In addition, increased BAALC expression was associated with shorter OS (28%±20% vs. 58±14%, p=0.054) and LFS (17%±18% vs. 55±14%, p=0.039). In the multivariate analysis for OS and LFS, including age, WBC, NPM1 mut, FLT3 -ITD, BAALC and miR-3151 expression levels as covariates, miR-3151 showed independent prognostic significance for OS (p=0.016; HR=2.52, 95% CI: 1.2-5.4),and a statistical trend for LFS (p=0.09). Patients with low expression of both miR-3151 and BAALC had better prognosis (OS: 66%±18% vs. 34±16%, p=0.027; LFS: 67%±20% vs. 27±16%, p=0.009).Interestingly, the combination of both miR-3151 and BAALC retained a significant prognostic value for LFS within the favorable molecular subgroup/ ELN favorable subgroup (patients harboring NPM1 mut without FLT3 -ITD or biallelic CEBPA mut; LFS: 44%±30% vs. 100%, p=0.017) and showed a trend in the unfavorable molecular subgroup/ELN Intermediate II OS: p=0.064 and LFS: p=0.072). In the validation set, miR-3151 overexpression confirmed its prognostic impact in patients in the univariate analysis for OS (45%±12% vs. 26±19%, p=0.039) and LFS (51%±14% vs. 30±24%, p=0.034) and in the multivariate analysis for OS (OS: p=0.038; HR 1.88, IC 95%: 1.06-3.34) and LFS (p= 0.014; HR 2.411 (1.198-4.855) Finally, a supervised analysis revealed that samples exhibiting high levels of miR-3151 expression had a distinctive miRNA signature including miR-509, miR-135a, miR-100*, miR-186*, let-7a* and miR-501. Conclusion miR-3151 is an independent prognostic marker in patients with IR-AML. The study of miR-3151 refines the molecular prognostic stratification of these patients and hence could be of help to guide therapy. Acknowledgements Marina Diaz-Beya is supported by Fundacion Espanola de Hematologia y Hemoterapia. This research is supported by Sociedad Espanola de Hematologia y Hemoterapia and by grants from Fondo de Investigaciones Sanitarias FIS-PI080158. Disclosures: No relevant conflicts of interest to declare.

Hydroxyurea (HU) is frequently given as treatment for myelofibrosis (MF), but data on its efficacy and tolerability are scarce. The results of HU therapy were evaluated in 40 patients with hyperproliferative manifestations of primary (n = 32), post-polycythemia vera (n = 6), or post-essential thrombocythemia (n = 2) myelofibrosis. Median interval between diagnosis and HU start was 6.2 months (range 0–141.7). Reasons for treatment were constitutional symptoms (55%), symptomatic splenomegaly (45%), thrombocytosis (40%), leukocytosis (28%), pruritus (10%), and bone pain (8%). The starting dose was 500 mg/day, subsequently adjusted to the individual efficacy and tolerability. Response was bone pain 100%, constitutional symptoms 82%, pruritus 50%, splenomegaly 40%, and anemia 12.5%. According to the International Working Group for Myelofibrosis Research and Treatment criteria, clinical improvement was achieved in 16 patients (40%). Median duration of response was 13.2 months (range 3–126.2). Worsening of the anemia or appearance of pancytopenia were observed in 18 patients, requiring administration of erythropoietin-stimulating agents (n = 17) and/or danazol (n = 9). Oral or leg ulcers appeared in five patients and one had gastrointestinal symptoms. HU is an effective and generally well-tolerated therapy for the hyperproliferative manifestations of MF. The accentuation of the anemia often induced by HU is usually manageable with concomitant treatment.

