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Patients with lower-risk myelodysplastic syndromes (LR-MDS) have a generally favorable prognosis; however, a small proportion of cases progress rapidly. This study aimed to define molecular biomarkers predictive of LR-MDS progression and to uncover cellular pathways contributing to malignant transformation. The mutational landscape was analyzed in 214 LR-MDS patients, and at least one mutation was detected in 137 patients (64%). Mutated RUNX1 was identified as the main molecular predictor of rapid progression by statistics and machine learning. To study the effect of mutated RUNX1 on pathway regulation, the expression profiles of CD34 + cells from LR-MDS patients with RUNX1 mutations were compared to those from patients without RUNX1 mutations. The data suggest that RUNX1-unmutated LR-MDS cells are protected by DNA damage response (DDR) mechanisms and cellular senescence as an antitumor cellular barrier, while RUNX1 mutations may be one of the triggers of malignant transformation. Dysregulated DDR and cellular senescence were also observed at the functional level by detecting γH2AX expression and β-galactosidase activity. Notably, the expression profiles of RUNX1-mutated LR-MDS resembled those of higher-risk MDS at diagnosis. This study demonstrates that incorporating molecular data improves LR-MDS risk stratification and that mutated RUNX1 is associated with a suppressed defense against LR-MDS progression.

Simple Summary Epigenetic drugs, such as azacitidine (AZA), hold promise in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), however, the mechanisms predicting the patients’ response to AZA is not completely understood. Quiescence of hematopoietic CD34+ progenitors has been proposed as a predictive factor for AZA therapy failure in MDS/AML patients, but the interplay between CD34+ cell cycle status and their metabolic signature in a predisposition to AZA (non)responsiveness remains unclear. Our data on patients with MDS or AML with myelodysplasia-related changes (AML-MRC) suggest that AZA-responders have actively cycling CD34+ cells poised for erythro-myeloid differentiation, with high metabolic activity controlling histone acetylation. Conversely, the patients who progressed early on AZA therapy revealed quiescence signature of their CD34+ cells, with signs of reduced metabolically-controlled acetylation of histones needed for transcription-permissive chromatin configuration. Our study delineates plasma citrate levels and CD34+ cells’ transcriptional signatures associated with cycling status and metabolic characteristics as factors predicting the response to AZA monotherapy in MDS/AML-MRC patients. Abstract To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.

Simple Summary Although lncRNAs have been increasingly recognized as regulators of hematopoiesis, only several studies addressed their role in myelodysplastic syndrome (MDS). By genome-wide profiling, we identified lncRNAs deregulated in various groups of MDS patients. We computationally constructed lncRNA-protein coding gene networks to associate deregulated lncRNAs with cellular processes involved in MDS. We showed that expression of H19, WT1-AS, TCL6, and LEF1-AS1 lncRNAs associate with higher-risk MDS and proposed processes related with these transcripts. Abstract Background: myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder with an incompletely known pathogenesis. Long noncoding RNAs (lncRNAs) play multiple roles in hematopoiesis and represent a new class of biomarkers and therapeutic targets, but information on their roles in MDS is limited. Aims: here, we aimed to characterize lncRNAs deregulated in MDS that may function in disease pathogenesis. In particular, we focused on the identification of lncRNAs that could serve as novel potential biomarkers of adverse outcomes in MDS. Methods: we performed microarray expression profiling of lncRNAs and protein-coding genes (PCGs) in the CD34+ bone marrow cells of MDS patients. Expression profiles were analyzed in relation to different aspects of the disease (i.e., diagnosis, disease subtypes, cytogenetic and mutational aberrations, and risk of progression). LncRNA-PCG networks were constructed to link deregulated lncRNAs with regulatory mechanisms associated with MDS. Results: we found several lncRNAs strongly associated with disease pathogenesis (e.g., H19, WT1-AS, TCL6, LEF1-AS1, EPB41L4A-AS1, PVT1, GAS5, and ZFAS1). Of these, downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with adverse outcomes in MDS patients. Multivariate analysis revealed that the predominant variables predictive of survival are blast count, H19 level, and TP53 mutation. Coexpression network data suggested that prognosis-related lncRNAs are predominantly related to cell adhesion and differentiation processes (H19 and WT1-AS) and mechanisms such as chromatin modification, cytokine response, and cell proliferation and death (LEF1-AS1 and TCL6). In addition, we observed that transcriptional regulation in the H19/IGF2 region is disrupted in higher-risk MDS, and discordant expression in this locus is associated with worse outcomes. Conclusions: we identified specific lncRNAs contributing to MDS pathogenesis and proposed cellular processes associated with these transcripts. Of the lncRNAs associated with patient prognosis, the level of H19 transcript might serve as a robust marker comparable to the clinical variables currently used for patient stratification.

Background: We aimed to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with myelodysplastic syndrome (MDS). Methods: Targeted enrichment resequencing of 84 DNA repair genes was initially performed on a screening cohort of MDS patients. Real-time polymerase chain reaction was used for genotyping selected SNPs in the validation cohort of patients. Results: A heterozygous frameshift mutation in the XRCC2 gene was identified. It leads to the formation of a truncated non-functional protein and decreased XRCC2 expression level. Decreased expression levels of all DNA repair genes functionally connected with mutated XRCC2 were also present. Moreover, a synonymous substitution in the PRKDC gene and 2 missense mutations in the SMUG1 and XRCC1 genes were also found. In the screening cohort, 6 candidate SNPs were associated with the tendency to develop MDS: rs4135113 (TDG, p = 0.03), rs12917 (MGMT, p = 0.003), rs2230641 (CCNH, p = 0.01), rs2228529 and rs2228526 (ERCC6, p = 0.04 and p = 0.03), and rs1799977 (MLH1, p = 0.04). In the validation cohort, only a polymorphism in MLH1 was significantly associated with development of MDS in patients with poor cytogenetics (p = 0.0004). Conclusion: Our study demonstrates that genetic variants are present in DNA repair genes of MDS patients and may be associated with susceptibility to MDS.

