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Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

Forty-eight patients with early myelodysplastic syndrome (MDS) without excess of blasts, with average initial serum ferritin levels of 2739.5 μg/L (range 825-11287 μg/L), were treated with deferiprone (L1) in a daily dose of 40-90 mg/kg. Median duration of chelation treatment was 10.9 months (range 4-24 months). Chelation was effective (maintained or decreased iron stores) in 16 out of 22 patients (73%) with serum ferritin levels2000 μg/L in contrast to only 12 out of 26 patients with serum ferritin levels2000 μg/L. Combination of L1 with recombinant human erythropoietin (rHuEPO) (30-40 kU/week) resulted in effective chelation in five additional patients with serum ferritin levels3000 μg/L. Incidence of adverse effects was comparable to that in thalassemic patients. Gastrointestinal symptoms represented the most frequent adverse effect of L1 therapy (37.5% of patients) that limited an effective escalation of the daily dose of the drug and led to discontinuation of the treatment for six patients. A decreased number of granulocytes was observed in five (13%) patients and agranulocytosis occurred in two patients (4%). Granulocyte counts were restored after cessation of L1 treatment and administration of granulocyte colony stimulating factor (G-CSF) in all but one patient. Administration of L1 in a daily dose of at least 75 mg/kg may represent an alternative approach in treatment of mild and moderate iron overload in MDS patients who cannot be treated with deferasirox (DFRA) or deferoxamine (DFO).

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.

We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia (RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell populations from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone marrow fraction/subject). The results were in agreement with the concept of increased apoptosis in low-risk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin A positive cells, and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for the investigation of difference between RA and RARS pathobiology.

Here we report response to treatment of chronic myeloid leukemia (CML) of five pregnant women during and after pregnancy. CML was diagnosed during pregnancy in three patients. Pregnancy was confirmed during CML in two patients: in one in the 21st week of pregnancy while on imatinib, in another in the 12th week during the interferon treatment. Interferon with leukapheresis when needed was applied in the 2nd and 3rd trimester. All patients except one achieved complete hematological response during pregnancy. After delivery four patients achieved partial cytogenetic response on imatinib and two patients achieved major molecular response after crossover to dasatinib.

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 Results: 558/228 significantly differentially expressed genes between CD14+ monocytes/CD3+ T-lymfocytes patients and control samples were identified (fold change >1.5; p Conclusion: Our results indicate that lenalidomide with a still unknown exact effect in 5q- causes significant changes in expression profile in many genes that are probably important in the pathogenesis of the disease and the treatment response. Significant decrease in relation to lenalidomide treatment was observed of tumor necrosis factor, interleukin 1 and transcription factor MYB. We plan next to monitor changes in gene expression during the course of therapy and comparing the gene expression in CD34+ cells before and after lenalidomide therapy. Supported by Celgene Corporation and grants NS/9634 MZCR, MZ0 UHKT2005 and MSM0021620808. Disclosures: No relevant conflicts of interest to declare.