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

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

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

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

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

Introduction: PiwiRNAs (piRNAs) are small non-coding RNAs processed by PIWI proteins, a subfamily of Argonaute proteins. The main functions of piRNAs include regulation of chromatin and genome organization, DNA methylation, transposon repression and regulation of protein synthesis. Until recently, PIWI/piRNA functions were believed to be limited to germinal stem cells and early embryonic development, where they play a crucial role in the regulation of genomic stability. However, piRNA expression has now been identified in some tumors, although the specific prognostic impact of piRNAs remains to be elucidated. We have examined the potential reactivation of the PIWI/piRNA pathway in classical Hodgkin lymphoma (cHL) and its impact on prognosis in cHL patients. Methods: Formalin fixed and paraffin embedded tumor samples from 96 cHL patients were studied. All patients were diagnosed and treated in a single institution. HIV+ was an exclusion criterion. Median age was 33 years (range, 15–89); 47.9% were male; 30.2% were EBV+. The most frequent histological subtype was nodular sclerosis (65.6%). First-line treatment consisted of ABVD-type therapies in 90% of patients. 12 reactive lymph nodes (RLNs) were used as controls. The PIWI proteins PIWIL1 and PIWIL2 were studied by immunohistochemistry. The expression levels of three piRNAs (piR-651, piR-20365, piR-20582) were analyzed by Real-time PCR. cDNA was synthesized using miScript II RT Kit and Real-time PCR was performed using miScript SYBR Green PCR Kit (Qiagen). PiRNA expression was correlated with treatment response, disease-free survival (DFS) and overall survival (OS). Paired serum samples from seven prospectively collected cHL patients at diagnosis and at complete response and from 10 healthy controls were used to study the expression of piRNAs in serum. In addition, 5 cHL cell lines were used for the functional analysis. RNA hybrid, Renilla/Luciferase assay and Western Blot were used to identify and validate the piRNA targets. Statistical analyses were performed using R v2.13. Results: PIWIL1 and PIWIL2 proteins were expressed in the cytoplasm of the Hodgkin/Reed Sternberg (HRS) cells, indicating that the piRNA pathway is active in cHL. This was corroborated by the finding that the 3 piRNAs were also expressed in all tumor samples and in the 5 cHL cell lines. piR-651 (p In situ hybridization with LNA probes (Exiqon) showed that piR-651 was highly expressed in the cytoplasm of HRS cells. We studied putative targets genes of piR-651 using RNA hybrid. We identified several candidate genes related to drug metabolism, including ABCC5, an efflux pump of multiple drugs including doxorubicin. We cloned the 3’UTR region of ABCC5 in the psicheck2 vector and performed a Renilla/Luciferase assay. We observed a significant increase of 28% (p=0.047) in the Renilla luciferase levels in the cells transfected with 200nM of piR-651 inhibitor compared to oligo control. To further validate this, we transfected 3 HL cell lines with piR-651 inhibitor or oligo control and analyzed the protein levels of ABCC5 by Western Blot, observing a mean increase of 18% in the cells transfected with piR-651 inhibitor. These results showed that piR-651 inhibits the translation of ABCC5. Conclusions:The PIWI/piRNA pathway is reactivated in cHL and the expression of piRNAs affects tumorogenesis and cHL patient outcome. piR-651 influences treatment response and survival through regulation of ABCC5. Our findings provide the groundwork for further investigation of the novel role of piRNAs in HL. Acknowledgments : AECC-Catalunya (2014), APIF-UB (AC). 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.

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

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

Introduction The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. Methods TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. Results The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p Conclusions MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. Acknowledgments SDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures: No relevant conflicts of interest to declare.

