ALBERT

Beschreibung: Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA) is a DNA demethylating agent that is effective in treating MDS and CMML. However, response rates are less than 50% and the basis for poor response is currently unknown. A patient's potential to respond cannot be currently determined until after multiple cycles of AZA treatment and alternative treatment options for poor responders are limited. To address these fundamental questions, we enrolled patients on a compassionate access program prior to the listing of AZA on the pharmaceuticals benefit scheme in Australia. We have collected bone marrow from 18 patients (10 MDS, 8 CMML) at seven different stages of treatment, starting from before treatment until after six cycles of AZA treatment, and isolated high-purity CD34+ HSPCs at each stage. 10 of these patients (5 MDS and 5 CMML) responded completely to AZA while 8 did not achieve complete response. We performed next-generation sequencing (RNA-seq) of these HSPCs to identify the basis of poor response to AZA therapy. Analysis of the RNA-seq data from pre-treatment HSPCs has revealed a striking differential expression of 1148 genes between patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA therapy (Figure 1A). Using a Fluidigm nanofluidic system, we have validated the differential expression of a subset of these genes between CR and non-CR patients in two independent cohorts, totalling 67 patients, from the U.K. and Sweden. We have additionally confirmed that our gene signature does not simply segregate patients based on disease severity or poor overall survival, but rather uniquely prognosticates best AZA response. Pathway analyses of the differentially expressed genes indicates that the HSPCs of non-CR patients have decreased cell cycle progression and DNA damage pathways, while concomitantly possessing increased signalling through integrin and mTOR/AKT pathways. Using computational methods, we have determined that the expression of 15 genes (within the 1148 gene set) is sufficient to separate CRs from non-CRs across independent cohorts (Figure 1B). We have also developed a predictive AZA response algorithm that utilises the expression of these genes to identify potential complete and non-complete responders to AZA with high specificity and sensitivity (Figure 1C). Furthermore, we have identified statistically significant correlations between recurrent DNA mutations in MDS and our prognostic gene signature (SF3B1 & TET2 with CR, STAG2 and NUP98 with non-CR, p

Beschreibung: The purpose of this study was to determine the facility and reliability of the World Health Organization (WHO) classification of myelodysplastic syndromes (MDSs) with several observers reviewing the same diagnostic specimens. We also wanted to determine if the WHO classification provided additional information about predictability of clinical response outcome. To accomplish these goals we reviewed 103 previously diagnosed cases of low-risk MDS. We found 92% interobserver agreement (P 〈 .001). Sixty-four of these patients had been entered into clinical trials using growth factors by the Nordic MDS Study Group. The WHO classification reliably predicted therapeutic response to the combination of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo). The response rate differed significantly between refractory anemia with ringed sideroblasts (RARS) and refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD/RS) with regard to therapeutic response (75% versus 9%; P = .003). Also, in the group of patients with less than 5% marrow blasts, there was a difference in median survival between patients with unilineage dysplasia (51% surviving at 67 months) and those with multilineage dysplasia (median survival, 28.5 months; P = .03). (Blood. 2004;103:3265-3270)

