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B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

The circulating tumor cells of chronic lymphocytic leukemia (CLL) are characterized by variably low surface IgM (sIgM) levels and signaling capacity, consequent to stimulation by tissue-based antigen. The variability is evident in both CLL subsets with unmutated and mutated immunoglobulin genes and affects tumor cell survival and proliferation. This appears clinically important because CLL with relatively higher sIgM levels/signaling capacity progress more rapidly than those with lower sIgM levels/signaling capacity (D'Avola, Blood 2016), likely due to a larger proliferative component at tissue sites. B-cell receptor (BCR)-signaling inhibitor ibrutinib exerts its therapeutic activity in CLL by irreversibly binding the Bruton's tyrosine kinase (BTK) at Cys481 in the active site. This leads to redistribution of CLL cells from tissue sites into peripheral blood and to a selective recovery of sIgM levels and function, consequent to release from antigen drive at tissue sites (Drennan, Clin Cancer Res 2019). We have now investigated the hypothesis that sIgM levels on the CLL cells affect duration of response to ibrutinib therapy possibly consequent to circumvention of the pharmacological BTK inhibition. Seventy CLL patients participating in the "real-world" observational study at the University of Southampton (NIHR/UKCRN ID: 31076) were investigated for phenotypic, molecular and functional characteristics associated with response to single-agent ibrutinib. Time to progression requiring a new treatment from ibrutinib start (TTNT) was used as primary endpoint to measure duration of response. The levels of sIgM were measured by flow cytometry. Anti-IgM induced signaling was determined by immunoblotting or iCa2+ mobilization. BTK and PLCγ2 mutations were determined using the Illumina NexteraXT kit and a MiSeq at the University of Southampton and validated independently at Karolinska Institutet. Our previous cut-offs of 50 (MFI) or 5 (iCa2+ % mobilization) were used to distinguish patients with high/low sIgM levels or signaling capacity, respectively. Median follow-up from ibrutinib start was 42 months (range 12-70). Pre-ibrutinib sIgM levels (range 7-618, median 65; high sIgM MFI 46/70, 66%) and sIgM signaling capacity (range 1-98; median 45; high sIgM signalers 56/70, 80%) were broadly heterogeneous. However, median values were significantly higher than the general CLL population at diagnosis, as expected in progressive CLL. By univariate analyses of clinical, phenotypic, FISH and immunogenetic characteristics for TTNT, high sIgM was the only parameter predicting shorter duration of response to ibrutinib (p=0.02). Only 2/24 CLL with low sIgM levels (CLLIgM_lo) had progressed (median TTNT not reached) compared to 15/46 CLL with high sIgM (CLLIgM_hi, 14% at 12 months, 18% at 24 months, 34% at 36 months, 44% at 42 months) during therapy. The levels of sIgM correlated significantly with sIgM signaling capacity prior to treatment (r= 0.68; p From in vitro analysis of 13 therapy-naive CLL samples, the degree of inhibition by ibrutinib of anti-IgM induced signals downstream to BTK correlated negatively with sIgM levels (r=-0.68,p=0.01) and signaling (r=-0.71,p=0.009). Anti-IgM induced signals were then measured in 12 patients (7 CLLIgM_hi, 5 CLLIgM_lo) after 1, 4 and 12 weeks of ibrutinib therapy. These patients had not acquired BTK or PLCγ2 mutations and BTK phosphorylation appeared completely inhibited at all time-points. Mean anti-IgM induced iCa2+ mobilization and ERK1/2 phosphorylation were significantly reduced but not abolished during therapy and degree of inhibition was variable between individuals. Remarkably, degree of inhibition of anti-IgM-induced iCa2+ mobilization and pERK1/2 was significantly lower in CLLIgM_hi than in CLLIgM_lo (p These results confirm that sIgM signaling is dependent on sIgM levels and that it can circumvent BTK blockade when sIgM levels are high. They suggest that high sIgM signaling can drive ibrutinib resistance despite ability of ibrutinib to fully occupy the BTK phosphorylation pocket. A possibility is that those CLL cells with high sIgM represent a potentially dangerous subpopulation equipped to migrate to tissue and to receive proliferative stimuli. These cells might be targeted by a combinatorial therapeutic approach with ibrutinib. Disclosures Johnson: Takeda: Honoraria; Kite: Honoraria; Bristol-Myers Squibb: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Epizyme: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria. Duncombe:Abbvie: Other: Advisory Board membership;Educational support; Gilead: Other: Advisory Board membership; Janssen: Other: Advisory Board membership;Educational support; Novartis: Other: Advisory Board membership. Scarfo:Janssen: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Sutton:Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria; Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding. Forconi:Roche: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Gilead Sciences: Research Funding.