TO THE EDITOR: Although the concept of somatic driver mutations in myeloproliferative neoplasms (MPNs) represented by polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis is well established,[1][1][⇓][2]-[3][3] the contribution of germline or co-occurring JAK2

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with a high risk of transformation to acute myeloid leukemia (AML). One of the processes implicated in MDS pathogenesis is RNA splicing. Its alterations are caused by somatic mutations in splicing factor genes. Mutations in SF3B1 (Splicing Factor 3b Subunit 1) gene are the most frequently found mutations in MDS. Circular RNAs (circRNAs) are covalently closed RNAs that are produced by back-splicing process. CircRNAs can regulate multiple biological processes through various molecular mechanisms, such as microRNA sponging. Their deregulation is frequently found in cancer. It is likely that they also contribute to the development of MDS, however, their role in MDS has not been researched yet. Therefore, our aim was to explore circRNA levels in MDS and analyze their association with patient prognosis. We further hypothesized that mutations in splicing factor genes can affect production of circRNAs and thus, we examined circRNA levels with respect to the mutational status. We explored transcriptome of 78 MDS patients, 7 AML patients, and 13 healthy donors using Illumina RNA-seq of total RNA isolated from CD34+ bone marrow cells. To associate circRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Of 8,620 circRNAs identified by RNA-seq, 204 circRNAs were deregulated in MDS (e.g., MENTRNL, EBF1, and PPM1L-derived circRNAs) and 246 circRNAs were altered between lower- and higher-risk patients (e.g., CHST15, TMTC2, and PDE3B-derived circRNAs). Most of the progression-related circRNAs (n = 234) showed elevated levels in higher-risk patients, suggesting that the back-splicing process might be stimulated during the disease progression. In MDS patients with SF3B1 mutations, other 40 circRNAs were deregulated (e.g., ZNF91, ZEB1, and ZNF124-derived circRNAs). This circRNA profile was substantially different from the profiles associated with the rest of recurrently mutated splicing factor genes (SRSF2, U2AF1, and ZRSR2). To study alterations in forward- and back-splicing in SF3B1-mutated patients, we examined transcriptional differences on the levels of whole genes, transcript variants, and circRNAs and searched for specificities in transcription within individual gene loci. A set of circRNAs whose levels differed specifically without affecting expression of corresponding forward-spliced mRNAs included several oncology/hematopoiesis-relevant genes (e.g., ATM, CBL, ERCC5, ETV6, FLT3, and MAPK6). Gene loci with changed expression of both, alternative mRNA transcripts and circRNAs, but stable transcription on whole gene level included for example CDK14, KDM1A, and ZEB1. Because ZEB1 (Zinc Finger E-Box Binding Homeobox 1) serves as an essential hematopoietic transcription factor, we focused on ZEB1-derived circRNAs (hsa_circ_0000228 and hsa_circ_0003793) in more detail. We demonstrated that upregulation of these circRNAs is SF3B1-specific and not related to any other clinical or molecular characteristics. Finally, using RNA-seq data from CRISPR/Cas9 edited K562 cells (Liberante FG, et al., Sci Rep. 2019; 9:2678), we confirmed that SF3B1 K700E mutation leads to strong upregulation of ZEB1 circRNAs. To conclude, this is an early report showing for the first time that the levels of specific circRNAs are altered in MDS. We demonstrated that particular circRNAs may have potential to become markers that would contribute to more accurate prognosis of MDS patients. Further, we identified circRNAs with deregulated levels specifically in MDS patients with SF3B1 mutation, suggesting that this mutation affects circRNA production. Supported by GA CR (N20-19162S) and MH CZ-DRO (UHKT, 00023736). Disclosures No relevant conflicts of interest to declare.

The karyotype of bone-marrow cells at the time of diagnosis is one of the most important prognostic factors in patients with myelodysplastic syndromes (MDS). In some cases, the acquisition of additional genetic aberrations (clonal evolution [CE]) associated with clinical progression may occur during the disease. We analyzed a cohort of 469 MDS patients using a combination of molecular cytogenomic methods to identify cryptic aberrations and to assess their potential role in CE. We confirmed CE in 36 (8%) patients. The analysis of bone-marrow samples with a combination of cytogenomic methods at diagnosis and after CE identified 214 chromosomal aberrations. The early genetic changes in the diagnostic samples were frequently MDS specific (17 MDS-specific/57 early changes). Most progression-related aberrations identified after CE were not MDS specific (131 non-MDS-specific/155 progression-related changes). Copy number neutral loss of heterozygosity (CN-LOH) was detected in 19% of patients. MDS-specific CN-LOH (4q, 17p) was identified in three patients, and probably pathogenic homozygous mutations were found in TET2 (4q24) and TP53 (17p13.1) genes. We observed a statistically significant difference in overall survival (OS) between the groups of patients divided according to their diagnostic cytogenomic findings, with worse OS in the group with complex karyotypes (P = .021). A combination of cytogenomic methods allowed us to detect many cryptic genomic changes and identify genes and genomic regions that may represent therapeutic targets in patients with progressive MDS.

