ALBERT

Description: Abstract 3589 Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In our study, B-CLL patients (N=71, F/M 31/40, median age 65) were subject to molecular analyses that found significant overexpression of MYB and miR-155 in B-CLL and physical association of MYB with the promoter of MIR155 host gene (MIR155HG, also known as B-cell integration cluster). In CD19+ B-cells derived from healthy control individuals (N=13), MYB and miR-155 expression was significantly lower. Next, we found that MYB positively regulates MIR155HG transcription in reporter assays. The endogenous chromatin structure of the MIR155HG promoter in B-CLL cells is characterized by spreading of active chromatin mark, histone H3K9 acetylation. Gene expression arrays on B-CLL patients (N=11) identified a set of predicted miR-155 target genes (N=94) that are downregulated. Their expression pattern displayed significant negative correlation with pri-miR-155 levels. Similarly, a number of MYB target genes were found deregulated (N=99) in B-CLL. Gene annotation of differentially expressed miR-155 and MYB targets in B-CLL revealed their biological functions as regulators of apoptosis (BCL2, API5), proliferation (CCND1, CCND2) and mediators of B-cell function (CD200, F11R). In addition, expression patterns of miR-155, MYB and their targets are currently being compared in a larger patient group with prognostic hallmarks of CLL (ZAP70, CD38, del(17)(p13.1), and IgVH). Our data collectively support novel candidate mechanism in B-CLL that includes MYB directly stimulating MIR155HG promoter coincident with its epigenetic dysregulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507). Disclosures: Trneny: ROCHE: 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: Background Chronic lymphocytic leukemia (CLL) in most patients is diagnosed with early stage disease identified incidentally on blood counts obtained for unrelated purposes. Immunophenotyping of peripheral blood (PB) is required for the diagnosis of CLL. A scoring system that helps in the differential diagnosis between CLL and other mature B-cell neoplasms (MBN) has been described twenty years ago (Matutes et al., Leukemia 1994; modified by Moreau et al., Am J Clin Pathol 1997). CLL/SLL typically demonstrates low-intensity staining for surface immunoglobulin, low or absent expression of CD22, CD79b and FMC7 and moderate to strong expression of CD5 and CD23. However, this phenotype is not entirely specific and some overlap in immunophenotype exists between CLL and non-CLL MBN. In particular, leukemic phase of CD5 positive mantle cell lymphoma (MCL) can be misdiagnosed as CLL. Recently, it has been shown that CD200 expression may help in differential diagnosis between CLL and other MBN. The present study aimed to prove CD200 usefulness in differentiating CLL from MCL on a series of consecutive patients and to investigate whether adding CD200 could improve the utility of Matutes scoring system, especially in atypical CLL. Methods Between January 2013 and March 2014, PB of consecutive patients with MBN was assessed in this study. Analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson) and samples were stained with panels of 4-color combinations of antibodies using a standard whole-blood assay. PB specimens were incubated with antibodies purchased from eBioscience (CD200 APC, clone OX-104), Immunotech (CD23, CD79b, FMC7), BD Biosciences (CD5, CD19), and DAKO (sIg). At least 5,000 B-cells were immediately acquired on flow cytometer. Diagnosis of CLL was made according to National Cancer Institute-Working Group criteria. Furthermore, tissue biopsies of 62 (31%) CLL cases were available for histological review, including all cases of atypical CLL. Diagnosis of MCL was based on morphology and immunohistochemical detection of cyclin D1 in tissue biopsies and further confirmed by detection of t(11;14) by FISH in selected cases. Results Table 1 provides details of the patient characteristics. In our series, CD200 was present on neoplastic B-cells of all 200 CLL cases (100%), whereas only 4 cases (8.7%) of MCL showed dim positivity of CD200. The remaining 42 cases (91.3%) of MCL were negative for CD200 expression. The revised Matutes score was calculated to classify CLL cases. All 179 cases of typical CLL (defined by a score ≥ 4) presented moderate to strong expression of CD200 (Median fluorescence intensity - MFI: median = 161). CD200 was also positive in all 21 cases of atypical CLL (defined by a score 〈 4), but showed lower intensity (MFI: median 128) than that observed in typical CLL (P = 0.02). Application of the Matutes scoring system to MCL cases showed that three cases scored 3 (6.5%), two cases scored 4 (4.3%) and none scored 5. Of note, CD200 was absent in two cases scoring 3 and was only dimly expressed in the remaining MCL cases scoring 3 or 4. Thus, the differential expression of CD200 in CLL and MCL retained even in those cases with otherwise indeterminate immunophenotype, therefore being particularly helpful for the distinction of atypical CLL and MCL. Conclusions Flow cytometry is an essential tool for the diagnosis of CLL. However, a significant immunophenotypic overlapping occurs especially between CLL and MCL cells. In this study, we investigated the expression of recently identified marker CD200 in PB of consecutive CLL and MCL patients. We have confirmed previous reports that CD200 is consistently expressed in all typical CLL. Furthermore, CD200 was expressed by all immunophenotypically atypical CLL cases. On the contrary, in MCL patients CD200 showed only a dim positivity in four subjects and was absent in the remaining 42. The inclusion of CD200 in the MBN routine flow cytometry panels facilitates the differential diagnosis between CLL and MCL and has a great impact on accurate diagnosis in cases with immunophenotypic aberrancies. This work was supported by grant RVO VFN64165 and PRVOUK P27/LF1/1 Table 1 MCL (46 pts.) CLL (200 pts.) Age (median, range) 66.7; 47.8-82.4 67.6; 32.2-90.7 Sex (F/M) 19/27 74/126 WBC x109/L (median, range) 10; 2.1-285.4 21.9; 2.8-375.2 % neoplastic B-cells of WBC (median, range) 17.1; 1.3-90.5 54; 1.7-94.7 CD200 MFI (median, range) 2.16; 1-53.2 147.5; 20.6-637 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 58 Introduction: Role of small non-coding microRNAs (miRNA) in hematopoiesis has been recently established by studies demonstrating increased levels of miR-155 in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is an ETS family transcription factor controling myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Our aim of this study is to determine mechanisms of miR-155 upregulation in CLL pathogenesis and the role of PU.1 downregulation in the process. Methods: miRNA and mRNA levels were determined by qPCR and Affymetrix mRNA expression profiling in peripheral blood mononuclear cells (PBMC) and in purified B cells. Control (N=13) and CLL patients (N=66) were studied. All patients were subgrouped according to cytogenetics (FISH) and Rai status. Protein-DNA localization assays were done using chromatin immunoprecipitation. Results: miR-155 is significantly upregulated in both CLL patient PBMCs and a subset of sorted B-cells whereas PU.1 and its target genes are repressed in all CLL subgroups. Indeed, expression profiling analysis of CLL samples identified a broad repression of ∼80 miR-155 targets (among them key transcriptional regulators FOS, SATB1, MEF2A, MYBL1, SIRT1, MECP2 and CEBPB) and ∼380 repressed PU.1 target genes, among them regulators of hematopoietic homeostasis (FOS, CSF1R, CSF2R, IL4R, IL21R) and apoptosis (BID, BIRC3). Next, we have studied the mechanism of miR-155 gene (also known as BIC) upregulation in CLL. Wehave newly identified a regulatory CpG island (BIC-CpG) upstream of miR-155 BIC gene that contains DNA binding motifs for E-box transcription factors and is not mutated in CLL patients. Two E-box-binding proteins, MYB and MYBL2, are significantly upregulated in CLL patient PBMCs as well as in a subset of sorted B-cells in all CLL subgroups. Furthermore in primary CLL cells, MYB protein presence is significantly enriched at BIC-CpG alongside a marked enrichment with transcriptionally active chromatin mark histone H3K9Acetyl. To further study the role of MYB in transactivation of the BIC-CpG we have prepared reporter constructs and found that MYB indeed activates BIC-CpG and downstream transcription. Apart from miR-155/BIC, expression profiling analysis identified additional ∼50 upregulated MYB targets, among them cancer-related genes such as CA1, MCM4, BCL2, PDCD4, and CXCR4. Functional assays using siRNA inhibition of PU.1 in normal PBMCs result in upregulation of miR-155 and MYB, indicating that silencing of PU.1 and upregulation of MYB and its target miR-155 may represent an important mechanism of CLL pathogenesis. Conclusion: Our data propose a mechanistic relationship between PU.1 and its negative regulator miR-155 in CLL. Our data also demonstrate that miR-155 is transcriptionally activated by MYB family of E-box binding proteins. Manipulation of these mechanistic relationships may harbor a potential for molecular therapies against CLL. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: 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.