Karyotype represents one of the most valuable prognostic markers for MDS patients with profound impact on differential diagnosis and therapeutic decisions despite of the fact that it is a heterogenous patient population with diverse pathogenesis and clinical course. In approximately 10–20% of MDS patients complex chromosomal aberrations (CCA; three or more numerical/structural cytogenetic aberrations) are present at diagnosis and this finding is associated with the treatment resistance and poor outcome. Precise identifications of chromosomal regions involved in CCA could help in detection of cryptic, recurrent, prognostically relevant aberrations and in identification of candidate genes involved in leukemogenesis and progression of the disease. In this study we used various modifications of molecular cytogenetic techniques to specify CCA more precisely and to determine exact localization and frequency of chromosomal breakpoints. During the last seven years we examined bone marrow samples of 630 adults with MDS and in 81 of them (12,9%) complex karyotypes were found (in 73 patients at diagnosis and in 8 cases during the disease progression). Out of 73 patients with CCA at diagnosis were 43 males and 30 females, age range 22 to 82 years (median 65,5 years). Primary MDS was diagnosed in 63 (86.3%) of them, in 10 cases therapy related MDS was ascertained. Diagnoses according to the WHO classifications: MDS NS 3x, RA 2x, RA/RCMD 6x, RCMD 5x, RAEB I 20x, RAEB II 20x, MDS-AML 16x and RARS 1x. 63 patients died, 10 patients are still alive (four of them after stem cell transplatation). Chromosomal aberrations found by G-banding were verified by FISH with appropriate locus specific probes (Abbott-Vysis, Des Plaines, Illinois, USA), and by multiplex FISH (mFISH/mBAND) with the “XCyte” probe kits (MetaSystems, Altlussheim, Germany). Deletion of 5q31 region was proved in 63 patients (86.3%). In 29 cases mFISH analyses showed that parts of deleted No. 5 were translocated into other chromosomes. The most recurrent chromosomal partners of No. 5 in unbalanced translocations were Nos. 17 (9x), 12 (5x), 7 (5x), 2 (4x) and 3 (4x). Monosomy of No. 5 was confirmed in one case only, thus it was proved that monosomy 5 is not separated diagnostic unit in CCA. Except No. 5 the most frequently involved in CCA were chromosomes 7 (36x), 3 (33x), 12 (31x), 17 (31x) and 11 (23x). Presence of CCA at diagnosis was connected with poor response to the therapy and with short overall survival. For survival analysis patients were classified into three groups according to the molecular cytogenetic findings: del(5)(q31) and additional CCA – 33 cases, deleted chromosome 5 involved in CCA – 29 cases, CCA without deletion of 5q – 10 cases. The worst overall survival was found in group 2 (median 3 months), followed by group 1 (median 5 months) and 3 (median 6 months). Patients with MDS and CCA should be considered as a unique entity with extremely poor prognosis. Exact molecular cytogenetic analysis by multiplex FISH of all chromosomal aberrations which are involved in CCA will lead to selection of the patients into new prognostic groups. Exploring the underlying mechanism of leukemogenesis could improve the therapeutic outcome for these patients and could contribute to the development of a new targeted treatment strategy.

Introduction: Inmyelodysplastic syndromes (MDS) the karyotype is one of the most important predictor of disease advancement, response to the treatment and patients' outcome. Clonal cytogenetic abnormalities are detected in the bone marrow cells in approximately 50% MDS patients. The interstitial deletion of the long arm of chromosome 5-del(5q)-is the most common finding, accounting for roughly 30% of abnormal karyotypes. According to IPSS-R, MDS with isolated del(5q) are associated with a favorable clinical course and are recognized as a specific subtype of MDS. However in some cases, acquisition of additional genetic aberrations may occur during the course of the disease and it has been proved it negatively influences outcome of MDS patients. The aim of the study was: (1) to evaluate the frequency of cytogenetic clonal evolution during the course of the disease in MDS patients with isolated del(5q), (2) to analyze the pattern of acquired cytogenetic abnormalities, and (3) to assess the impact of clonal evolution on transformation to acute myeloid leukemia (AML) and/or overall survival (OS). Patients and Methods: A detailed retrospective and/or prospective genome-wide analysis of fixed bone-marrow cells of 184 adults with del(5q), identified with conventional G-banding at the diagnosis of MDS, was performed during the course of their disease. The chromosomal aberrations were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods using the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with CytoChip Cancer SNP 180K (BlueGnome, Cambridge, UK) or with Illumina Human CytoSNP-12 arrays (Illumina, San Diego, CA). Amplicon deep sequencing of TP53 mutations (exons 4-11) was performed on a Roche 454 GS Junior system (Roche, Indianapolis, IN) using oligonucleotide primer plate assays validated according to the IRON-II (Interlaboratory Robustness Of Next generation sequencing). Results: Cytogenetic clonal evolution was observed in 21/184 MDS patients with isolated del(5q) (11.4%). Abnormalities most frequently acquired during the course of the disease were non-balanced