Azacitidine (AZA) is a hypomethylating drug used to treat disorders associated with myelodysplasia and related neoplasms. Approximately 50 % of patients do not respond to AZA and have very poor outcomes. There is thus great interest in identifying predictive biomarkers for AZA responsiveness. We searched for specific genes whose expression level was associated with response status. Using microarrays, we analyzed gene expression patterns in bone marrow CD34+ cells in serial samples from 32 patients with myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia with myelodysplasia-related changes before and during the AZA therapy. At baseline, a comparison of the responders and non-responders showed 52 differentially expressed genes (P

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p &lt, 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

We aimed to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with myelodysplastic syndrome (MDS).Targeted enrichment resequencing of 84 DNA repair genes was initially performed on a screening cohort of MDS patients. Real-time polymerase chain reaction was used for genotyping selected SNPs in the validation cohort of patients.A heterozygous frameshift mutation in the XRCC2 gene was identified. It leads to the formation of a truncated non-functional protein and decreased XRCC2 expression level. Decreased expression levels of all DNA repair genes functionally connected with mutated XRCC2 were also present. Moreover, a synonymous substitution in the PRKDC gene and 2 missense mutations in the SMUG1 and XRCC1 genes were also found. In the screening cohort, 6 candidate SNPs were associated with the tendency to develop MDS: rs4135113 (TDG, p = 0.03), rs12917 (MGMT, p = 0.003), rs2230641 (CCNH, p = 0.01), rs2228529 and rs2228526 (ERCC6, p = 0.04 and p = 0.03), and rs1799977 (MLH1, p = 0.04). In the validation cohort, only a polymorphism in MLH1 was significantly associated with development of MDS in patients with poor cytogenetics (p = 0.0004).Our study demonstrates that genetic variants are present in DNA repair genes of MDS patients and may be associated with susceptibility to MDS.

BACKGROUND The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests that defects in DNA repair mechanisms. We monitored DNA repair pathways in MDS and their alterations during disease progression. METHODS Expression profiling of DNA repair genes was performed on CD34+ cells, and paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 was done on histology samples. RESULTS RAD51 and XRCC2 showed differential expression between low-risk and high-risk MDS (P

