ALBERT

Description: Introduction. Fli1 (Friend leukemia virus integration 1) together with other transcription factors induces the megakaryocytic differentiation of MEP (megakarycytic and erythroid progenitor). Refractory anemia and thrombocythemia is typical for 5q- syndrome. We found increased mRNA level of Fli1 in mononuclear bone marrow cells of 5q- syndrome patients in comparison with healthy controls (Neuwirtova et al., Ann Hematol 2013). The reason of the elevated Fli1 in 5q- syndrome is haploinsufficiency of microRNA-145, which targets Fli1 mRNA (Kumar et al., Blood 2011). Due to haploinsufficiency of RPS14 in 5q- syndrome non-consumed ribosomal proteins cause ribosomal stress and inactivate HDM2 in erythroblasts. E3 ubiquitin ligase HDM2 regulates p53 level by p53 degradation in proteasome. Inactivated HDM2 in erythroid precursors of 5q- syndrome leads to apoptosis of erythroblasts and to anemia. Why ribosomal stress does not cause thrombocytopenia and ineffective megakaryopoiesis as well? Our previous results support significant role of Fli1 in this process. Fli1 binds to promoter of the HDM2 gene and increases its transcription (Truong et al., Oncogene 2005). The increased activity of HDM2 in megakaryocytes inspite of ribosomal stress maintains p53 regulation and its degradation in proteasome. Megakaryopoiesis remains effective. Why it is not the case in erythroid precursors? To answer this question it was necessary to detect Fli1 as the protein and to determine in which cells Fli1 is present. Material and Methods. Twenty-three control representative bone marrow trephine biopsies of patients from controls (8 negative staging biopsies in lymphoma) and of patients with various hematological diagnoses (7 MDS with normal chromosome 5, 4 MPN, 3 AML and 1 RARS-T) and from 15 patients with 5q- syndrome were examined. In 13 patients with 5q- syndrome, samples taken before and 6 months after lenalidomide (Revlimid) therapy were available. The expression of Fli1 protein was investigated by immunohistochemistry (IHC). Expression of Fli1 on erythroid precursors was studied by double staining IHC procedure utilizing antibodies against Fli1 and either glycophorin A or E-cadherin known as reliable markers for erythroid precursors. Results. Nuclear expression of Fli1 was demonstrated in normal as well as in dysplastic megakaryocytes, in most cells of granulocytic series and lymphocytes. No staining for Fli1 was seen in erythroblasts and proerythroblasts visualized by expression of either glycophorin A or E-cadherin both in 5q- syndrome and controls. There were no significant differences in Fli1 expression between samples taken before and after lenalidomide treatment.The used IHC technique does not permit quantitative analysis of Fli1 protein levels. This fact could explain why we did not find any difference in Fli1 protein labeling in megakaryocytes before and after lenalidomide treatment while Fli1 mRNA level was decreased in majority of 5q- syndrome patients after six months of this therapy. Conclusion. Fli1 expression was found in normal as well as in dysplastic megakaryocytes. However, no Fli1 positivity was found in erythroid precursors in both 5q- syndrome and controls. Negativity of Fli1 expression in erythroid precursors in 5q- syndrome support our hypothesis of protective role of Fli1 against apoptosis under ribosomal stress in megakaryocytes in contrast to erythroblasts lacking Fli1. This protective role of Fli1 in megakaryocytes consists in Fli1 potentiation of expression of the E3 ubiquitine ligase HDM2 (Truong et al., Oncogene 2005). The presence of increased Fli1 in megakaryocytes helps to explain effective megakaryopoiesis in 5q- syndrome and is the answer to the question in the title of our abstract. Supported by Ministry of Health, Czech Republic-conceptual development of research organization Institute of Hematology and Blood Transfusion 00023736, RVO-VFN64165 and PRVOUK P-27/LF1/2. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND AND AIMS The high incidence of mutations in patients with myelodysplastic syndrome (MDS) strongly suggests an implication of defective DNA repair mechanisms in the MDS pathogenesis. Based on the hypothesis that genetic changes are amplified during evolution of MDS-cell clone and disease progression; we investigated abnormalities in DNA repair gene expressions and monitored their possible development during MDS progression. Further, we focused on sequencing of selected DNA repair genes and searched for their mutations associated with the disease. METHODS First, gene expression of 84 DNA repair genes in bone marrow (BM) CD34+ cells of 18 MDS patients was measured by RT² Profiler PCR Arrays (Qiagen). Validation of expression data in selected genes was performed on a cohort of 100 MDS patients. Moreover, paired samples from 15 patients with disease progression were used for monitoring of RAD51 and XRCC2 gene expressions in the course of disease. Mutational analysis was performed on 84 DNA repair genes in 16 patients by targeting next generation sequencing (NGS) (SeqCap EZ System, NimbleGen). Detected mutation was confirmed by Sanger sequencing. Multivariate analysis using a Cox regression model to determine the independent impact of each variable (BM blasts, hemoglobin, neutrophils, platelet count, karyotype, and gene expressions of RAD51 and XRCC2) examined for overall survival (OS) was done. RESULTS RAD51 (p

Description: The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)–independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC50 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

Description: Abstract 4641 Introduction: The 5q- syndrome constitutes a World Health Organization (WHO)-defined category of myelodysplastic syndromes (MDS) with the following hematological features: macrocytic anemia, erythroid hypoplasia, normal or elevated platelet count, hypolobulated megakaryocytes and isolated del(5q). Its pathogenesis remains uncertain. Lenalidomide has shown impressive rate of erythroid and in some cases even cytogenetic response. Lenalidomide is anti-angiogenic, anti-adhesive, affects important cytokine circuits and can directly induce growth arrest and apoptosis in certain types of malignant cells. There is also a potent immunomodulatory effect including costimulation of CD4+ and CD8+ T-cells that are partially activated via the T-cell receptor and enhancement of NK cell-mediated lysis. The mechanism of lenalidomide on 5q- syndromes patients is not known, but appears to be suppression of the 5q- clone, resulting in transfusion-independence and a rise in hemoglobin levels. The aim of this study is to investigate the direct effects of lenalidomide on gene expression in isolated CD3+ T-lymphocytes and CD14+ monocytes from 5q- syndromes. Patients and Methods: Six lenalidomide responding patients (three females and three males) with 5q- syndromes (aged 55 to 75 years) and six healthy controls (aged 32 to 74 years) were included in the study. Samples of peripheral blood were collected before the treatment and then at the time of first erytroid response (2-5 months). HumanRef-8 v2 Expression Bead Chips (Illumina) were used to generate expression profiles. The raw data were normalized with the R software, lumi package. Normalized data were filtered by detection p-value 1.5; p

Description: Introduction Treatment with 5-azacytidine (5-AC) is indicated for high-risk MDS patients. Besides the inhibitory effects of 5-AC on DNA and RNA methylation, 5-AC has been recently shown to induce DNA damage and apoptosis in cultured cells. However, in vivo effects of 5-AC remain to be elucidated. Several recent publications implicate aberrant bone marrow (BM) microenvironment and inflammation-related changes in the occurrence and/or progression of the MDS. To provide more insights into this emerging concept, we assessed: i) the extent to which inflammation related cytokines may contribute to MDS progression, and ii) potential changes of cytokine abundance in response to 5-AC therapy. Patients and methods We have collected BM samples from 30 high-risk MDS patients (IPSS int II or IPSS high, 16 females, 14 males) treated by 5-AC at the Hematology Clinic, General University Hospital in Prague. Patients' mean age was 72y (range 55-85) and the WHO 2008 diagnoses were: 15 RAEB II, 5 RAEB I, 2 CMML