Beschreibung: Immune aplastic anemia (AA) is a life-threatening bone marrow failure syndrome in which the hematopoietic stem cells are destroyed, leading to pancytopenia. Although the exact biological process leading to AA remains largely unknown, bone marrow destruction is thought to be mediated by an autologous T cell response. We hypothesized that the autoimmune process in AA would create a T cell fingerprint unique to aplastic anemia. To decipher this signature, we collected an international, multi-centre cohort of 245 AA-samples from bone marrow and peripheral blood profiled with T-cell receptor beta (TCRβ) -sequencing. CD8+ T cell- and MNC-sorted samples from 153 clinically annotated AA patients were obtained from diagnosis, during remission and at relapse. To compare AA to similar diseases, we gathered 116 samples from other bone marrow failure syndromes, including MDS, PNH and hypoplastic LGL, and 45 samples from other autoimmune disorders. As healthy controls, we profiled 60 CD4+ and CD8+ T cells and utilized 786 MNC samples from public data repositories. To gain insight into T cell phenotypes, we also profiled 6 longitudinal samples with scRNA+TCRαβ-sequencing. As there are 1x1012-16 different TCRs and most of them are exclusive to individuals (private), we reasoned that by studying the most biologically interesting clonotypes from each individual, we could explain differences in disease severities, variation in treatment responses and pathogenesis. From all subjects, we selected private response clonotypes: highly expanded clonotypes (at least 1% of the total repertoire), convergent clonotypes (in which multiple nucleotide sequences converge to encode the same amino acid sequence) and from patients with AA, treatment-responding clonotypes (clonotypes that were suppressed/expanded after immune therapy). To analyse epitope-specificities of these clonotypes we leveraged TCRGP, our recently described Gaussian process method that can predict if TCRs recognize previously known epitopes. Clonotypes recognising viral epitopes (CMV, EBV and Influenza A) were enriched among private response clonotypes in comparison to the total repertoire (Fisher's exact test, p=2e-16), indicating that our filtering strategy indeed enriched for epitope-specific clonotypes. Of interest, the healthy donors' private response clonotypes showed more anti-viral clonotypes than did AA-patients (p=0.003), suggesting that in AA the epitope-specifities of private response clonotypes are not driven by common viral antigens. To identify specifities against unknown epitopes of the private response AA clones, we used an unsupervised learning strategy, GLIPH,that groups TCRs recognising the same epitope based on amino acid level similarities. Clonotypes in AA showed high convergence in their epitope-targets, as 1709 of 5744 (29.75%) clonotypes formed a single, potentially epitope-specific cluster that was not viral-specific. Similar analysis of control samples resulted in fewer clones clustering to the most prominent cluster (23.20%, p=1.63e-10), suggesting for a more homogenous target population within AA patients' clones. After showing sequence-level similarity of the private response clonotypes in AA, we aimed to link these pathological clonotypes to transcriptomes at the single-cell level using scRNA+TCRαβ-sequenced samples. The cells of the private response clonotypes showed multiple T cell phenotypes, but most cells (47.13%) in the bone marrow environment were recently activated CD8+ effector phenotype, marked by expression of GZMH, GNLY and PRF1. In comparison, the anti-viral clonotypes were mostly (37.3%) central memory phenotype (CCR7, TCF7). In serially sampled patients, anti-thymocyte globulin treatment suppressed private response clonotypes in a responding patient (55.03% of T cells to 12.79%), while the amount of these clonotypes increased in a non-responding patient (18.65% to 37.86%), where treatment mostly affected the viral-specific clonotypes. In summary, our data suggest that the private response clonotypes in immune AA patients may recognise a common antigen, which was not predicted to be viral. Further, at the single-cell level AA signature clonotypes are of effector phenotype and fluctuate following immunosuppressive treatment. Monitoring of these clonotypes throughout treatment may provide insight into disease biology and variation in treatment responses. Figure Disclosures Blombery: Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