Background and Aims A part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia (AML). The progression may be predicted in some of these patients by determining mutations associated with myeloid malignancies at the time of diagnosis. We focused to find out dysregulated pathways caused by a well-defined mutation associated with disease progression. Methods We examined 158 samples from LR-MDS patients at diagnosis using TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to identify the mutational profile. We applied RNA-seq (NEB; Illumina) on bone marrow CD34+ cells of 5 LR-MDS patients with RUNX1 mutation and 6 LR-MDS patients without mutations. We performed differential gene expression analysis, gene set enrichment analysis (GSEA), Reactome biological pathway and Gene Ontology (GO) annotations. Results Forty out of 158 patients (25%) progressed during the follow-up period (median: 45.1 months). Mutation in RUNX1 gene was detected in 10 patients. Using RNA-seq, significantly dysregulated expression was observed in 986 genes (FDR2). Patients with mutation in RUNX1 gene had significant down regulation of expression genes involved in cellular senescence induced by DNA Damage/Telomere Stress (FDR=1.4e-11) and Oxidative Stress (FDR=3.6e-9), HDACs deacetylate histones (FDR =1.1e-13) and Senescence Associated Secretory Phenotype (FDR=1.4e-11) according to Reactome pathway database. There were 21 genes involved in the regulation of these pathways, of which 10 were part of all described pathways and belonged to the Nucleosome Assembly according to GO biological process with 33 down regulated genes (FDR=6.3e-28). According to GSEA, GO chromatin silencing gene set (Figure 1) was down regulated in the patients with RUNX1 mutation (p2); however, they did not form a specific pathway. The up-regulated genes were classified as so-called leukemia-associated antigens (LAAs) such as PRAME (adjusted p=6.9e-09; logFC=6.8), WT1AS (adjusted p=4.4e-07; logFC=5.7), BAALC (adjusted p=3.5e-06; logFC=2.1), WT1 (adjusted p=6.8e-07; logFC=3.2), FLT3 (adjusted p=0.004; logFC=1.3) and LEF1 (adjusted p=5.3e-08; logFC=-4.2). Conclusions To study the transcriptome of the hematopoietic stem cells of LR-MDS patients, we used gene expression profiling and identified biological processes in relation to mutations in RUNX1 gene that were detected in patients with disease progression. According to our results, we suppose that cells of LR-MDS patients with no mutations are maintained in senescent state or apoptosis in opposite to RUNX1 mutated cells. Finding out of dysregulated senescence was reinforced by other dysregulated pathways such as HDACs deacetylate histones, chromatin structural changes and senescence-associated secretory phenotype. About cellular senescence is known that may act as a strong tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. We suppose that in precancerous cells of LR-MDS patients apoptosis or cellular senescence limits the replicative capacity of cells, thus preventing the proliferation. The disturbed state of cellular senescence in RUNX1 gene mutated hematopoietic stem cell can escape proliferation control and then can contribute to the disease progression in these patients. Supported by grants NV18-03-00227 and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a tendency to transform to acute myeloid leukemia. Several prognostic systems based almost entirely on clinicopathological characteristics have been developed. However, considering large heterogeneity of MDS, there is an ongoing substantial effort to identify new molecular markers, which would improve patient stratification. It has recently been discovered that various types of small noncoding RNAs (sncRNAs) are exported into blood circulation. Encapsulated in extracellular vesicles (EVs) or as a part of various molecular complexes, they play important roles in long distance cell-to-cell communication. Their secretion appears to be controlled and specific and thus may reflect (patho)physiological processes occurring in different cells. As diagnostic procedures move from bone marrow biopsies towards less invasive techniques, circulating sncRNAs have become of particular interest as potential novel semi-invasive biomarkers. Aims: We investigated circulating sncRNAs in MDS on the genome-wide level to better understand MDS pathogenesis and to search specific RNAs applicable as potential biomarkers. We analyzed paired samples from the whole plasma and EV fractions to define which of these two materials is better source of relevant sncRNA-based markers of MDS. Methods: We performed small RNA-seq in paired samples (plasma and EVs) in 42 patients and 17 healthy controls. To associate sncRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Overall survival (OS)‐associated sncRNAs were identified by performing univariate Cox regression along with a permutation test using BRB‐ArrayTools. Multivariate Cox regression analysis with backward variable selection was done to identify independent variables associated with patient survival. Results: Data analysis showed that the most abundant category of RNAs were miRNAs (51 %), followed by rRNAs, piRNAs, tRNAs, and mRNAs. Sample clustering revealed that RNA content of EV cargo is more homogenous than of plasma. Differential analysis identified a striking disbalance in RNA content between plasma and EVs in MDS patients (> 400 significantly deregulated sncRNAs) whereas these sncRNA profiles remained similar in controls. Such disproportion may suggest that mechanisms of sncRNA export into blood circulation may be specifically affected in MDS. As a result, these changes might substantially affect cell-to-cell communication of blood cells, contributing to the disease development. Further, we analyzed levels of individual sncRNAs and found that many hematopoiesis-related miRNAs were significantly increased in MDS patients compared to healthy controls, mostly both in plasma and EVs (e.g., miR-34a-5p, miR-125a-5p, and miR-150). Several miRNAs (e.g., miR-103b, miR-107, and miR-221) showed different levels between early and advanced MDS. Interestingly, the majority of miRNAs from the 14q32 miRNA cluster were specifically increased in early MDS. Additionally, we investigated possible impacts of somatic mutations on expression of circulating sncRNAs. However, only low numbers of circulating sncRNAs significantly associated with mutational status, suggesting no fundamental effects of somatic mutations on sncRNA export from MDS cells. Survival analysis identified sncRNAs with the highest level of association with OS (plasma: miR-1260b, miR-3191-3p, and miR-328-3p; EVs: miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p). To better stratify MDS patients, we defined risk prediction scores (separately for plasma and EVs) that combined effects of the above mentioned sncRNAs. The results showed that EV-combined score significantly increased the predictive power of the survival risk model (Figure 1). Moreover, Cox multivariate analysis revealed that this score is an independent variable the most significantly associated with OS (HR = 2.942, 95% CI 1.786 to 4.849, p < 0.001). Conclusions: Our data demonstrate that profile of circulating sncRNAs is specific in MDS and changes with the disease progression. Monitoring of sncRNA levels in EVs provides better prognostic performance for patient survival (compared to whole plasma), probably due to higher homogeneity of the EV fraction. Supported by grants 16-33617A and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Myelodysplastic syndromes (MDS) represent a heterogeneous spectrum of hematopoietic stem cell disorders characterized by inefficient hematopoiesis, peripheral blood cytopenia, dysplasia in one or more myeloid cell lineages, and a tendency to evolve into acute myeloid leukemia (AML). Hypomethylating agent azacitidine (AZA) is a standard therapy for patients with higher-risk MDS and AML with myelodysplasia-related changes (AML-MRC). The DLK1-DIO3 region is located on chromosome 14q32 and contains one of the largest miRNA clusters in the genome. Functions of these miRNAs have been linked to apoptosis and proliferation. Their expression is under the control of a differentially methylated region (DMR) located in the promoter of MEG3 gene. Significant overexpression of these miRNAs has previously been found in MDS and acute promyelocytic leukemia (APL). In APL, this upregulation has been linked to hypermethylation of MEG3-DMR. AIMS: We examined (i) expression of the 14q32 miRNAs and mRNAs (DLK1, RTL1, DIO3, MEG3, and MEG8) and (ii) DNA methylation of MEG3-DMR in patients with higher-risk MDS and AML-MRC before and during AZA treatment with the aim to reveal whether the overexpression of these miRNAs is epigenetically regulated in myelodysplasia. METHODS: The study comprised 24 paired samples of CD34+ bone marrow cells obtained from 12 patients with higher-risk MDS and AML-MRC before and during AZA therapy. miRNA profiling was done using Agilent microarrays and mRNA levels of the genes located within the 14q32 region were assessed by Illumina microarrays. Measurement of MEG3 level by RT-qPCR confirmed the microarray results on an independent cohort (81 MDS and 14 AML-MRC patients, and 13 controls). Methylation status of MEG3 promoter was investigated by amplicon bisulfite sequencing. RESULTS: We studied levels of miRNAs and mRNAs encoded in the DLK1-DIO3 region and found that 50 % of untreated patients showed strong upregulation of expression activity within the locus. Following AZA therapy, we observed a significant reduction in the miRNA/mRNA expression to nearly normal levels, suggesting that expression activity might be affected by hypomethylation. Interestingly, expression levels of miRNAs and mRNAs positively correlated, indicating that transcriptional activity was regulated uniformly throughout the region. Evaluation of expression data with clinical features in the pretreatment samples revealed that expression activity was related to BM blast count, patient diagnosis, and outcome. High expression was significantly associated with the diagnosis of AML-MRC and poor outcome, whereas low expression with MDS and favorable outcome. No chromosomal abnormality or cytogenetic risk category was linked to the expression changes. In the patients before treatment, we revealed a significant hypermethylation of a part of the MEG3-DMR that closely preceded the MEG3 start codon. This hypermethylation was seen in 50 % of the patients and negatively correlated with expression activity. Interestingly, the hypermethylation was associated with longer progression-free survival after initiation of AZA therapy (hazard ratio = 0.220, P = 0.03). Following AZA therapy, we identified an apparent decrease of methylation in another part of MEG3-DMR. This hypomethylation might be attributed to the hypomethylating effect of AZA and could therefore influence the deregulated expression of the miRNA cluster. Finally, we investigated the potential application of the deregulation observed in this locus for prognostic purposes regarding the responsiveness to AZA treatment. Although, we identified changes in expression and methylation levels before AZA therapy, these changes were not associated with future response status. CONLUSIONS: Here, we correlated changes in expression of 14q32 miRNAs and mRNAs with specific methylation changes in its regulatory sequences in higher-risk MDS and AML-MRC patients treated with AZA. We thoroughly investigated a complex regulatory mechanism within this region and based on our data, we assumed that epigenetic alterations affect expression activity in the locus. However, also other mechanisms such as influence of some transcription factors may play yet unrecognized roles in the resulting state. Supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 (Ministry of Health of the Czech Republic). Disclosures No relevant conflicts of interest to declare.