BACKGROUND: Systemic Anaplastic Large Cell Lymphoma (sALCL) is comprised of two distinct T-cell non-Hodgkin lymphoma entities: ALK-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL. These entities are characterized by either the presence of absence of an ALK-translocation. It has been reported that ALK+ ALCL has a better prognosis compared to ALK- ALCL, with a 5-year overall survival (OS) of 70-80% versus 15-45%, respectively. Furthermore, more than 25% of ALK+ ALCL patients undergo relapse. sALCL is a genetically heterogeneous disease whose genomic characterization has been improved through the implementation of high-throughput technologies. Despite this, the prognostic value of somatic mutations has been poorly described. Here we present the analysis of genetic aberrations of sALCL, shedding light on disease pathogenesis, novel diagnostic biomarkers and prognostic markers for the detection of refractory and/or relapsed patients which could hold clinical implications. METHODS: Formalin-fixed paraffin-embedded (FFPE) and/or fresh frozen tissue and related clinical information for 82 sALCL patients (47 ALK+ and 35 ALK-) were obtained after written informed consent from 5 centres across Europe. Using deep targeted DNA sequencing, the entire coding region of 275 selected genes (QIAseq Targeted DNA - Human Comprehensive Cancer Panel, Qiagen, Germany) was investigated in our retrospective cohort of patient' samples as well as 6 ALCL cell lines (Karpas-299, SU-DHL-1, DEL, SR786, FEPD and MAC2a). The average depth achieved across all the sequenced samples was approximately 2000x allowing the detection of aberrations with low allele frequencies ( RESULTS: ALK- patients in our cohort have a worse prognosis than ALK+, whereby the 7y-OS was 45.1% and 73.2%, respectively, although the 7 year progression free survival (PFS) was comparable at 57.1% for ALK+ and 42.4% for ALK- patients. As previously reported, ALK+ patients were significantly younger in our series than ALK- patients with an average age of 23.5 and 55.2 years, respectively. Among the 275 genes sequenced, 148 (53.8%) genes harbor at least one mutation throughout the entire patient and cell line cohort; almost 1/3 of mutated genes were shared between ALK- and ALK+ ALCL patients. On average 4.2 mutations per patient were detected in ALK- ALCL, a higher level than observed in ALK+ samples (2.7), most likely due to the oncogenic-driving role of the ALK-translocation in this group. Seventy-two out of 82 (88%) patients carried at least one single mutation within the genes analysed in our panel. The most recurrent genes mutated were TP53 in both sALCL categories (16% of sALCL patients), followed by LRP1B being prevalently mutated in ALK+ disease (15% of sALCL patients). As expected, STAT3 and JAK1 were mutated solely in the ALK- ALCL cohort and represent the most commonly mutated genes in this group. Prognostically, the most recurrent genes mutated in patients with a dismal outcome (dead and/or relapsed patients) were TP53, STAT3, EPHA5, JAK1, LRP1B, KMT2D, PRDM1 and SOCS1. Comparing samples at diagnosis versus relapse highlighted two key findings: firstly, the clonal expansion of TP53 mutated clones in relapsed ALCL patients and secondly, the acquired mutations in EPHA5 which were only detected at relapse. These data suggest their possible use as biomarkers associated with clonal evolution. CONCLUSION: This study provides information regarding the genetic landscape in sALCL across 275 select genes. We have confirmed the importance of known frequently mutated genes (STAT3 and JAK1) and describe novel recurrently mutated genes (LRP1B, EPHA5 and KMT2D). Mutations in STAT3 were more recurrent in ALK- ALCL patients with shorter OS, and clones harboring mutated TP53 and EPHA5 were detectable more often in relapsed sALCL thereby suggesting a possible driving role. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

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.