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

Abstract 3873 Introduction: Disruption of normal DNA methylation, including both gene specific hypermethylation and genome-wide hypomethylation, is found in most malignant tumors. Most epigenetic studies in chronic lymphocytic leukemia (CLL) have been focused in CpG islands and gene promoter regions, and have identified hypomethylated genes, such as BCL2 or TCL1, and hypermethylated genes, such as GRM7. However, the quantification of overall methylation measured as levels of 5-methylCytosine (5mC) has been poorly explored. As compared to their normal counterparts (CD19+ B cells), overall hypomethylation has been observed in CLL neoplastic cells. Importantly, the overall methylation varies among patients but its clinical significance has not been widely investigated. In addition, it is known that microRNA (miRNA) expression is altered in CLL, and that and epigenetic mechanisms, such as methylation, can affect miRNA expression. Aim: To investigate the prognostic impact of overall methylation in patients with CLL and to analyze the correlation of 5mC levels with miRNAs expression. Methods: We analyzed 73 CLL patients (median age, 69 [range, 34–86]; 43% males) diagnosed in our institution between 1992 and 2007. The median follow up was 10.5 years. The level of global methylation in total DNA was estimated after determination of percentage of 5mC using anti-5mC monoclonal antibodies (MethylFlash Methylated DNA Quantification Kit, Epigentek). The expression of 377 mature miRNAs was analyzed using TaqMan Array Human MicroRNA A Card v2.0 (Applied Biosystems). Statistical analysis was performed with SPSS version 15.0.1 and R software version 2.9.0. MaxStat package of R were used to determine the optimal cutoffs and Quantitative trail function in BRB array tools to correlate miRNA expression and methylation levels. Results: The analysis of methylation levels showed a wide distribution of methylation degree among patients (median: 3.02%, range: 0.58–6.14%). From the clinical standpoint, methylation levels were only correlated with Binet clinical stage, patients with C stage showing a higher degree of methylation (p=0.015). Using MaxStat, we identified two cutoffs which classified patients as having low, medium or high degree of methylation. Mean progression-free survival (PFS) was 8.4 years (95% CI: 6.4–10.4), 6.2 years (95% CI: 4.7–7.7) and 3.2 years (95% CI: 2.4–4.8) for patients with low, medium, and high methylation levels, respectively (p=0.013). In the multivariate analysis for PFS (including ZAP70, IGHV, Age≤65, cytogenetics and global methylation), high ZAP70 expression (HR: 3; 95%CI: 1.1–7.9; p=0.026) and high global methylation (HR: 5.4 95%CI: 1.7–17.1; p=0.004) were independent unfavorable prognostic factors, while a significant trend was observed for high-risk cytogenetics (17p-, 11q-, +12) (p=0.054). Interestingly, methylation levels retained its prognostic significance in subgroup analysis: clinical stage A (p=0.06) and B/C (p=0.009); mutated (p=0.008) and unmutated IGHV (p=0.028); low (p=0.028) and high ZAP70 (p=0.001); and low-risk (normal karyotype, 13q-)(p=0.008) and high-risk (17p-, 11q-, +12) cytogenetics (p=0.001). Finally, we identified a 4-miRNA signature associated with global methylation levels: miR-103 (Spearman correlation [SC]: −0.821;p=0.03), miR-132 (SC: 0.786;p=0.05), miR-494 (SC: −0.786; p=0.02), and miR-193a-5p (SC: 0.786; p=0.05). Interestingly, miR-103, miR-132 and miR-494 are located in subtelomeric regions, which are known to be more susceptible to overall methylation changes. Conclusions: In this study, the degree of global DNA methylation was an independent prognostic factor for PFS in patients with CLL. The analysis of overall methylation could be useful not only for the prognosis of patients with CLL but also in the monitoring of clinical trials in which hypomethylating agents (e.g., decitabine) are being investigated as CLL therapy. The correlation between overall methylation levels and certain miRNAs may be a surrogate marker of epigenetic lesions and deserves further investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2551 Background: The presence of multilineage dysplasia (MLD) is the hallmark