Background and Aims A part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia. These patients have more aggressive type of disease than is expected based on their classification according to the International Prognostic Scoring System. Alongside, recent advances in DNA sequencing have enabled the identification of a large number of mutated genes in more than 80% of MDS patients. We focused on the identification of mutational profile that would be predictive of disease progression in LR-MDS patients. Methods We examined samples from 73 LR-MDS patients by TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to determinate the mutational profile. In 32 (44%) patients, paired samples processed at the time of diagnosis and during progression were analyzed. The median age of patients was 64.5 years (range, 29-83 years). The median observation period was 33.4 months (range, 4.5-195.9 months) from diagnosis, 36 (49%) patients remained alive including 8 who underwent HSCT. The FASTQ files were analyzed by NextGENe V2.4.2.2 (SoftGenetics). The survival distributions were estimated using the Kaplan-Meier method and prognostic factor analysis was performed with Cox's multivariate regression method. The Mann-Whitney test was used to determine the group independence. Results In total, 70% of patients had a recurrent mutation in at least one gene of the panel (87.5% of patients with progression and 56% without progression). The patients, who progressed, carried on average of 2.4 mutations and unprogressed patients 1.9 mutations. Most of the mutations detected in LR-MDS were in SF3B1 (30.1% of patients) and DNMT3A (24.7% of patients) genes but had no effect on overall survival (OS). Multivariate analysis demonstrated that RUNX1 and TP53 mutations were the strongest independent prognostic factors for OS (HR: 14.9; p=0.0001/HR:6.3; p=0.0007) at the time of diagnosis and also for progression-free survival (PFS) (HR: 3.6; p=0.05/HR:3.3; p=0.05). Moreover, mutations in these genes were detectable in 59.4% of patients with disease progression and only in 4.8% of patients without progression, who however were followed shortly (4.7 months). The median OS was 27.9 and 39.6 months in the patients with mutations of RUNX1 and TP53 genes compared to 84.2 months (p Conclusions The goal of the study was to detect mutations in relevant genes that might predict disease progression up to 6 years from the time of diagnosis in LR-MDS patients and comparison of mutant allele frequencies in selected cases between the time of diagnosis and disease progression. We found that 60% of patients who progressed, carried mutations in RUNX1 and TP53 genes at the time of diagnosis. On the contrary, other mutations detectable at the time of diagnosis had no effect on OS and PFS. The VAF of mutations detected at the diagnosis increased at the time of progression. The NRAS and PTPN11 genes contained most of the new mutations acquired during progression. In conclusion, the results indicate that routine monitoring for mutations in LR-MDS should be performed to refine the risk prediction of disease progression. Supported by AZV grants (16-33485A, 16-31689A, 17-31398A and NV18-03-00227), RVO-VFN64165 and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by dysplastic and ineffective blood cell production and a tendency for transformation to acute myeloid leukemia (AML). Because of the heterogeneity of MDS, the development of additional molecular tools able to refine the Revised International Prognosis Scoring System (IPSS-R), predict outcome and monitor the response to treatment would be highly beneficial. Long noncoding RNAs (lncRNAs) are protein-noncoding RNAs that play multiple roles in hematopoietic differentiation. They contribute to the pathogenesis of hematologic malignancies, representing a new class of potential biomarkers and therapeutic targets. AIMS: In this study, we aimed to identify lncRNAs with deregulated expression in MDS that could serve as novel potential molecular markers of poor outcome. METHODS: The discovery cohort included 54 MDS patients, 14 AML patients with myelodysplasia-related changes (AML-MRC), and 9 controls. Agilent microarrays were used to profile lncRNAs expression in CD34+ bone marrow (BM) cells. Data analyses were focused on the identification of lncRNAs with altered levels between MDS patients (i) with (very) low vs. (very) high risk IPSS-R, and (ii) with progression-free survival (PFS) shorter vs. longer than 2 years. RT-qPCR was applied to validate expression levels of selected deregulated lncRNAs (H19, TCL6, LEF1-AS1, and WT1-AS) and levels of their protein-coding counterparts (LEF1 and WT1) in an independent validation cohort (79 MDS and 14 AML-MRC patients, and 13 controls). Further, we examined whether the expression of H19, TCL6, LEF1, and WT1 changed in patients treated with azacitidine (AZA). For this purpose, we retrieved data from our previous microarray analysis (GEO GSE77750) performed on a cohort of 19 MDS, 3 chronic myelomonocytic leukemia (CMML), 10 AML-MRC patients and 10 controls. RESULTS: In the MDS patients with higher risk IPSS-R, 23 lncRNAs were downregulated (e.g., CXADRP3, LEF1-AS1, PCED1B-AS1, SLC8A-AS1, and TCL6) and 35 lncRNAs were upregulated (e.g., BCAR4, FAM225A, FARP1-AS1, H19, and RBPMS-AS1) vs. lower risk IPSS-R (|logFC| ≥ 1, p ≤ 0.01). Furthermore, we identified significant upregulation of e.g. H19, SATB2-AS1, and WT1-AS in patients with PFS < 2 years vs. those with longer survival. For subsequent study, we selected H19, LEF1-AS1, TCL6, and WT1-AS lncRNAs based on their known association with hematopoiesis. In the validation cohort, we proved that downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with poor PFS and OS (p < 0.01, Figure 1). Further, we correlated lncRNA expression levels with multiple clinical parameters (sex, age, BM blasts, hemoglobin, neutrophils, platelets, and IPSS-R-based cytogenetics) and found out that only level of H19 was independent from all these variables whereas expression of other lncRNAs was associated with BM blast count. Multivariate analysis confirmed that H19 level, BM blasts, and platelets remained independent factors for OS and PFS in MDS (p < 0.05). Importantly, WT1-AS level, platelets, and hemoglobin were identified as independent variables for survival of MDS patients with BM blast counts < 5 % (p < 0.05). Additionally, we measured levels of appropriate protein-coding counterparts of LEF1-AS1 (i.e. LEF1) and WT1-AS (i.e. WT1) and found strong positive correlation between these pairs (p < 0.001), indicating their co-transcriptional regulation. Because expression of the studied lncRNAs significantly varied between lower and higher risk MDS patients, we further investigated impact of AZA treatment on the expression of H19, TCL6, LEF1, and WT1 in higher risk disease. However, we detected neither any difference in their expression between responders and nonresponders nor any significant return to normal expression levels caused by AZA therapy. CONLUSIONS: Our data proved that particular lncRNAs are specifically deregulated in higher risk MDS. Altered transcript levels of these lncRNAs may serve as independent markers of poor survival, even in the patients with low BM blast counts, providing additional information about prognosis of MDS patients beyond the currently used scoring system. The work was supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Introduction: Chromothripsis is a recently identified genomic instability phenomenon that plays a role in the genesis and progression of cancer. It is a one-step catastrophic genomic event involving multiple chromosomal breakages and random DNA rejoining. This genetic abnormality can affect an entire chromosome, a chromosomal arm, or a single chromosomal region. Chromothripsis is associated with highly complex karyotypes and a very poor prognosis, and has been detected in a wide range of tumor entities, including hematological malignancies. However, this complex genomic abnormality has not been comprehensively studied in patients with myelodysplastic syndromes (MDS). The aim of the study was to assess the incidence, associated genetic features, and clinical significance of chromothripsis in a large homogeneous cohort of patients newly diagnosed with high-risk MDS and complex karyotypes. Methods: A detailed genome-wide analysis of fixed bone-marrow cells from adults with complex karyotypes (≥ 3 aberrations), identified with conventional G-banding at the diagnosis of MDS, was performed. The complex rearrangements were studied with integrative genetic methodologies: fluorescence in situ hybridization (FISH) with Vysis DNA probes (Abbott, Des Plaines, IL), multicolor FISH (mFISH) and/or multicolor banding (mBAND) methods with the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany), and array-based comparative genomic hybridization with CytoChip Cancer SNP 180K (Illumina, San Diego, CA) or the SurePrint G3 Cancer CGH+SNP 4x180K Microarray (Agilent, Santa Clara, CA). A mutational analysis of the TP53 gene was also performed in selected cases using amplicon-based deep sequencing on a 454 GS Junior System (Roche, Basel, Switzerland) or the TruSight Myeloid Sequencing Panel on MiSeq sequencing instruments (Illumina). Results: In total, 265 patients with complex karyotypes and newly diagnosed high-risk MDS were included (131 females, 134 males; median age, 70 years). The hallmarks of chromothripsis were detected in 67.7% of cases, in both the main clones and one or more subclones. At the cytogenetic level, chromothripsis was apparent as multiple deletions, insertions, ring chromosomes, amplification of individual genes or chromosomal regions, and/or the formation of chaotically reassembled chromosomes. Chromothripsis affected almost all the chromosomes, except the Y chromosome. The most frequently involved were chromosomes 5 (33.3% of events), 7 (28.1% of events), 17 (18.9% of events), 11 and 12 (15.6% of events each). In samples with signs of chromothripsis, the higher frequency of aberrations on chromosome 17p was observed (loss of heterozygosity [LOH], copy-number neutral LOH [cnLOH] and/or homozygous mutation of TP53) (p = 0.05). Patients with chromothripsis had significantly worse overall survival (OS; median, 3 months). We also investigated the effect of chromothripsis on the survival of 183 patients treated with azacytidine. In this cohort, the median OS of chromothripsis-positive patients was 10.1 months (mean, 12.2 months), whereas the median OS of chromothripsis-negative patients was 17.3 months (mean, 31.0 months). Conclusions: Our results demonstrate that chromothripsis is a frequent genomic abnormality in patients with high-risk MDS, which influences the patient's prognosis and disease biology. Chromothripsis was associated with a higher frequency of LOH/cnLOH 17p, rapid disease progression, and short survival. The adverse outcomes may be attributable to the effects on the functions of many important genes. The OS of patients with high-risk MDS with chromotripsis may be slightly improved by azacytidine treatment, but the prognosis of these patients remains very poor. Therefore, a better understanding of the mechanistic basis of chromothripsis is extremely important and could lead to the development of new treatment strategies based on drugs that target the genes present in amplified or deleted regions and/or the DNA damage response pathways. This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, AZV 16-27790A , Progres Q26 and Q28/LF1, GACR 18-01687S, MHCR 00023736 and UNCE/MED/016 . Disclosures No relevant conflicts of interest to declare.