II, 2 RCMD, 1 U-MDS/MPN and 5 AML/MDS with 〈 30% myeloblasts. We analyzed BM aspirates collected before 5-AC therapy and at day 7 after the completion of respectively the 4th and 8th cycle at which time initial response was also assessed. BM plasma was immediately separated from cells and kept in liquid nitrogen until the time of analysis. As controls we used BM samples from 4 healthy subjects (males, mean age 42y, range 32-59), along with BM samples from 6 low-risk 5q- MDS patients (females, mean age 68y, range 46-80). For the presence of inflammation-related cytokines, BM plasma was analyzed using Human Inflammation 11-Plex (IFNγ, IL1α, IL1β, IL6, IL8, IL10, IL12p70, IL27, IP10, MCP1, and TNFα; YSLBio) via flow cytometry. For the purposes of data analysis, 5-AC treated patients were divided into 2 groups depending on their response to therapy: responders (hematological improvement, partial remission, complete remission, complete remission with incomplete BM recovery) and non-responders (stable disease, progressive disease). Obtained cytokine values were transformed using Box-Cox procedure, and repeated measurements, analyzed using linear models with mixed effects. Comparisons of 5-AC-treated patients, 5q- MDS low-risk patients and controls were subjected to a Kruskal-Wallis test. P-values less than 0.05 were considered as statistically significant. Analyses were conducted using the R statistical package, version 3.1.2, R Core Team (2014). Results Among the 11 cytokines analyzed, 3 (IL27, IP10 and MCP1) displayed significantly altered levels when comparing high-risk 5-AC treated patients, low-risk MDS patients and healthy controls. First, IL27 was elevated in low-risk MDS in comparison to 5-AC or healthy controls (p = 0.041); Figure 1. For IP10, 5-AC MDS patients before therapy showed higher levels (p = 0.005) compared to the low-risk group and healthy controls, respectively. The difference for IP10 was also significant after 4 cycles of 5-AC therapy (p = 0.005), but insignificant after 8 cycles (p = 0.288). The difference in IP10 levels between the two treated groups (4 vs. 8 cycles) were not significant, likely reflecting insufficient sample size, thereby masking the presumably lower levels of IP10 in responders (Figure 2). Further, the 5-AC treated patients showed higher levels of MCP1 than MDS low-risk patients and healthy controls, a difference apparent before therapy (p = 0.011), after 4 cycles (p = 0.003), but not after 8 cycles of therapy (p = 0.058). Also, MCP1 levels changed (p = 0.030) during the treatment, yet irrespective of clinical responses to therapy (Figure 3). Conclusions The IL27 level was higher in low-risk MDS patients compared to high-risk MDS 5-AC patients. Levels of IP10 and MCP1 were higher in high-risk MDS 5-AC patients. Levels of MCP1 changed significantly during the 8 cycles of 5-AC therapy. The observed correlation of IP10 with responses to 5-AC therapy should be further validated. Acknowledgment and Institutional support: This study was supported by grant from Internal Grant Agency of Ministry of Health of the Czech republic (Project NT14174-3) and by Institutional grant (Project RVO 68378050). Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Anemia represents the main therapeutic challenge in pts with lower-risk MDS (Fenaux P, Adès L. Blood. 2013;121:4280-6). Prospective studies evaluating LEN for the treatment of red blood cell transfusion-dependent pts showed significant clinical activity in both non-del(5q) and del(5q) International Prognostic Scoring System-defined lower-risk MDS (Raza A, et al. Blood. 2008;111:86-93; Santini V, et al. Blood. 2014;124:abstract 409; List A, et al. N Engl J Med. 2006;355:1456-65; Fenaux P, et al. Blood. 2011;118:3765-76). Hematologic adverse events (AEs) are common, but manageable, with LEN treatment (Giagounidis A, et al. Ann Hematol. 