rearrangements involving chromosomes 5, 7, 3, 17, 12 and 11. Deleted chromosome 5 was highly unstable and was often involved in different types of cryptic unbalanced rearrangements (translocations, insertions). Fragmentation of 5q was present as well.Clonal evolution was frequently associated with TP53 mutations and/or unbalanced aberrations of 17p.The median time between the first and the last evaluation was 23 months (range 2-83 months). Clonal evolution was detected between 2 and 83 months (median 14 months) after first cytogenetic evaluation. Median OS was 35 months (range 6-89 months), median survival from the first emergence of cytogenetic clonal evolution was 7.5 months (range 1-31 months). Progression to RAEB 1 or RAEB 2 was detected in 10 patients, 11 patients transformed into AML. Conclusions: Clonal evolution was detected in 11.4% of patients with MDS and isolated del(5q). The pattern of acquired abnormalities was similar to the changes previously described in high risk MDS at primary evaluation. The emergence of specific clones with additional genetic aberrations frequently correlated with altered clinical parameters. Cytogenetic clonal evolution was strongly associated with higher frequency of loss of heterozygosity (LOH) of 17p and/or TP53 mutations, shorter OS, rapid disease progression and transformation to AML. This data substantiate a need for regular molecular cytogenetic monitoring to guide clinical treatment decision in MDS with isolated del(5q). This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, PRVOUK-P27/LF1/1, and MHCR 00023736. Disclosures No relevant conflicts of interest to declare.

Introduction: Complex chromosomal aberrations (CCAs) are seen in approximately 20% of patients with newly-diagnosed MDS and are associated with poor prognosis. Bone marrow cells of MDS patients with CCAs are characterized by a high degree of genomic instability, which is connected with an increased risk of formation of different subclones with additional aberrations. The phenomenon of clonal evolution can be observed either at diagnosis or during the disease progression. At diagnosis it is manifested by the presence of two or more related subclones derived from one founder clone. These subclones can obtain a proliferative advantage leading to the clonal expansion. The aim of the study was to perform detailed genome-wide analyses of bone marrow cells of previously untreated MDS patients with CCAs, to investigate the clonal heterogeneity and to assess the frequency and clinical significance of related cytogenetic subclones with complex karyotypes. Methods: A comprehensive molecular cytogenetic analysis of fixed bone-marrow cells from 182 adults with CCAs (³3 aberrations) identified with conventional G-banding in the diagnosis of MDS was performed. The CCAs were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with CytoChip Cancer SNP 180K (BlueGnome, Cambridge, UK) or with Illumina Human CytoSNP-12 arrays (Illumina, San Diego, CA). Results: Cytogenetic subclones were detected in 143 of 183 patients with complex karyotypes (78.6%). Among them, 98/183 cases (53.8%) displayed a defined number of subclones, whereas in 45/183 patients (24.7%) a precise definition of the individual subclones was not possible due to the high number of heterogeneous findings. Therefore these patients were subsumed as having composite karyotype. In 138/143 cases with clonal heterogeneity (94.9%) subclones showed related karyotypes. Twelve patients had one clone with an interstitial 5q deletion as the sole abnormality and other subclones with additional changes. One of these 12 cases displayed a subclone with complex karyotype in which deleted chromosome 5 was involved in an unbalanced translocation. Five patients (5.1%) had a combination of related clones with del(5q) and additional changes and unrelated subclones without this aberration. Complex aberrations were often associated with the loss of heterozygosity (LOH) of 17p (44%), however a difference in the frequency of LOH 17p in groups with and without clonal heterogeneity was not ascertained (p = 0.782). Moreover, the overall survival (OS) of patients with clonal heterogeneity did not differ from that of patients with one homogeneous complex aberrant clone (p = 0.391). Median OS in both groups was three months only. Conclusions: The results of this study confirmed clonal heterogeneity at the cytogenetic level as a frequent finding in patients with MDS and complex karyotypes. Although clonal diversity has been implicated as a driving mechanism of tumor development and progression and is usually associated with more aggressive disease, in this cohort of MDS cases with complex karyotypes, the presence of different subclones did not affect patients’ overall survival. It seems that the dismal prognosis of patients with CCAs is generally correlated with overall genomic instability and the complexity of aberrations. This may be manifested at different genetic levels by various phenomena such as increasing number of aberrations, accumulation of gene mutations and/or losses of heterozygosity of tumor-suppressor genes (including LOH TP53), involvement of deleted chromosome 5 in unbalanced rearrangements, extensive chromosome shattering, and clonal heterogeneity as well. Supported by RVO-VFN64165, GACR P302/12/G157/1, PRVOUK-P27/LF1/1, and MHCR 00023736. Disclosures No relevant conflicts of interest to declare.