// Monika Belickova 1 , Jitka Vesela 1 , Anna Jonasova 2 , Barbora Pejsova 1 , Hana Votavova 1 , Michaela Dostalova Merkerova 1 , Zuzana Zemanova 3 , Jana Brezinova 1 , Dana Mikulenkova 1 , Marie Lauermannova 1 , Jan Valka 1 , Kyra Michalova 1, 3 , Radana Neuwirtova 2 , Jaroslav Cermak 1 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic 2 First Department of Medicine, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic 3 Center of Oncocytogenetics, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic Correspondence to: Monika Belickova, email: monika.belickova@uhkt.cz Keywords: myelodysplastic syndrome, mutational status, TP53, prognosis, variant allele frequency Received: January 03, 2016 Accepted: April 16, 2016 Published: May 06, 2016 ABSTRACT TP53 mutations are frequently detected in patients with higher-risk myelodysplastic syndromes (MDS); however, the clinical impact of these mutations on the disease course of patients with lower-risk MDS is unclear. In this study of 154 lower-risk MDS patients, TP53 mutations were identified in 13% of patients, with prevalence in patients with del(5q) (23.6%) compared to non-del(5q) (3.8%). Two-thirds of the mutations were detected at the time of diagnosis, and one-third were detected during the course of the disease. Multivariate analysis demonstrated that a TP53 mutation was the strongest independent prognostic factor for overall survival (OS) (HR: 4.39) and progression-free survival (PFS) (HR: 3.74). Evaluation of OS determined a TP53 variant allele frequency (VAF) threshold of 6% as an optimal cut-off for patient stratification. The median OS was 43.5 months in patients with mutations detected at the time of diagnosis and a mutational burden of > 6% VAF compared to 138 months (HR 12.2; p = 0.003) in patients without mutations; similarly, the median PFS was 20.2 months versus 116.6 months (HR 79.5; p < 0.0001). In contrast, patients with a mutational burden of < 6% VAF were stable for long periods without progression and had no significant impact on PFS or OS. Additionally, we found a high correlation in the mutational data from cells of the peripheral blood and those of the bone marrow, indicating that peripheral blood is a reliable source for mutation monitoring. Our results indicate that the clinical impact of TP53 mutations in lower-risk MDS patients depends on the level of mutational burden.