2008;87:345-52). However, there has been no direct comparison of safety profiles in non-del(5q) and del(5q) pts. This pooled analysis compared the incidence of AEs in LEN-treated lower-risk MDS pts with or without del(5q). Methods: This retrospective analysis of pooled data from 7 prospective clinical trials compared the incidence of AEs in LEN-treated lower-risk MDS pts with or without del(5q). The non-del(5q) group included 416 pts from 4 studies: MDS-005 (n = 160), MDS-002 (n = 215), MDS-001 (n = 24), and PK-002 (n = 17). The del(5q) group included 243 pts from 5 studies: MDS-003 (n = 148), MDS-004 (n = 69), MDS-007 (n = 11), MDS-001 (n = 8), and PK-002 (n = 7). A TEAE was defined as an AE that began or worsened in severity on or after the first dose of LEN through to 28 days after the last dose of LEN. Pts received the recommended starting dose of 10 mg LEN for ≥ 1 cycle; in study MDS-005, pts with impaired creatinine clearance (CrCl; ≥ 40 to 〈 60 mL/min) had a LEN 5 mg starting dose in order to achieve a similar area under the curve as pts with normal CrCl who were receiving LEN 10 mg. Results: Among the LEN-treated lower-risk MDS pts with or without del(5q) in this pooled analysis, the most commonly reported TEAEs (any grade) occurring in ≥ 5% of pts were hematologic: neutropenia [49.3% vs 73.7% for non-del(5q) vs del(5q), respectively], thrombocytopenia (37.3% vs 64.2%), and anemia (16.8% vs 20.2%). Overall, 84.6% of non-del(5q) pts and 96.3% of del(5q) pts experienced grade 3-4 hematologic TEAEs, including neutropenia [45.2% vs 72.0% for non-del(5q) and del(5q), respectively], thrombocytopenia (31.3% vs 52.7%), and anemia (11.8% vs 12.8%) (Table). Non-hematologic TEAEs were similar for both non-del(5q) and del(5q) pts, except deep-vein thrombosis (1.2% vs 4.9%, respectively) and hypertension (0.2% vs 3.7%). Acute myeloid leukemia was reported as a TEAE in 3 non-del(5q) and 9 del(5q) pts. Bleeding events (any grade) occurring concurrently with grade 3-4 thrombocytopenia were observed in 20.7% of non-del(5q) and 24.4% of del(5q) pts. Infection (any grade) occurring concurrently with grade 3-4 neutropenia was observed in 33.6% of non-del(5q) and 54.0% of del(5q) pts. Analysis of grade 3-4 hematologic TEAEs for pts receiving long-term (〉 12 months) LEN treatment by time of onset (0 to 6, 〉 6 to 12, and 〉 12 to 18 months) showed that onset rates of grade 3-4 neutropenia during the first 6 months were higher versus rates at 〉 6 to 12 months for non-del(5q) (42.9% vs 19.5%, respectively) and del(5q) pts (65.4% vs 21.3%). Rates decreased similarly for thrombocytopenia in non-del(5q) (13.0% vs 5.2%) and del(5q) pts (40.4% vs 6.6%). At 〉 12 to 18 months, onset rates of neutropenia and thrombocytopenia for non-del(5q) pts were 15.6% and 9.1%, respectively; rates for del(5q) pts during this period were 23.5% and 4.4%. Grade 3-4 TEAEs resulted in discontinuation of LEN in 27.4% of non-del(5q) and 20.6% of del(5q) pts (Table); however, the criteria for discontinuation differed between studies. Conclusions: In this analysis of pooled data from 7 studies, the safety profiles of LEN-treated lower-risk MDS pts were similar between non-del(5q) and del(5q) pts. Neutropenia and thrombocytopenia were the most common TEAEs in both groups; however, the frequency of these TEAEs was lower in non-del(5q) pts. Among non-del(5q) and del(5q) pts receiving long-term treatment with LEN, onset rates of thrombocytopenia and neutropenia were lower at 〉 6 to 12 months versus the first 6 months of treatment. In summary, TEAEs in lower-risk MDS pts with or without del(5q) treated with LEN 10 mg for ≥ 1 cycle are predictable, well characterized, and clinically manageable. Disclosures Almeida: Shire: Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Celgene: Consultancy; Novartis: Consultancy. Off Label Use: Lenalidomide used to treat MDS patients without del(5q). Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Vey:Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria. Giagounidis:Celgene Corporation: Honoraria. Hellström-Lindberg:Celgene Corporation: Research Funding. Mufti:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Skikne:Celgene Corporation: Employment, Equity Ownership. Hoenekopp:Celgene International: Employment, Equity Ownership. Séguy:Celgene International: Employment. Zhong:Celgene Corporation: Employment, Equity Ownership. Fenaux:CELGENE: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; JANSSEN: Honoraria, Research Funding.