Background Aberrations of chromosome 5 are common cytogenetic findings in bone marrow cells of patients with myelodysplastic syndromes (MDS). The most frequent is the interstitial deletion of the long arm of chromosome 5 [del(5q)] - occurring as sole abnormality or in combination with additional aberrations in ∼30% of primary and up to 50% of secondary MDS. Extent and breakpoints of deleted segment differs among patients. A critical area deleted in all cases (the common deleted region, CDR) has been localized between bands 5q31.1 and 5q32. Loss of the whole chromosome 5 was described in about 3-8% MDS cases. However, in recent studies it has been shown that many suspected monosomies 5 are in fact cryptic translocations or insertions, undetectable by conventional G-banding. The aim of the study was to perform detailed genome wide analyses of bone marrow cells of MDS patients with suspected monosomy 5 detected by conventional G-banding at diagnosis, to verify the loss of genetic material of chromosome 5, to compare the breakpoints and extent of chromosome 5 deletions in individual patients, and to assess real existence and clinical significance of monosomy 5 in MDS. Patients and methods In the period 2002–2012, bone-marrow samples from 870 adults with newly diagnosed MDS were examined and 80 patients (9.2%) with suspected monosomy 5 were identified (50 males, 30 females; median age 67 years). Karyotypes were analyzed by conventional G-banding, FISH with Vysis DNA probes (Abbott, Des Plaines, IL), and mFISH/mBAND (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with oligonucleotide CGH-SNP arrays (BlueGnome, Cambridge, UK) or with SNP arrays (Illumina, San Diego, CA). Results All 80 patients presented a complex karyotype with confirmed del(5)(q31). The most frequent breakpoints on chromosome 5 involved bands 5q14.3, 5q34, 5q33.3, 5q11.2, and 5q13.2. On 5p, breaks occurred sporadically (recurrent breakpoints at 5p12, 5p14.1, and 5p15.3). The extent of del(5q) ranged from 23.55 Mb to 131.4 Mb (whole arm). The most proximal and most distal breakpoints were located at 5q11.1 and 5q35.3, respectively. The CDR occurred between bands 5q31.1 and 5q32 (15.1 Mb). No patient with pure monosomy 5 was found. In all cases, parts of the deleted chromosome 5 were fragmented and inserted elsewhere in the genome. A commonly retained region conserved in all patients was established at 5p11.1–p14.2 (22.31 Mb). The most recurrent partners of deleted chromosome 5 in cryptic unbalanced rearrangements were chromosomes 17, 3, 7, and 18. Patients with deleted chromosome 5 involved in complex aberrations have an extremely poor prognosis (in the present cohort, a median OS of only two months). Discussion and conclusions Based on these results, we believe that pure monosomy 5, quoted in the literature, in MDS does not actually exist. It seems that the primary interstitial deletion of 5q is likely the cause of increased genomic instability, which may, under certain circumstances, lead to fragmentation and disintegration of the whole chromosome 5 and to the emergence of additional complex rearrangements. Mechanism responsible for fragmentation of deleted chromosome 5 remains unclear. One of possible explanations might be the phenomenon called chromothripsis, whereby one or more chromosomes or chromosomal regions shatter into pieces in a single catastrophic event. Some of these pieces are then stitched haphazardly together by DNA repair machinery, whereas some of them are lost. This process is resulting in severe genomic damage and fast disease progression. MDS patients with deleted chromosome 5 involved in complex rearrangements should be considered as a unique entity with extremely poor prognosis. Supported by grants RVO-VFN64165/2012, GACR P302/12G157/1, PRVOUK-P27/LF1/1 and MHCR 00023736. Disclosures: No relevant conflicts of interest to declare.