Beschreibung: Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment. Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR. Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression (

Beschreibung: Abstract 1723 Background: Two multicenter studies (MDS-003/-004) found LEN leads to RBC transfusion independence (TI) in 〉 50% of pts with RBC transfusion dependent Low-/Int-1-risk MDS with del5q (List A et al. NEJM 2006;355: 1456–65; Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). RBC-TI ≥ 8 wks with LEN was associated with significantly reduced risk of AML progression and death (Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). Alternative therapy is required for pts failing LEN therapy. Aims: To assess predictive factors of LEN response and long term outcomes (especially after primary or secondary LEN failure) of pts 〈 65 yrs included in MDS-003/-004; ie, those in whom intensive therapies including allogeneic stem cell transplantation (ASCT) may be considered. Methods: LEN was administered as follows (all 28-d cycles): 5 mg/d on d 1–28 and 10 mg/d on d 1–21 or 1–28. RBC-TI ≥ 26 wks and cytogenetic response (CyR; IWG 2000) are reported. Overall survival (OS) and AML progression were assessed using Kaplan-Meier method. Response rates and outcomes in pts 〈 65 yrs were retrospectively compared with pts ≥ 65 yrs. Primary failure was defined as lack of RBC-TI with LEN treatment and secondary failure as relapse after achievement of RBC-TI ≥ 26 wks. Cox proportional hazards models were used to evaluate the effect of potential risk factors (ie, age, sex, time since diagnosis, FAB classification, LEN dose, IPSS risk, WPSS risk, cytogenetics, bone marrow blast %, transfusion burden, no. of cytopenias, hemoglobin level, platelet and neutrophil counts, RBC-TI ≥ 26 wks [time-dependent variable] and CyR [categorical variable]) on OS and AML progression. Logistic model was used to evaluate the effect of potential risk factors on achievement of RBC-TI ≥ 26 wks. Results: The trials included 97 (33.9%) pts 〈 65 yrs. Of these, 73.2% were female; 20.6% were IPSS Low-, 52.6% Int-1-, and 4.1% Int-2-risk; 30.9% had del5q with ≥ 1 additional cytogenetic abnormality (8.2% had complex cytogenetics). At baseline (BL), median time since diagnosis was 2.4 yrs (range 0.2–20.7) and median RBC transfusion requirement was 6 units/8 wks (range 1–15). In pts ≥ 65 yrs (n = 189) most BL characteristics were similar except IPSS risk, which was lower (36.5% Low-, 37.0% Int-1-, 5.8% Int-2-risk; p =.012). RBC-TI ≥ 26 wks was achieved by 54 (55.7%) pts 〈 65 yrs (vs 49.7% pts ≥ 65 yrs; p =.563). The median duration of RBC-TI ≥ 26 wks in responders was not estimable in pts 〈 65 yrs or ≥ 65 yrs (log-rank p =.879). None of the potential risk factors assessed was a significant predictor of RBC-TI ≥ 26 wks in pts 〈 65 yrs, possibly due to small pt number. In pts 〈 65 yrs with available follow-up cytogenetics (n = 71), CyR was achieved by 32 (45.1%) pts (vs 64.5% pts ≥ 65 yrs; p =.014). At time of data cutoff, 51 (52.6%) pts 〈 65 yrs were alive (vs 36.0% pts ≥ 65 yrs; p =.008); 29 (29.9%) pts progressed to AML (vs 20.1% pts ≥ 65 yrs; p =.077). The 1-, 2-, and 3-yr AML-progression rates were 9.7%, 15.4%, and 24.0% in pts 〈 65 yrs; and 6.0%, 17.9%, and 22.3% in pts ≥ 65 yrs (log-rank p =.308). The 1-, 2-, and 3-yr OS rates were 91.7%, 78.1%, and 66.4% in pts 〈 65 yrs; and 83.1%, 65.2%, and 49.9% in pts ≥ 65 yrs (log-rank p