We and others have shown that deregulation of microRNAs (miRNAs) is associated with the biology of B cell malignancies, including regulation of B cell proliferation and survival (Musilova & Mraz, Leukemia, 2015). We focused on studying miRNAs that associate with the aggressiveness of FL and its transformation to diffuse large B cell lymphoma (DLBCL). First, we analyzed the expression of 380 miRNAs (TaqMan Arrays, ABI) in 8 paired primary samples of FL that subsequently transformed to DLBCL. We identified statistically significant changes (P1.8) in the expression of 5 miRNAs. The most significant change was the down-regulation of miR-150 (~5 fold, P=0.01). Similarly, we observed significantly reduced miR-150 levels in an independent cohort of non-paired samples of FL before vs. after transformation to DLBCL, and miR-150 was significantly less expressed in de novo DLBCL in comparison with FL. MicroRNA miR-150 is of particular interest as we have shown that its expression determines BCR signaling propensity in chronic lymphocytic leukemia (CLL) B cells, and low levels associated with worse survival (Mraz et al., Blood, 2014). Therefore, we analyzed miR-150 expression in a cohort of 89 FL samples. We noticed that miR-150 expression was lower in samples from patients with a FLIPI score ≥3 (P=0.03), and with high Ki67 positivity (>20%; P=0.003). Moreover, FL patients with low miR-150 levels ( To determine the potential reason for variable miR-150 levels in FL B cells, we tested the effect of microenvironmental interactions on its expression. In this experiment, a short term (48hrs) co-culture of B cell lymphoma cells with stromal cells (HS-5) led to down-regulation of miR-150 levels (P CONCLUSION: Low miR-150 levels associate with a shorter overall survival in FL. This could be used as a reasonable prognostic marker since high miRNA stability allows reliable analyses of miR-150 levels from formalin-fixed, paraffin-embedded (FFPE) samples. Interactions with stromal cells and/or soluble microenvironmental factors down-modulate miR-150 levels in B cells, which support their BCR signalling potential. We are further investigating to what extent the miR-150 down-regulation is causally connected with the aggressiveness and/or transformation of FL. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; the research grant GACR (16-13334Y); the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein; Masaryk university as part of the project "New approaches in research, diagnostics and therapy of hematological malignancies III", number MUNI/A/1028/2015 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2016; the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16); the Ministry of Education, Youth and Sports of the Czech Republic, grant nr. LD15144 (COST CZ); the research grant TACR (TEO2000058/2014-2019); and EHA Research Fellowship award granted by the European Hematology Association. G.P. is a city of Ostrava scholarship holder. Disclosures No relevant conflicts of interest to declare.

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab. We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz Disclosures No relevant conflicts of interest to declare.

The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

Abnormalities of TP53 and ATM genes are well established adverse prognostic markers in CLL. Mutations in splicing factor SF3B1 have recently been described as recurrent and predominantly subclonal aberration. Previous studies inconsistently suggested mutual exclusivity or partial overlap with TP53 mutations. Concerning ATM defects, the association between SF3B1 mutations and del(11q) was reported, but relation to ATM mutations remains unclear. The aims were: (a) to assess association between SF3B1 mutations and the most adverse classic genetic lesions represented by TP53 mutations and del(11q); (b) to investigate association between SF3B1 mutations and ATM mutations in a subset of ATM-characterized patients, and (c) to delineate SF3B1 mutation profile and proportion. We used Sanger sequencing of SF3B1 hot-spot exons 14-16, FASAY analysis of TP53 exons 4-10 and resequencing microarray for ATM mutation detection (all 62 coding exons). We analyzed unfavorable cohort of 338 patients characterized by prevalence of unmutated IGHV (72%). At the time of analysis, 82.5% of the patients were previously untreated. We observed SF3B1 mutation in 17.5% (59/338), TP53 mutation in 20% (68/338), and del(11q) in 27.5% (93/338) of cases. All these genetic defects were significantly more frequent in treated patients (SF3B1: P=0.008, TP53: P Our study indicates that SF3B1 mutations frequently overlap with mutations in the DNA damage response pathway genes at least in prognostically unfavorable cohort. It seems that SF3B1 mutations do not lead to complete protein loss indicating rather active involvement of mutated SF3B1 in splicing processes in CLL cells. The work was supported by grants NT13519-4, NT11218-6, MSM0021622430, MUNI/A/0723/2012. Disclosures: No relevant conflicts of interest to declare.