Introduction The binding of immunomodulatory drugs (IMiDs), including lenalidomide, to CRBN is associated with cytotoxicity of IMiDs used in the treatment of multiple myeloma, myelodysplastic syndromes (MDS) and lymphomas. Cereblon (CRBN) is named for its putative role in cerebral development, especially in memory and learning. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4 (CUL4) and regulator of cullin 1 (ROC1). This complex regulates DNA repair, DNA replication and transcription. CRBN is a primary target of thalidomide teratogenicity (Ito et al., Science 2010; 327:1345-1350). Down-regulation of the CRBN expression is associated with the development of marked IMiDs resistance in human multiple myeloma cells (Zhu et al., Blood 2011; 118: 4771-4779; Lopez Girona et al., Leukemia 2012; 26: 2326-2335; Heintel et al., Br J Haematol 2013; 161: 695-700; Broyl et al., Blood 2013; 121: 624-627; Lodé et al., Br J Haematol 2013; Jul 17. doi: 10.1111/bjh.12478). CRBN expression is thus required for the antimyeloma activity of IMiDs. Aims To gain insight into the role of CRBN in lower risk myelodysplastic syndromes with or without 5q deletion and into the mechanisms of lenalidomide action, we study CRBN expression in these two groups of lower risk MDS patients and in healthy controls. We further study the expression of DDB1 and IRF4 (interferon regulatory factor 4, one of target genes of CRBN). Methods Mononuclear cells were isolated both from bone marrow [23 low risk MDS patients with 5q- deletion, 37 low-risk MDS patients with normal chromosome 5 (non5q-) and 24 healthy controls] and from peripheral blood [38 patients with 5q- , 52 non 5q- patients and 25 healthy controls] by Ficoll-Paque PLUS gradient separation, and washed with phosphate-buffered saline. The rest of the red cells were lysed. Total RNA was isolated using RNA isolation solvent and complementary DNA was synthesized from total RNA using SuperScript II reverse transcriptase. Relative levels of the CRBN, DDB1 and IRF4 mRNAs were determined by TaqMan-based quantitative real-time PCR and by calculation to the level of housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiments were performed in duplicate. Informed consent was obtained from all patients and healthy controls. Evaluation of 2-ddCt indicates the fold change in gene expression relative to the control. Results The median of CRBN mRNA levels in total RNA isolated from peripheral blood mononuclear cells was the highest (3.6) in lower risk MDS with 5q deletion, lower (1.5) in non5q- MDS and lowest (1.2) in healthy controls. Similar results were obtained in bone marrow mononuclear cells (medians 3.3 in 5q- syndrome, 2.2 in non5q- MDS and 1.3 in healthy controls). The differences between the 3 groups were statistically significant (p˂0.05, Mann-Whitney test) with the exception of non5q- MDS in comparison with healthy controls in mononuclear cells of peripheral blood. Very similar results were obtained in the analysis of DDB1 and IRF4 mRNAs levels, which correlated with CRBN levels. We analyzed CRBN mRNA levels before and in the course of the lenalidomide treatment of 5q- low risk MDS (6 patients). We obtained high CRBN mRNA levels before and during the treatment by lenalidomide in all lenalidomide responders (4 patients). We found a sharp decrease of CRBN mRNA in mononuclear cells of bone marrow in two patients who stopped responding to lenalidomide therapy and subsequently progressed to higher risk MDS. A/G polymorphism located at -29 nt of the 5x UTR of CRBN does not act as a biomarker of response to treatment with lenalidomide in 5q- syndrome. Conclusions 5q- low risk MDS patients have the highest levels of CRBN mRNA in comparison to lower risk myelodysplastic syndromes with normal chromosome 5 and to healthy controls. DDB1 and IRF4 mRNAs levels correlate with CRBN levels. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of the therapy. Significant decrease of CRBN levels during treatment by lenalidomide is associated with loss of response to treatment and disease progression. Similarly to the treatment of multiple myeloma, these results suggest that high levels of CRBN mRNA are necessary for the efficacy of lenalidomide in low risk 5q- patients. Supported by the MH CR grant NT/13836-4/2012, PRVOUK-27/LF1/2 and by the MH CR project for development of research institute (UHKT). Disclosures: No relevant conflicts of interest to declare.