to define acute myeloid leukemia (AML) with myelodysplasia-related changes. The recognition by the WHO classification of this category as a separate entity implies the presence of differential biological and clinical features. While this seems to be confirmed for patients with AML and MLD associated with high risk cytogenetic abnormalities, the prognostic significance of MLD in cases with intermediate risk karyotype is unclear. Recent studies have shown that MLD has no prognostic impact in patients with mutated NPM1, but the prognosis of patients with intermediate-risk cytogenetics and MLD harboring a wild-type configuration of NPM1 is uncertain. Objective: The aim of this study was to analyze the presence of MLD and its prognostic value in AML patients with intermediate risk cytogenetics (IR-AML) and wild-type NPM1. Patients and methods: One-hundred eighty-two patients diagnosed with de novo IR-AML and wild-type NPM1 treated with the same chemotherapy protocol (CETLAM-2003) were included (M/F:101/81; median age: 55.5, 18–73). Bone marrow aspirate smears performed at diagnosis were reviewed by a panel of expert cytologists, and the cases were categorized as having MLD by applying strictly the WHO criteria (Harris et al, WHO 2008, MLD-1). Additionally, cases with dysplasia in ≥50% of the cells in one cell line, and between 30% and 50% in another cell line, were classified as having significant dysplasia (MLD-2). Results: The degree of dysplasia was evaluable in 139 of the 182 cases, observing strictly WHO-defined multilineage dysplasia (MLD-1) in 45 (25%), significant dysplasia (MLD-2) in 17 additional patients (9%), and absence of MLD in 80 (44%). In 43 (30%) cases dysplasia could not be thoroughly quantified in all hematopoietic lines, mostly due to the lack of enough evaluable precursors in a context of massive blast infiltration. No major differences concerning main characteristics were observed between patients with or without MLD, including age, leucocyte count at presentation, degree of BM involvement, proportion of normal karyotype, and frequency of mutations of FLT3 (internal tandem duplication), CEBPA, and partial tandem duplication of MLL gene. Since the outcome of patients with MLD-2 was similar to that of patients with stringent WHO MLD, and markedly inferior to patients lacking MLD (table 1), these two groups were merged in a unique MLD group (MLD-1+MLD-2). Thus, MLD was associated with a lower probability to achieve complete remission (CR) after frontline induction chemotherapy (66% vs. 80%, p=0.033). In addition, patients with MLD showed a trend to an inferior overall survival (OS at 5 years: 39.2±5% vs. 22.9±8.1%, p=0.064). Of note, a multivariate analysis identified increasing age, higher leucocyte count at presentation, and presence of MLD (hazard ratio, 95% CI: 1.569,1.056–2.331; p=0.026) as the only variables associated with a shortened survival; this analysis also included presence of FLT3-ITD and cytogenetics (normal karyotype vs. other intermediate-risk abnormalities). Age was the only variable with independent prognostic value on leukemia-free survival in this cohort of patients, whereas MLD did not reach statistical significance. Interestingly, the outcome of patients with MLD-AML with a minimum CR duration of 3 months was significantly better after allogeneic stem cell transplantation in first CR compared to other post-remission treatments (5-year OS: 82±12% vs. 31±9%, p=0.006), suggesting that alloHSCT could partially overcome the adverse prognosis associated with this AML category. Conclusions: The presence of MLD confers adverse prognosis to patients with IR-AML and wild-type NPM1. Presence of significant dysplasia in two hematopoietic cell lines without fulfilling stringent WHO criteria was associated to an outcome similar to that of patients with WHO-defined criteria, an observation that leads to a wider interpretation of significant MLD in AML. These results have an important clinical relevance and should lead to the search of new genetic and epigenetic markers associated with MLD in this setting. This study has been partially supported by grant n° 03/0423 from Instituto de Salud Carlos III/FIS0. Disclosures: No relevant conflicts of interest to declare.