Description: Abstract 1790 Background: The immunochemotherapy regimen composed of fludarabine, cyclophosphamide and rituximab (FCR) has emerged as highly effective frontline or second line therapy for chronic lymphocytic leukemia (CLL). This regimen may be however associated with prolonged cytopenia and the risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Aims and methods: In our retrospective single center analysis, we evaluated the efficacy and the toxicity of FC or FCR regimen in unselected population of CLL patients with treatment indication. The overall survival (OS) and progression free survival (PFS) was calculated for all patients as intent to treat analysis. The prolonged cytopenia was defined as cytopenia (grade 2–4 according to CTCAE v.4 ) developing during of after the last cycle of FC/FCR and persisting two or more months. Cytopenia was evaluated in patients with follow-up at least 6 months after this treatment. Patients were excluded from analysis of cytopenia if they underwent immediate other treatment (antibody maintenance, high dose therapy with autologous stem cell transplantation (ASCT) consolidation, or they received other therapy due to unsatisfactory response to FCR). Patients with missing laboratory data after FC(R) were also excluded. Kaplan Maier curves for PFS and OS were calculated and log rank test was used for survival comparison. Results: Altogether, 252 patients started the treatment with FC or FCR in the years 2000–2012 at our institution. There were 86 (34%) women and 166 (66%) men with a median age of 62 years (31–87) at the time of FC(R) therapy. 52 (21%) pts received FC regimen, including 40 pts treated in first line therapy and 12 pts in second line therapy. FCR therapy was administered in 200 pts (79%): 153 pts received FCR as first line therapy, 38 pts as second line therapy and 8 pts as third or fouth line therapy. The median number of FC cycles was 5 (1–8) with or without R. The estimated OS for the first line therapy was 87,5% in FCR group vs 80% at 3y in FC group (p ns) (Hallek,CLL8: 87% vs 83%) and PFS was 70% in FCR group vs 50% in FC group (p=0,004) with the median of follow-up 45 months. Altogether 184 pts fulfill the criteria for cytopenia analysis. The most frequent immediate subsequent therapy considered as exclusion for this analysis was ASCT consolidation (n 20). Out of 184 pts, 146 recieved FC(R) as 1st line treatment and 38 subsequent therapy. The prolonged cytopenia was observed in 54 pts (29%), 42 (29%) in 1st line group and 12 (32%) in subsequent line group. Median duration of cytopenia was 8 m (2–65), 29 out of 54 patients have had persistent cytopenia at the time of last follow up. The cumulative probability to develop cytopenia was 30.3% at 2y among all pts and 29.7% among first line FCR treated pts. There was no significant difference between FC and FCR treated pts. Eleven pts developed MDS/AML, 7 cases were observed in the followed group of 184 pts (with probability 6.1% at 6y), in all cases the cytopenia preceded the MDS onset, 6y probability to develop MDS was 25.2% for patients who develop prolonged cytopenia after FC(R). Moreover 2 MDS and 1 AML were observed among 20 pts treated with ASCT (6y probability 5.6%, 8y probability 22.5%). The OS probability from 1stcycle of FC(R) was significantly better for pts without cytopenia (75.5% vs 57.5% at 5y, p