We used microarray profiling to investigate the direct effects of lenalidomide on gene expression in isolated CD14(+) monocytes from 6 patients with del(5q). Our data demonstrate that changes in genes involved the tumor necrosis factor (TNF) signaling pathway and the bone marrow stroma, suggesting that treatment with lenalidomide may help restore the damaged niche and suppress the TNF signaling pathway.Lenalidomide is an effective treatment for patients with del(5q) and myelodysplastic syndrome (MDS) The exact mechanism of lenalidomide function and its impact on the prognosis of patients is not known exactly.We used gene expression profiling to study the effect of lenalidomide therapy in peripheral blood CD14(+) monocytes of 6 patients with del(5q) and MDS.After lenalidomide treatment, genes involved in the tumor necrosis factor (TNF) signaling pathway that were upregulated in the patients before treatment decreased to the healthy control baseline expression level. This change in gene expression, in conjunction with increased expression of repressed genes that affect the stem cell niche (ie, CXCR4 and CRTAP), may exert a positive effect on treated patients. In contrast, we found that increased expression of the ARPC1B gene may have a negative impact on the stability of patient remission.The observed changes in gene expression described here may contribute to the identification of pathways that are affected by lenalidomide, which may help to explain the effects of this drug.

Background Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. High-risk MDS patients are treated by hypomethylating agents, of which they benefit significantly. However, only half of the patients respond positively to the treatment. Aberrant DNA methylation and mRNA expression in MDS were documented in several studies, but their prognostic impact in response to hypomethylating therapy is still unclear. The aim of the project was to find a relationship between methylation and expression status prior to azacytidine (AZA) treatment and the overall survival and clinical response of MDS patients. Methods We performed methylation and expression profiling in CD34+ cells from 30 samples from MDS patients before AZA treatment and after 4-8 treatment cycles. HumanMethylation27 BeadChips and HumanHT-12 v4 Expression BeadChips (Illumina) were used to generate profiles. DNA and RNA were isolated from same CD34+ cells separated from bone marrow by magnetic beads. The β-values represent quantitative measurements of DNA methylation levels of specific CpGs, and range from 0 for completely unmethylated to 1 for completely methylated DNA. The nonparametric Mann-Whitney test was used for comparison of β-values and expression levels between responders and nonresponders. Results To determine whether DNA methylation and expression might predict a response to AZA treatment, we compared methylation and expression status at baseline with clinical responses in 30 MDS patients. Twelve patients of 30 (40%) achieved complete remission or partial remission, 10 had stable disease (33.3%), and 8 showed progression (26.7%). Median survival after initiation of AZA treatment in progression patient group was 8.7 months, stable group 21.2 months, and group with complete or partial remission 24.5 months. We found significant differences in methylation status in 20 genes (p Conclusions Our finding of different DNA methylation levels at baseline between groups of responders and nonresponders as well as detection of decreased methylation after AZA treatment in the group of patients with clinical response may represent useful prediction markers of treatment success. However, the data require detailed examination along with confirmative cohort of patients. Supported by grant (NT/13899, NT/14377, NT/14539, NT/13847) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures: No relevant conflicts of interest to declare.

Abstract 5023 A direct effects of lenalidomide on gene expression in 5q- patients was studied using HumanRef-8 v2 Expression BeadChips (Illumina). Expression profiles of 6 patients (before treatment and at the time of the first erytroid response) and 6 healthy controls were investigated from CD14+ monocytes of peripheral blood. Differentially expressed genes were identified by Significance Analysis of Microarrays (SAM). Simultaneously, selected genes (TNF, JUN, IL1) were monitored in the course of treatment using Real-Time PCR with Taqman Gene Expression Assays. A comparison of gene expression levels before and during lenalidomide treatment revealed 97 differentially expressed genes (FC >2; p To conclude, described changes in genes expression may contribute to identification of the pathways affected by lenalidomide and to the explanation of some effects of this drug that have not been fully understood yet. Supported by grants NS/9634 MZCR, UHKT2005 00023736, MSM0021620808 and COST EUGESMA Disclosures: No relevant conflicts of interest to declare.