Abstract 2788 Poster Board II-764 Introduction. Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic erythropoiesis and on the contrary thrombocythemia with effective though dysplastic megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common progenitor MEP (T.P.McDonald et al., Exp. Hematology 1993). There are two key transcription factors which together with other transcription factors and relevant cytokines and receptors determine the hemopoietic differentiation of the common stem cell: erythroid Krüppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1 (FLi-1) for megakaryopoiesis (Pilar Frontelo et al., Blood 2007; G.A.Blobel, Blood 2007; F.Bouilloux et al., Blood 2008). There is functional cross antagonism between FLi-1 and EKLF (J.Starck et al., Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H.Huang et al., ASH Abstracts 2008). The question is whether both factors play any role in 5q- syndrome. Methods. FLi-1 and EKLF gene expressions were determined in mononuclear cells isolated from the whole blood or bone marrow using Ficoll-Paque PLUS. Expression of both factors was measured by quantitative real-time PCR. RT-PCR products were verified by electrophoresis and direct sequencing. The assays were performed for sample in duplicate. Glyceraldehyd-3-phosphate dehydrogenase (GAPDH), FLi-1 and EKLF were amplified in 25 μl reaction mixture containing 12.5 μl SYBR Green JumpStart Taq Ready Mix, 2.5 μl 2 μM FLi-1 or EKLF forward and reverse primers, 0,25 μl internal reference dye and 1 μl cDNA. Relative levels of FLi-1 and EKLF mRNAs were calculated to the level of housekeeping GAPDH mRNA. Results. FLi-1 and EKLF were measured in blood mononuclear cells of 8 patients fulfilling all criterias of 5q- syndrome. FLi1mRNA/GAPDHmRNA was higher in all samples, average value was 0.0930 (0.0242-0.4274) compared to control value 0.0194. FLi1mRNA/GAPDHmRNA in bone marrow mononuclear cells of 7 patients with 5q- syndrome was higher in all samples but one. The average value was 0.0827 (0.0070-0.2554) compared to healthy controls 0.0044. EKLF gives very low values in the majority of patients′ blood and bone marrow samples as well as in healthy controls. The evaluation is therefore less reliable then FLi-1 assessment. EKLFmRNA/GAPDHmRNA in blood was 0.0004 (0.0-0.0023) compared to the control 0.0222. The results of EKLF in 5 bone marrow samples are inconsistent. Three are lower than the control (0.0068), 1 of remaining 2 samples is extremely high (0.3491). It is interesting that this patient is the only one who responded to erythropoietin and is transfusion independent. Summary. Our preliminary results with FLi-1 and EKLF gene expression measurement are in agreement with expected findings: increased FLi-1 expression corresponds to thrombocytemia in 5q- syndrome patients and expression of EKLF, lower than in controls would correspond to anemia in these patients. However, EKLF values are less reliable because of very low values in patients as well as in controls and because of inconsistent results in bone marrow samples. We prepare to follow both factors in 5q- patients after the treatment with lenalidomide. Lenalidomide improves anemia in 5q- syndrome patients and temporarily causes decrease of thrombocytes (A.List et al., N.Engl.J.Med. 2005, 2006). Inhibition of phosphatases by lenalidomide (S.Wei et al., Proc.Natl.Acad.Sci.USA 2009) can stop FLi-1 dephosphorylation which leads to FLi-1 inactivation. Hypotetically inactive FLi-1 would enable EKLF to induce MEP into erythroid lineage. Supported by MSM 0021620808 Disclosures: No relevant conflicts of interest to declare.