Description: Introduction: Somatic mutation detection in myelodysplastic syndrome (MDS) is very important in deciphering clonal pathogenesis of every patient and if determined correctly will become useful tool in followup studies such as testing individual susceptibility to epigenetic therapy with azacitidine (AZA). While some patients respond to AZA by restoring hematologic parameters, others progress to AML. Recent identification of quite heterogeneous sets of mutated genes (Bejar R et al. 2013) suggested that: patients with specific mutation pattern/s may respond to epigenetic therapy differently. Aim: We herein set to determine mutation profiles of MDS cohort indicated to and treated by AZA and utilized TrueSight DNA amplicon NGS sequencing approach containing 54 genes all previously associated with MDS or AML. Patients: We analyzed immunomagnetically CD3-depleted bone marrows of two MDS patients - AZA responders. First patient (male, 68y), was diagnosed with RAEB2, IPSS int-2, transfusion dependent (4 TU/Mo), intermediate cytogenetics (tri21). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) the patient responded by partial remission, and AZA was discontinued after 17 cycles. Twelve months after discontinuation he progressed and AZA was readministered for additional 3 cycles and the patient achieved again partial remission. Analyzed are samples after 11 (P394) and 20 (P1380) cycles of Vidaza. Second patient (female, 64y), was diagnosed with RAEB2, IPSS high, transfusion dependent (2 TU/Mo), favorable cytogenetics (46XX). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) she responded by hematology improvement and later by partial remission. Analyzed is a sample after 4 (P1510) cycles of Vidaza. As negative controls we used two normal donor bone marrows from 41y male and 32y female. As a positive control we also used: 1 MDS/AML cell line MOLM-13 with previously identified mutations of CBL and FLT3 (DSMZ; ACC 554). Methods and approach: Samples were sequenced on Illumina MiSeq sequencer. The mapping was performed using Burrows-Wheeler Aligner algorithm. Illumina Somatic variant caller was used to identify mutations. Then we applied following filters on the data: sequencing coverage should be higher than 1000 per mutation (~80% data left), mutation should be heterozygous (~95% data left), mutation frequency should be higher than 10% (~10% data left), Illumina Somatic variant caller should flag the mutation as "PASS" (~50% data left), mutation should not be synonymous (~75% data left) and mutation should be exonic (~40% data left). These filters were also applied to find mutations in the two control samples. Those mutations which were identified also in the control samples were removed from the analysis of patient samples (~50% data left). Results: the MDS/AML cell line MOLM-13 contained mutations (SNVs or InDels) in ABL1 (SNV/frequency=46.5%), ASXL1 (SNV/49.8%), CEBPA (In/47.9%), HRAS (SNV/54.5%), TET2 (SNV/49.7%), and as expected also in the genes encoding CBL (delta-exon8/52.4%) and FLT3 (ITD/50.6%). Patient' sample P1510 contained mutations in CBL (SNV/67.9%), CUX1 (SNV/51.8%), IKZF1 (2 different SNVs/41.9 and 50.7%), KDM6A (SNV/51.6%), SF3B1 (SNV/38.9%), and SMC3 (SNV/33.1%). Patient samples P394 and P1380 contained mutations in the ASXL1 (SNV/ 35.5% and 32.6% respectively), CUX1 (2x SNVs, first SNV/46.1-〉64.5%, second 48.4-〉57.9%), and IKZF1 (SNV, 50.4-〉44.5%) in similar frequencies in the sample before and after 2.5 years (including 9 cycles of AZA) suggesting limited genetic heterogeneity in this AZA-responding patient. Consequently, to gain more insight into how AZA modulates mutation pattern in MDS, we now analyze a set of fourty nine additional patients before and following at least 4 cycles on AZA treatment. Conclusions: Our data support use of immunomagnetic CD3-depletion of bone marrow and addition of normal control samples in the sequencing of MDS patient samples and support this approach for testing genetic heterogeneity during MDS disease course upon AZA treatment. Disclosures Stopka: GAČR P305/12/1033 and UNCE 204021: Research Funding; Celgene: Research Funding; PersMed ltd.: Equity Ownership. Vargova:GAČR P305/12/1033 and P305/11/1745: Research Funding; UNCE 204021: Research Funding; PRVOUK P24/LF1/3: Research Funding. Kulvait:PersMed ltd.: Equity Ownership. Jonasova:PRVOUK P24/LF1/1: Research Funding; Celgene: Research Funding.