Beschreibung: Abstract 3450 Introduction: Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML), suggesting the presence of tumor suppressor(s) at the locus. Two different critical deleted regions (CDRs) have been identified on 5q. One region is linked to de novo AML and high-risk MDS (CDR1 at 5q31), and a second region to the low-risk MDS 5q- syndrome (CDR2 at 5q32–33). However, definitive identification of putative tumor suppressor genes remains controversial. Since it has recently been shown that several types of hematological malignancies have globally altered expression of non-coding RNAs (ncRNAs), we therefore searched for candidate ncRNA genes in the CDR1 region. Methods: Cell lines were treated with either 5-azacytidine or 5-aza-2`-deoxycytidine. Upregulated microRNAs (miRs) were identified by microarray analysis and confirmed by RT-qPCR analysis. The transcription start site was determined by 5`RACE and promoter methylation status by methylation specific melting curves, pyrosequencing and bisulfite sequencing. ncRNA expression and processing were analysed by RT-qPCR, Northern blotting, and siRNA mediated knock down of Drosha. Results: We identified the putative miR886, which became induced by azanucleoside treatment in an AML cell line, suggesting that it was regulated by promoter methylation. We found that the processing of miR886 was independent of Drosha, and northern blotting showed that this was a different type of longer ncRNA, annotated vtRNA2-1. These observations were supported by the results from three others groups (Nandy, J Mol Biol 2009; Stadler, Mol Biol Evol 2009; Lee, RNA 2011). The gene that encodes VTRNA2-1 is embedded in a CpG island. The CpG island is fully methylated in 4 different myeloid cell lines (HL60, NB4, U937 and F36P) and these cell lines have no expression of vtRNA2-1. Treatment with 5-azacytidine and 5-aza-2`-deoxycytidine derepressed expression of vtRNA2-1 in the cell lines. vtRNA2-1 is expressed in hematopoietic tissue (including CD34+ cells) from healthy individuals. Surprisingly, 75% of these carry a monoallelicly methylated VTRNA2-1 promoter while 25% carry an unmethylated VTRNA2-1 promoter. The methylation status of VTRNA2-1 was examined in bone marrow mononuclear cells from 101 AML patients taken at the time of diagnosis. 38 (38%) of these patients carried a hypomethylated promoter, 53 (52%) an intermediate methylated promoter and 10 (10%) a hypermethylated promoter. AML patients with hypomethylation of VTRNA2-1 have a significant better prognosis than patients with intermediate- or hypermethylation of the promotor (p=0.001). VTRNA2-1 methylation was independently associated with a poor survival in Cox proportional-hazards analysis (p=0.043), when testing against age, cytogenetic risk classification and leukocyte count at diagnosis. Discussion: It has previously been shown that vtRNA2-1 may be involved in the regulation of the double stranded RNA dependent kinase, PKR. Down regulation of vtRNA2-1 leads to activation of PKR and its downstream targets including NFkB (Lee, RNA 2011). Furthermore, PKR has previously been shown to alter response to chemotherapeutic agents by promoting cell survival (Pataer, Cancer Biol Ther 2009). Since constitutive PKR and NFkB activity are well documented features in AML, we speculate whether this may at least in part be mediated via loss of vtRNA2-1 expression. Here, we show that VTRNA2-1 may be directly implicated in AML, that expression of vtRNA2-1 is regulated by promoter methylation. Interestingly, we found that the majority of the healthy Danish population (∼75%) carry a monoallelically silenced VTRNA2-1 in normal hematopoietic cells. Our data suggest that the gene dosage of this particular type of ncRNA may play an important role in tumor progression or response to therapy since patients with hypomethylation of both alleles of the VTRNA2-1 promoter have a significantly better prognosis, while those that gain hypermethylation of the second VTRNA2-1 copy have a poorer outcome. Our data, combined with the previous findings, suggest that VTRNA2-1 is a novel tumor suppressor, located on chromosome 5q31.1, which probably acts through PKR. Disclosures: Jones: Eli Lilly: Consultancy.