Description: Role of small non-coding microRNAs (miR) in hematopoiesis has been recently established by work demonstrating critical importance of miR-223 for granulocytic development (Johnnidis 2008) and miR-155 in lymphocytic differentiation (Thai 2007) in mice. The importance of miR-155 is further supported by several independent studies demonstrating its increased levels in human diffuse large B-cell lymphoma (Eis 2005, Kluvier 2005, Volinia 2006, Lawrie 2007, Rai 2008) and in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is ETS family transcription factor involved in myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Studying primary CLL (N=27) and normal (N=13) peripheral blood mononuclear cells, we found significantly increased miR-155 levels and also demonstrate that PU.1 levels are significantly decreased. Next, we have tested genes that are targets of both miR-155 and PU.1 including CSF1R and FOS and again demonstrated their significantly decreased expression in CLL. Using gene expression profiling of CLL peripheral lymphocyte samples compared to normal controls, we have determined significant downregulation of the PU.1 mRNA and of numerous miR-155 targets previously reported to be required for myelo-lymphoid differentiation. These findings suggest that the relationship between PU.1 and miR-155 becomes disrupted in CLL. This in turn leads to overexpression of miR-155, reduced levels of PU.1 and of numerous miR-155 targets (N=2481 and N=400 respectively) and to inhibition of PU.1 regulated differentiation and proliferation programs. Next, we correlated negative cytogenetic prognostic features (p53 and ATM gene deletion detected by FISH) of the CLL patients with the miR-155 and PU.1 levels, In a relatively small-size patient cohort studied so far, the low ratios of PU.1/miR- 155 levels are similarly distributed among low, intermediate and high risk CLL patients, suggesting that inverse correlation of PU.1 and miR-155 represents a hallmark of CLL and likely represents a primary event during CLL pathogenesis.

Description: Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.