Abstract 1623 Poster Board I-649 Clonal cytogenetic abnormalities are detected in approximately 50% of patients with primary MDS and have a strong impact on disease outcome. More prevalent are unbalanced chromosomal aberrations reflecting a gain or loss of genetic material, whereas balanced rearrangements are rather rare. The most recurrent structural karyotypic abnormality is an interstitial deletion of the long arm of chromosome 5 occurring in 30% of primary MDS cases, either as isolated aberration (10%) or combined with additional cytogenetic changes (20%). An isolated del(5q) is generally associated with a better prognosis, whereas patients with one or more additional chromosomal abnormalities have a significantly shorter overall survival. The worst prognosis is observed in patients with del(5q) occurring in the context of a complex chromosomal aberrations (CCA; 33 abnormalities). Identifications of chromosomal regions involved in CCA in patients with MDS is very important and could yield detection of cryptic, recurrent, prognostically relevant aberrations and identification of candidate genes involved especially in progression of the disease. Detailed molecular cytogenetic analyses of CCA might lead to better understanding of molecular etiology of MDS with its therapeutic consequences. The aim of this study was to assess prognostic relevance of CCA in a cohort of patients with primary MDS and del(5q), to evaluate involvement of specific chromosomes and/or chromosomal regions in CCA and to establish the exact localization of chromosomal breakpoints. During 2002 and 2009, we examined bone marrow samples of 720 adults with MDS. Out of these, a total of 70 patients with primary disease and del(5q) associated with the CCA were identified at the time of initial diagnosis - 32 women (46%) and 38 men (54%), with a median age of 66 years (range 22 - 83). Diagnoses according to the WHO classification were as follows: RA (3%), RCMD (14%), RAEB I (27%), RAEB II (35%), MDS-AML (17%), CMML (3%) and MDS-U (1%). For precise analyses of chromosomal regions and breakpoints involved in CCA, various modifications of molecular cytogenetic techniques were used: FISH with locus specific probes (Abbott Vysis), CGH, mFISH/mBAND (MetaSystems) and/or array CGH (BlueGnome, NimbleGene). In 40 cases, mFISH analyses showed that parts of deleted chromosome 5 were translocated to other chromosomes. Fragmentation of 5q was also frequently found. Interestingly, no patient with monosomy 5 was identified in this study. Even in patients with suspect monosomy 5 detected by conventional cytogenetics, further detailed molecular cytogenetic analyses revealed parts of chromosome 5 material were retained in means of insertions within CCA. The most recurrent partners of del(5q) in unbalanced translocations were chromosomes 12 (23%), 3 (21%) and 17 (21%). The most common chromosome 5 breakpoints were 5q33 (47%), 5q13 (39%) and 5q11 (13%), the most frequently encountered deletion was q13q33 (30%). Except the chromosome 5, most often in CCA involved chromosomes were 17 (53%), 12 (49%), 7 (47%), 3 (43%) and 11 (31%), and the most recurrent breakpoints were at regions 12p11 (16%), 7q11 (10%), 7q21 (9%) and 7p11 (9%). Finding of CCA at diagnosis were associated with poor response to the therapy and short overall survival (median 4 months). Patients were classified into two groups according to the results of molecular cytogenetic analyses: group A (n=30) – del(5q) and additional CCA, and group B (n=40) – deleted chromosome 5 involved in CCA. The shortest overall survival was found in group B (median 3 months versus 5 months in group A). In conclusion, the involvement of specific chromosomes and/or chromosomal regions in CCA associated with del(5q) is nonrandom. Deleted chromosome 5 is very unstable and is often involved in different types of cryptic unbalanced rearrangements (translocation, insertions). Monosomy of chromosome 5, quoted in the literature, does not actually exist in MDS. Patients with del(5q) involved in CCA should be considered as a unique entity with extremely poor prognosis. Supported by grants NR/9227-3, NR/9481-3, MZO VFN2005, MSM 0021620808 and MSM LC535 Disclosures No relevant conflicts of interest to declare.