BACKGROUND AND AIMS Recent results have indicated that the TP53 mutations had a negative prognostic relevance for lower-risk myelodysplastic syndrome (MDS) patients but the relations between the presence of mutations and treatment has not been clarified yet. Therefore, we analyzed the prevalence of TP53 mutations in lower-risk MDS patients and determined the proportion of TP53 mutated bone marrow cells during the disease. We further observed mutual relation of treatment and mutated clones and defined impact of mutations on survival of patients. METHODS TP53 mutations were analyzed in DNA from bone marrow of 141 low risk or intermediate-1 risk MDS patients included into the cohort according to the International Prognostic Scoring System using primers designed within the IRON-II study research consortium (Roche Applied Science) on the GS Junior system. If mutations were detected, we searched for mutations at different timepoints of the disease and overall 210 samples were examined for mutations. Ten of the 16 patients with TP53 mutation received lenalidomide and/or 5-azacytidine. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. RESULTS The overall incidence of TP53 mutations was 11.3 % (16/141), three patients harbored two mutated clones. The mutation frequency in patients with and without del(5q) was 21% (13/62) and 3.8% (3/79), respectively. During the observation period, 37 patients (26.2%) died, the data obtained from remaining patients were censored either at the last date they were known to be alive (94 patients - 66.7%) or at the time of stem cell transplantation (HSCT) (10 patients - 7.1%). The median overall survival was 164.2 months in TP53 unmutated group and 84.5 in mutated group respectively calculated by Kaplan-Meier Survival Curves and the Log-Rank Test (P=0.18). Mutations were detectable in eight of the sixteen available patients (50%) at the time of diagnosis and remaining patients (50 %) displayed mutations in an average 35.1 months (range 12-69 months) from diagnosis. Three patients with mutational burden below 5% are alive more than 3 years without signs of disease progression and three patients with clone size above 40% died. The mutated clone disappeared after bone marrow transplantation in one patient. The proportion of TP53 mutated cells decreased radically after successful treatment with azacytidine in two patients. The clone size increased in four patients treated with lenalidomide and all of them died. Two patients with mutated clone sizes below 20% are still under observation. CONCLUSIONS We confirmed the higher frequency of TP53 mutations in lower- risk MDS with del(5q) in a large cohort of patients. Patients with the TP53 mutations had shorter survival compared to those without mutation. The mutations were detected in a half of the patients at the time of diagnosis and approximately three years after diagnosis in remaining patients. The presence of TP53 mutation as an adverse prognostic factor should be routinely investigated in lower risk MDS patients at the time of the diagnosis as well as repeatedly in the course of the disease. Supported by grant (NT/13899, NT/14377, NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

BACKGROUND AND AIMS Approximately 30% of patients with myelodysplastic syndrome (MDS) progress to acute myeloid leukemia (AML) within a few months up to several years. So far, blast counts and cytogenetic abnormalities are known to be major determinants of the risk for AML transformation; however, little is known about the molecular genetic events associated with MDS progression. Alongside, recent advances in DNA sequencing have enabled the identification a large number of new mutated genes in 90% of MDS patients. We focused on the identification of prognostic factors for patients who progress, namely, creating a specific mutational profile predicting disease progression. METHODS We examined paired samples from 23 patients by TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) for formation of mutational profile. Patients were evaluated at the time of diagnosis and during progression to more advanced MDS subtype or to AML. Selected mutations were confirmed by Sanger sequencing. The survival distributions were estimated using the Kaplan-Meier method and prognostic factor analysis was performed with Cox`s multivariate regression method. RESULTS A total of 43 mutations at diagnosis and 56 mutations in progression across 54 genes were identified in the patient cohort. Patients carried at average 2.4 mutations, two patients carried five mutations and only one patient had no detectable mutation. The variant allele frequency (VAF) of mutations detected at the time of diagnosis increased during disease progression, except of mutations in SFR3B1 and TET2 genes. The most common acquired mutation at the time of progression was found in PTPN11 gene. 57% of patients harbored mutations in RUNX1, TP53, PTPN11 or NRAS gene at the time of diagnosis and 13 % of patients evolved these mutations in the course of disease. The median overall survival (OS) was 9.9 months in patients with this combination of mutations compared to 34 months (HR 4.0; p=0.01) in patients without mutations above mentioned. Multivariate analysis demonstrated that the combination of RUNX1, TP53, PTPN11 or NRASmutations was the strongest independent prognostic factor for OS (HR: 20.9; p=0.003) at the time of diagnosis. CONCLUSIONS Comparison of mutational profiles of MDS patients at diagnosis and during progression suggested candidate genes involved in AML transformation. Our study demonstrates that mutated RUNX1, TP53, PTPN11 and NRAS are most associated with the disease progression. Mutations in these genes are often detectable with low VAF already at the time of diagnosis and in some patients are detectable only at the time of progression. On the contrary, individual SF3B1 or TET2 mutations are associated with delayed progression and longer OS, which corresponds to a good prognostic value of these mutations. In conclusion, the results indicate that routine monitoring for mutations in MDS should be performed to refine the risk prediction of disease progression. Supported by AZV grants (16-33485A and 16-31689A) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Background Interactions between genetic variants and risk factors in myelodysplastic syndromes are poorly understood. In this case–control study, we analyzed 1 421 single nucleotide polymorphisms in 408 genes involved in cancer-related pathways in 198 patients and 292 controls. Methods The Illumina SNP Cancer Panel was used for genotyping of samples. The chi-squared, p-values, odds ratios and upper and lower limits of the 95% confidence interval were calculated for all the SNPs that passed the quality control filtering. Results Gene-based analysis showed nine candidate single nucleotide polymorphisms significantly associated with the disease susceptibility (q-value . Four of these polymorphisms were located in oxidative damage/DNA repair genes (LIG1, RAD52, MSH3 and GPX3), which may play important roles in the pathobiology of myelodysplastic syndromes. Two of nine candidate polymorphisms were located in transmembrane transporters (ABCB1 and SLC4A2), contributing to individual variability in drug responses and patient prognoses. Moreover, the variations in the ROS1 and STK6 genes were associated with the overall survival of patients. Conclusions Our association study identified genetic variants in Czech population that may serve as potential markers for myelodysplastic syndromes.