Abstract 3895 Background Unique miRNA expression signature is associated with CLL prognostic factors and disease progression (Calin et al., 2005; Mraz et al., 2009), and thus contributes to CLL pathogenesis. The mechanism through which miRNA expression is deregulated in cancer, including CLL, is not well characterized. However, the presence of mutations and single nucleotide polymorphisms (SNPs) in miRNA genes has been described (Calin et al., 2005; Ryan et al., 2010). Nevertheless, the impact and frequency of such variations in CLL patients have yet to be elucidated. Aim We searched for sequence variations in 109 pre-miRNAs with possible role in CLL pathogenesis. Additionally, we studied the effect of variations detected in miR-29b/29c genes on their expression in more details. Methods A custom resequencing microarray (Affymetrix) contained probes for 109 pre-miRNAs incl. ∼20 nt from 5′ and 3′ ends of pri-miRNA. MiRNAs were amplified by long range PCR from genomic DNA isolated from peripheral blood cells of 98 high-risk CLL patients (81.6 % unmut IgHV; 37.8 % mut TP53). Amplicons were pooled, fragmented and co-hybridized on the array according to the manufacturer's protocol. The presence of variations detected by microarray was confirmed by Sanger sequencing. The germline/somatic origin of novel variations was performed by direct sequencing of DNA isolated from buccal swabs of the patient carrying the variation. Pri-miR-29c and pri-miR-29b-2 were analysed by Sanger sequencing in additional cohort of 213 CLL patients. The effect of variations detected in miR-29 on their expression was studied by Real-Time PCR (TaqMan miRNA Assays, ABI) in 107 CLL patients. RNAfold tool was used to study the effect of variations on miR secondary structures. To see the difference between allelic frequencies of SNPs detected in miRNAs in CLL patients and healthy population, data from the 1000 genomes project (n=1092 samples) were used. Results Within 98 CLL patients, resequencing microarrays detected 18 annotated SNPs (NCBI dbSNP Build 134, 135) in 15 miRNAs, and importantly, 5 novel variations in 5 miRNAs (pri-miR-29a, pre-miR-16–1, pre-miR-372, pre-miR-106b, mature miR-142–3p). To see whether the novel variation in pre-miR-16–1 is a true mutation or a very rare SNP, 193 CLL patients (105 pts. with del13q) were further analysed. Surprisingly, neither novel variation detected by microarray nor known mutation in pri-miR-16–1 previously detected in two CLL patients (Calin et al., 2005), were found. MiR-29 family, which is directly implied in CLL pathogenesis (Pekarsky et al., 2006; Santanam et al., 2010), has been analysed in more details. In the studied cohort of 213 CLL patients, 7 SNPs and one novel variation were found in pri-miR-29b-2 and two SNPs were found in pri-miR-29c. As shown by RNAfold, miR secondary structure was affected by 14 variations in total, suggesting their possible subsequent relevance for miR stability and processing. Moreover, the insertion in pri-miR-29b lowered its expression (fold change 0.7), but only in patients with unmut IgHV (p=0.036). All detected variations were of germline origin except for miR-655, whose mutation was present only in one B-cell subclone in a biclonal CLL case. The allelic frequency of miR-SNPs differed significantly in 8/62 SNPs (p Conclusion We herein confirm that both SNPs and new sequence variations, most of which were present outside mature miRNAs, are frequent in CLL patients. We also show that miR-16 variations are extremely rare ( Disclosures: No relevant conflicts of interest to declare.