Description: Deletion of the long arm of chromosome 20 - del(20q) - is a recurrent abnormality observed in various myeloid disorders, including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), or acute myeloid leukemia (AML). It is a primary cytogenetic aberration, occurring often as a sole abnormality or the first abnormality in complex karyotypes, therefore it is assumed to play a key role in pathogenesis of myeloid malignancies. The proximal breakpoints of the deletion are consistently located in the 20q11.21 band, and the distal breakpoints span from 20q13.13 to band 20q13.33. In our previous study we showed a fusion of the ASXL1 and TSHZ2 genes resulting from an isochromosome of a deleted 20q in a patient with MDS (Brezinova et al Br J Haematol 2014). The ASXL1 gene is one of the most frequently mutated genes in myeloid disorders, mutations are generally associated with more aggressive course of the disease and poor clinical outcome. The aim of this study was to determine the frequency of ASXL1 breakpoints and/or ASXL1/TSHZ2 fusion in del(20q) cases. Fluorescence in situ hybridizations (FISH) with locus specific probes for 20q11 and 20q12 regions (Abbott, Des Plaines, IL; Kreatech Diagnostics, Amsterdam, The Netherlands) confirmed the cytogenetically observed deletions of 20q in a cohort of 20 patients with myeloid malignancies (MDS 13x, MPN 3x, AML 2x, myelofibrosis 1x, thrombocytopenia 1x). In 15 patients deletion of 20q was a sole aberration, in 2 patients a variant of del(20q), an isochromosome of deleted 20q, was detected by FISH with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott). Metaphase FISH mapping with a set of 7 bacterial artificial chromosome (BAC) probes (BlueGnome, Cambridge, UK) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe (Kreatech Diagnostics) as a control was used for determination of the breakpoints. Array comparative genomic hybridization (CytoChip Cancer 180K, BlueGnome) was performed on DNA samples of bone marrow cells of 11 patients with suspected ASXL1 gene deletion to find out the gene copy number variation. A weak signal of RP11-358N2 BAC probe (30.93-31.12 Mb from telomere on the short arm, 20q11.21) was observed in 6 patients(30%), suggesting the proximal breakpoint of the deletion in the ASXL1 gene (30.95-31.03 Mb). However, the distal breakpoint in the TSHZ2 gene (51.80-52.11 Mb; 20q13.2) was found in one patient only, as previously reported. In 6 patients (30%) the signal of RP11-358N2 BAC probe was not present on the derivative chromosome confirming the ASXL1 gene deletion. In the remaining 8 patients the proximal breakpoint of the deletion was determined distally to the ASXL1 gene, in 3 patients into 9.3 Mb region between the MAPRE and the PTPRT genes. None but 1 patient had the distal breakpoint localized in the TSHZ2 gene. Three patients died in a group of 12 patients with ASXL1 gene alteration (deletion or partial deletion). Using combination of molecular cytogenetic methods we proved that the extent of 20q deletions varied among the patients with a frequent proximal breakpoint in the ASXL1 gene. In our cohort, the ASXL1 gene was altered (deleted or partially deleted) in totally 60% of patients. The determination of the ASXL1 gene alteration in del(20q) cases may have a clinical and prognostic impact, but the relations with clinical data should be studied in a larger cohort of patients. Supported by MHCR project for conceptual development of research organization 00023736, RVO-VFN64165/2012, and GACR P302/12/G157/1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndrome (MDS) is often manifested by anemia due to ineffective erythropoiesis. Upon transformation to MDS/AML the uniform population of leukemic blasts