Beschreibung: AML and high risk MDS are two myeloid malignancies with poor prognosis. Epigenetic changes such as silencing of tumor suppressor genes by promoter hypermethylation play a role in pathogenesis and disease progression. There is no consensus regarding the prognostic implications of epigenetic alterations in AML. We analyzed 108 de novo AML (median age 62 years, range 23–85), and 47 patients with MDS-related disease (19 MDS with IPSS INT-2 or High, 28 MDS-AML, median age 77 years, range 54–83), with respect to clinical and prognostic parameters and hypermethylation of the promoter region of three tumor suppressor genes; HIC, CDKN2b(p15) and CDH. All patients were eligible for and received standard induction chemotherapy. Methylation analysis was performed by Denaturing Gradient Gel Electrophoresis (DGGE) following bisulfite treatment and promoter specific PCR. A complete remission (CR) was achieved by 81 and 43 percent in the de novo AML group and the MDS group respectively. Hypermethylation of CDH or of multiple genes has previously been reported to correlate negatively to the probability of CR after intensive chemotherapy in the MDS cohort (p=0.008 and p=0.05, respectively). This was not true for the de novo AML patients (p=0.748 and p=0.681, respectively). The incidence of hypermethylation was significantly higher in de novo AML where 89% had at least one methylated gene compared to 66% in MDS (p=0.006). The mean number of methylated genes were 1.8 in de novo AML compared to 1.0 in MDS (p=0.002). In de novo AML the number of methylated genes decreased with increasing age (p=0.041) and an opposing trend was seen in MDS (p=0.15). Hypermethylation of all three genes was associated with higher age in MDS (p=0.040) whereas a trend towards the opposite was seen in de novo AML (p=0.084). In de novo AML the occurrence of FLT3-TKD was associated with hypermethylation of all three genes (p=0.011). Kaplan-Meier curves showed that hypermethylation of CDH and p15 together was associated with better overall survival (OS) in the de novo AML patients (p=0.001) (fig 1A). Median OS was 1.4 months for cases with no methylated genes and 13.5 and 14.5 months for those with one or two methylated genes, respectively. In contrast, in the MDS cohort, hypermethylation of CDH and p15 was a negative prognostic factor (p=0.002) (fig 1B). Median OS was 2.9 months for cases with two methylated genes and 12.7 and 9.4 for 0–1 methylated genes, respectively. The significance of these findings were retained after correction for age in a Cox regression analysis, HR 0.20 (p=0.001) for de novo AML and HR 2.96 (p=0.015) for MDS. We conclude that de novo AML and high risk MDS/MDS-AML show significant differences with respect to DNA promoter hypermethylation. Also, the correlation between hypermethylation and age as well as the effect on OS and complete remission rate differs between the de novo AML and the MDS cohort, respectively. This indicates epigenetic differences that may explain some of the clinical and morphological differences between the diseases. We also for the first time describe a relationship between FLT3-TKD and hypermethylation in AML. Fig 1A: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in de novo AML. Fig 1B: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in high risk MDS. Fig 1A:. Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in de novo AML. . / Fig 1B: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in high risk MDS.

Beschreibung: The del(5q) is the most commonly reported deletion in de novo MDS and is found in 10–15% of all patients. Our group demonstrated haploinsufficiency for the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q- syndrome (Boultwood et al, Br J Haematol2007, 139:578–89). Haploinsufficiency of RPS14 has been shown to be the mechanism underlying the erythroid defect in this disorder (Ebert et al, Nature2008, 451:335–9). We have recently shown that haploinsufficiency of RPS14 in patients with the 5q- syndrome is associated with deregulated expression of ribosomal- and translation-related genes, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis (Pellagatti et al, Br J Haematol2008, 142:57–64). The del(5q) in the 5q-syndrome is cytogenetically indistinguishable from the del(5q) found in other MDS and in the vast majority of these patients the CDR of the 5q- syndrome will be deleted (and therefore one allele of RPS14 will be lost). We are investigating the hypothesis that haploinsufficiency of RPS14 and consequent deregulated ribosome biogenesis may also play a role in the pathogenesis of non-5q- syndrome MDS patients with del(5q). Using Affymetrix U133 Plus2.0 arrays, we have studied the expression profiles of a group of 579 ribosomal- and translation-related genes in the CD34+ cells of 21 non-5q- syndrome MDS patients with del(5q) and 95 MDS patients without del(5q). 168 of 579 ribosomal-and translation-related probe sets were found to be significantly differentially expressed between these two groups, with approximately 90% of these showing lower expression levels in patients with del(5q). Hierarchical clustering using this set of 168 genes gave a good separation between patients with and without the del(5q). RPS14 was one of the most significant differentially expressed genes, with lower expression levels in patients with del(5q) confirming its haploinsufficient status in these patients. Other significant differentially expressed genes include the ribosomal protein RPL22L1, and the translation initiation factors EIF4EBP3 and EIF4B. Interestingly, when samples from 16 patients with 5q- syndrome were included in the analysis, hierarchical clustering using significantly differentially expressed ribosomal- and translation-related genes showed that most patients with 5q- syndrome and most patients with del(5q) clustered together. We are currently using polysome profile analysis on bone marrow cells to examine the levels of the 40S ribosomal subunit in patients with del(5q) and without del(5q). Our results support the hypothesis that haploinsufficiency of RPS14 and deregulation of ribosomal- and translation-related genes contribute to disease pathogenesis in MDS patients with del(5q). An exciting possibility is that other MDS with the del(5q) and the 5q- syndrome share a related molecular basis in that they are all disorders of defective ribosomal biogenesis.