Abstract 1561 In recent years, the microRNA (miRNA) pathway has emerged as a crucial regulation system in tumorogenesis. miRNA expression is deregulated in the tumor, and miRNAs can function both as oncogenes and tumor suppressor genes. miR-SNPs are a novel class of single nucleotide polymorphisms that can affect miRNA biogenesis and target sites and can alter the expression and functions of miRNAs. We have evaluated 9 miR-SNPs and investigated whether a distinct haplotype of miR-SNPs predicts clinical outcome in HL. One hundred and forty-one adult patients (median age, 32 yrs; range, 13–89; males 51.1%) diagnosed with HL in our institution between September 1995 and June 2005 were included in the study. Distribution according to histology: nodular sclerosis (58.9%), mixed cellularity (17.7%), lymphocyte rich (6.4%), lymphoid depletion (4.3%), and nodular lymphocyte predominant (7.1%). Epstein-Barr Virus was present in 38.1% of the samples. SNP analysis was performed by allelic discrimination on ABI Prism 7500 (TaqMan assays) in DNA obtained from formalin-fixed, paraffin-embedded lymph nodes. We examined 9 miR-SNPs: 4 in miRNA genes (MIR196A2 rs11614913; MIR149 rs2292832; MIR423 rs6505162; MIR146 rs2910164); 2 in miRNA binding sites (KRT81 rs3660; FAM179B rs1053667); and 3 in miRNA-processing machinery (XPO5 rs11077; RAN rs14035; TRBPrs784567). miR-SNP genotypes were correlated with probability of treatment failure, treatment-related toxicity, disease-free survival (DFS) and overall survival (OS). The median follow-up was 50 months (range, 1–143). Of 141 patients, 119 (84.4%) achieved complete response, 7 (5%) showed a partial response, and 14 (9.9%) were chemoresistant. We observed an increased probability of treatment failure in patients carrying the XPO5 AA or CC genotype (P=0.036). In 14 patients with neurological toxicity, an association was observed with the KRT81 genotype (P=0.047). In 7 patients with bleomicine-associated pulmonary toxicity, we observed an association with the XPO5 genotype (P=0.048). XPO5 and TRBP genotypes emerged as significant markers for DFS. Mean DFS for 57 patients (56%) with the XPO5 AC genotype was 111 months vs 82 months for patients with the AA or CC genotype (P=0.044). Mean DFS for 37 patients (31.6%) with the TRBP CC genotype was 124 months vs 90 months for patients with the TT or TC genotype (P=0.022). A trend towards an association between the MIR196A2 genotype and DFS was also observed (P=0.07). Only the XPO5 genotype was associated with OS. Mean OS for 66 patients (54%) with the XPO5 AC genotype was 134 months vs 111 months for patients with the AA or CC genotype (P=0.038). Given the evidence for the influence of TRBP and XPO5 as individual markers, we then investigated the combined effect of these miR-SNPs on DFS and OS. We found a significant correlation between the TRBP/XPO5 haplotype and DFS (P=0.005) and OS (P=0.005). Patients with the XPO5 AA/CC and TRBP TT/TC genotypes had the worst prognosis (DFS: 71 vs 114 months; OS: 95 vs 135 months). In the multivariate analyses, the TRBP/XPO5 haplotype (OR, 2.977; 95%CI, 1.1–7.4; P=0.01) emerged as an independent variable for DFS. Only the Hasenclever prognostic index (OR, 5.7; 95%CI, 2–18; P=0.004) emerged as an independent variable for OS, but we observed a trend towards significance for the TRBP/XPO5 haplotype as well (P=0.06). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of HL, specifically in the detection of chemoresistant patients and patients likely to relapse. The TRBP/XPO5 haplotype has surfaced as a promising prognostic factor that warrants further investigation to confirm its role as a biomarker in HL. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Disclosures: No relevant conflicts of interest to declare.