overgrow dysplastic bone marrow. Hematopoiesis is regulated by transcription factors GATA-1 and PU.1 that interact and mutually inhibit each other in progenitor cells to guide multilineage commitment and subsequent lineage differentiation. Expression of PU.1 is controlled by several transcription factors including PU.1 itself at distal URE enhancer. It has been well established that underexpression of PU.1 in progenitor cells leads to AML (Rosenbauer F et al. 2004). In addition, co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents induction of differentiation programs regulated by these transcription factors. In our laboratory, we recently observed that MDS/AML erythroblasts display repressive histone modifications and methylation status of PU.1 gene that respond to 5-azacitidine leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000) however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. Hypothesis GATA-1 inhibits PU.1 levels directly and modulates its transcriptional outcome in early erythroblasts. We also hypothesize that GATA-1-mediated repression of PU.1 transcription is delayed and this may play a role in ineffective erythropoiesis. Material and Methods Cell lines: MDS-derived OCI-M2 EL and other two human ELs (HEL, K562) and one murine EL (MEL); all co-expressing GATA-1 and PU.1. Patients: MDS patients (N=5) with rather advanced disease; MDS/AML (4) and RAEBI (1). Four received AZA; response: PR (2), SD (2) with HI. Median OS〉24 Mo. For chromatin immunoprecipitation (ChIP) analysis either cell lines or CD19/CD3-depleted bone marrow cells were used. Results Direct association of GATA-1 with PU.1 gene was demonstrated in all three human ELs using ChIP. Occupancy of GATA-1 was detected upstream the PU.1 promoter and distally at GATA-1 binding sites or at PU.1 binding sites together with PU.1. Comparable data documenting occupancy of GATA-1 at PU.1 gene were observed also in MEL cells and in normal murine fetal erythroblasts using ChIP-sequencing. To test how GATA-1 regulates PU.1 expression we overexpressed GATA-1 in erythroblasts and tested expression of PU.1, histone H3 modification (near GATA-1 occupancy) and cell growth. We found that GATA-1 inhibited PU.1 expression, facilitated enrichment of repressive modifications at PU.1 gene (H3K9Me, H3K27Me) while depleted activation modifications (H3K9Ac, H3K4Me), and also inhibited cell growth. Next, we tested effects of GATA-1 knockdown using siRNA. Indeed, inhibition of GATA-1 expression in erythroblasts leads to increase in PU.1 level as well as of its targets (CEBPA, MAC1). Using Luciferase assay we confirmed that both endogenously produced PU.1 and GATA-1 are capable to stimulate exogenously inserted reporters. Next, we compared chromatin structure of PU.1 gene between data from ELs, normal controls and high risk MDS. Our data revealed that PU.1 gene in MDS is enriched with repressive modifications (H3K9Me, H3K27Me) while depleted with activation modifications (H3K9Ac, H3K4Me) suggesting defects in dynamic regulation of PU.1 expression in MDS. Conclusion Our data from ELs provide a) evidence of GATA-1-mediated repression of PU.1 gene in erythroblasts and that b) manipulation of GATA-1 affected PU.1 level in opposite direction. In high risk MDS, the chromatin structure of PU.1 gene displays accumulation of repressive epigenetic marks that are responsive to AZA. We think that during early erythroid differentiation GATA-1 binds and represses PU.1 gene, however this is not fully completed in MDS and therefore erythroid differentiation is not efficient. Grants: P301/12/P380, P305/12/1033, NT14174-3/2013, UNCE204021, FR-TI2/509, SVV-2013-266509, PRVOUK-P24/LF1/3 Disclosures: No relevant conflicts of interest to declare.