Beschreibung: Abstract 2394 Introduction: We have recently shown that the allelic methylation level of the non-coding RNA, VTRNA2-1, predict outcome in acute myeloid leukemia (AML) (Treppendahl et al., 2012). VTRNA2-1 is located on chromosome 5q31.1, in the commonly deleted region for higher risk myelodysplastic syndrome (MDS) and de novo AML. Around 75% of the healthy Danish population carries a monoallelically methylated VTRNA2-1 promoter while the rest carries 2 unmethylated promoters. A hypermethylated promoter was only observed in patients. These interindividual differences in the methylation pattern are intriguing, and our data suggest that the gene dosage of this particular type of ncRNA may play an important role in tumor progression or response to therapy, since AML patients with hypomethylation of both alleles of the VTRNA2-1 promoter have a significantly better prognosis, while those with hypermethylation or loss of the second VTRNA2-1 copy have a poorer outcome(Treppendahl et al., 2012). Accordingly, we speculated if the allelic methylation levels of VTRNA2-1 also predict outcome in higher risk MDS patients. Methods: Bone marrow mononuclear cells from primary higher risk MDS patients (IPSS category INT2 and HIGH risk) and peripheral blood mononuclear cells, sampled during treatment with azacytidine, were analyzed for promoter methylation by pyrosequencing and methylation specific melting curve analysis. Results: Bone mononuclear cells from 57 higher risk MDS patients, never treated with azacytidine, were examined for VTRNA2-1 promoter methylation. 18 (32%) cases carried an unmethylated promoter (less than 15% methylation), 31 (54%) cases carried an intermediate methylated promoter (15–41% methylation) and 8 (14%) cases carried a hypermethylated promoter (more than 41% methylation). Patients with hypomethylation of the VTRNA2-1 promoter have a considerable better prognosis than those with intermediate or hypermethylation of the VTRNA2-1 promoter (P=0.026). Interestingly, in an azacytidine treated cohort the survival benefit of having an unmethylated promoter disappeared and a tendency towards a better survival of the methylated cases were observed (N=28, P=0.180).The in vivo effect of azacytidine on VTRNA2-1 methylation were examined in a small cohort of MDS patients (N=6), showing that azacytidine can induce demethylation of the VTRNA2-1 promoter in peripheral blood mononuclear cells. Discussion: Our studies show, that the allelic methylation of VTRNA2-1 can predict outcome, not only in AML patients, but also in higher risk MDS patients not treated with azacytidine. Patients with hypomethylation of the VTRNA2-1 promoter have a considerable better outcome than those with intermediate methylation or hypermethylation of the promoter. Interestingly, in the group of azacytidine treated patients there was no difference in survival between cases with and without methylation of the VTRNA2-1 promoter These data could indicate that patients with methylation of the VTRNA2-1 promoter might have a survival benefit of the azacytidine treatment. Thus we suggest that constitutive interindividual differences in the methylation of VTRNA2-1 potentially can be used as a pretreatment marker for selecting patients who will have a survival benefit of azacytidine treatment. This is to our best knowledge the first study identifying a potential epigenetic marker for selecting patients with benefit of treatment with azacytidine before treatment start. Our study is conducted in a quite heterogeneous and small patient cohort, and it has to be verified in a more homogenously treated larger group of MDS patients, ideally in a prospective clinical trial. Disclosures: No relevant conflicts of interest to declare.