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Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q). Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of < 5% in the bone marrow, a platelet count > 50.000/µl and absolute neutrophil counts of > 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment. Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. Disclosures Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

Abstract 3801 Background Aberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q). Methods All patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25). DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/). Results Using a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score. CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria. Conclusions We present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients. Disclosures: Nowak: Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.