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

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

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

Abstract 3885 Background: In recent years, microRNAs (miRNAs) have emerged as key regulators of carcinogenesis. miRNA expression is deregulated in multiple hematological malignancies. One of the mechanisms that can affect miRNA expression is methylation in the promoter regions of miRNA genes. The main objective of the present study was to identify tumor suppressor miRNAs that are silenced by alterations in gene methylation in Hodgkin Lymphoma (HL). In addition, since demethylating agents could affect miRNA expression, we evaluated the in vitro effectiveness of 5-Aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, in HL cell lines. Methods: To detect miRNAs regulated by methylation, we analyzed the expression of 670 mature miRNAs in two HL cell lines, L-428 and L-1236, before and after AZA treatment, by TaqMan Human MicroRNA Arrays V2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2-ΔΔCt method using RNU48 as endogenous control. To validate the methylation status, genomic DNA samples from four HL cell lines (L-428, L-1236, HDMYZ and L-540) and peripheral B-cells from healthy donors were modified by sodium bisulfite using the EZ DNA Methylation kit (Zymo Research). The DNA methylation status of these samples was analyzed by methylation specific PCR (MSP) after sodium bisulfite modification of DNA. To evaluate AZA in vitro effectiveness, we treated the four HL cell lines daily with 20 nM, 250 nM, 1 μM and 5 μM of AZA or DMSO (vehicle control), and we assayed proliferation with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) after 72 hours of treatment. Results: After AZA treatment in the L-428 and L-1236 HL cell lines, 32 miRNAs (of 670 analyzed) were expressed de novo, 15 of which were present in both cell lines. miR-34a, miR-203, miR-342, miR-105, miR-490, miR-375 and the miR-500 family had well-defined CpG islands in their promoter regions. The MSP analysis showed that miR-34a, miR-203, miR-490 and miR-375 were methylated in L-428 and L-1236 HL cell lines, but not in the normal B-cells. miR-342 was methylated in L-1236 HL cell line, but not in L-428 nor normal B-cells. To further validate this we analyzed the methylation pattern in HDMYZ and L-540 HL cell lines and found that only miR-203, miR-375 and miR-490 were methylated in all 4 HL cell lines analyzed. We observed an AZA dose-dependent reduction in proliferation in all four HL cell lines. After 72 hours of treatment with AZA at 5μM, proliferation significantly decreased by 29% in L-428, 36% in L-1236, 28% in HDMYZ and 22% in L-540. Moreover, the analysis of miRNA levels during the treatment showed a re-expression of methylated miRNAs after 48 hours. The methylation analysis of these miRNAs in laser captured microdisected HL cells from patients and functional analysis are ongoing and complete data will be reported. Conclusions: In summary, HL exhibits a characteristic epigenetic pattern which includes the methylation of key miRNAs during the carcinogenesis process, such as miR-34a and miR-203, which have previously been shown to act as tumor suppressors in lung, colorectal and other cancers. Moreover, we present evidence that demethylating agents allow the re-expression of these tumor suppressor miRNAs, suggesting that they could be a promising novel treatment for HL patients. Supported by FIS grant (PS09/00547). Disclosures: No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous category of lymphoid tumors that comprises different clinical forms not fully recognized in the WHO classification. In this regard, extranodal (EN) DLBCLs have particular clinicobiological features and outcome, sometimes related to the specific site where the lymphoma arises. Nowadays, rituximab plus chemotherapy (CT) is the gold-standard in the treatment of DLBCL. However, the superiority of rituximab-CT (R-CT) over CT alone has not been addressed for all the clinical subsets of the disease and, in fact, the clinical role of the new therapies might be different for primary nodal or EN DLBCLs. The aim of this study was to assess the impact of rituximab in patients suffering from nodal or EN DLBCL. Two-hundred and thirty non-immunocompromised patients (112M/118F; median age, 61 years) diagnosed with CD20+DLBCL in a single institution between 1997 and 2006 (five years before and after establishing R-CT as the standard treatment in DLBCL) and treated with adriamycin-containing regimens were the subject of the present study. The series included 148 primary nodal and 82 EN DLBCL. Patients with primary CNS lymphoma were excluded and lymphomas arising at Waldeyer ring were considered as nodal DLBCL. The main EN sites were GI (n=26), bone (n=13), soft tissue (n=13), lung/pleura (n=9), liver (n=9), and other (n=12). Main clinico-biological and evolutive variables were analyzed. One hundred nineteen patients received only CT and 111 R-CT. Eighty-seven cases with available information were assigned to germinal center B-cell-like (GCB) (41%) or non-GCB (59%) groups according to the Hans method (Blood2004;103:275–82) based on CD10, BCL6 and MUM1 expression. Main initial features, including the primary nodal or EN origin, international prognostic index (IPI), and GCB/non-GCB categories were similar for CT and R-CT groups. No correlation was observed between the GCB/non-GCB groups and the primary site of the tumor, although nodal lymphomas more frequently expressed MUM1 than EN (69% vs. 31%, respectively; p=0.01). CR rate and 5-year overall survival (OS) according to the treatment arm (CT vs. R-CT) is detailed for the whole series and for the nodal and EN groups in the table and OS curves depicted in the figure. In the whole series, variables predicting poor OS in the multivariate analysis were high-risk IPI (RR 2.5; p Complete response CR (%) 5-years OS (%) CT R-CT CT R-CT *p Figure Figure

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

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown. We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients. The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6; 9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival OR: 2.04; P = 0.001), shorter leukemia-free survival (OR: 2.56; P < 0.001) and a higher cumulative incidence of relapse (OR: 1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004). Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature., Marina Diaz-Beya is supported by ISCII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (IP: Dr. Jordi Esteve), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding). Anna Cordeiro is an APIF fellow of the University of Barcelona