Abstract 3859 Chronic lymphocytic leukemia (CLL) is the first disease in which miRNAs (hsa-miR-15a-16–1) were directly linked to cancer pathogenesis (Calin et al. PNAS, 2002). We and others have also shown that expression of certain miRNAs associates with disease activity in patients with CLL (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012; Mraz et al. Leukemia, 2009). Moreover, patients with more aggressive disease have CLL cells that generally express unmutated IGHV and/or ZAP-70 and have a miRNA expression profile that differs from that of CLL cells from patients with indolent disease (Calin et al. NEJM, 2005). However, we still have very limited understanding of how miRNAs affect CLL cell-biology and expression of genes that play a critical role in either promoting or arresting the disease. We used pooled samples from 10 CLL patients to screen (TaqMan miRNA Cards-ABI, 750 miRNAs) for abundantly expressed miRNAs that could hypothetically influence CLL B cell biology. We identified miR-150 as the most abundant miRNA in CLL cells and also as being strongly expressed when compared to CD19+ blood lymphocytes of normal adults (N=5, P=0.008). This miRNA already has been reported to influence the differentiation and gene expression of normal B cells (Xiao et al. Cell, 2007) suggesting its possible relevance for CLL B cell biology. We examined additional CLL cell samples (N=168) and confirmed high miR-150 levels and also noted heterogeneity in its expression between CLL cells of patients with aggressive versus indolent disease. In our cohort, CLL cells of patients that expressed ZAP-70 (20% cut-off, N=74) or had unmutated IGHV (N=72) expressed significantly lower median-levels of miR-150 (fold change −1.7 and −2.0 respectively, p10.0). Importantly, cells with higher expression of GAB1 or FOXP1 were more responsive to BCR stimulation in vitro (Pmedian) were associated with significantly longer OS (not-reached vs. 13.9 years, N=168, P=0.006). Additionally, the expression level of miR-150 was an independent predictor of OS and time to first treatment (TTFT) in multivariate analyses, which included IGHV status, ZAP-70, CD38, Rai stage, gender, and age (OS HR: 3.4 [CI 1.4–8.6], P=0.009; TTFT HR: 2.3 [CI 1.3–4.2], P=0.004). We conclude that there is an inverse association between high-risk disease and expression of miR-150, which may reflect its capacity to regulate the expression of genes encoding proteins that may contribute to BCR-signaling and/or survival of CLL B cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2872 Introduction: The DNA damage response axis plays a crucial role in chemoresistance in CLL, as indicated by the prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM). These deletions coincide with mutations in the remaining allele, although frequency, especially for ATM varies. Functional read-outs of the p53 axis might add clinical relevant information on the actual DNA damage response. Currently, different p53 function analyses are being developed. These assays are either based on measurement of (i) RNA expression levels of a single gene (RT-PCRp21) or gene sets (RT-MLPA), or protein expression levels (FACSp53-p21) after DNA damage by irradiation or etoposide/nutlin exposition or (ii) gene expression levels at base-line (RT-PCR miR34a). To what extent these different assays correlate is currently unknown. Aim: Detailed side-by-side analysis of available p53 functional assays in well characterized CLL samples Methods: Freshly frozen PBMC's of 15 different CLL samples (CD19/CD5>90%) were exchanged between 5 research groups that developed the following p53 functional assays: RT-PCRp21 (Ulm), RT-MLPA (bax, puma, p21 and CD95; Amsterdam), FACSp53-p21 (Paris) and RT-PCR miR34a (Salzburg/Brno). FISH-analysis (11q, 17p, 13q and 12) was performed on all samples. In addition mutations in TP53 were determined by FASAY, DHPLC and Sanger sequencing and ATM mutations were assessed by Sanger sequencing. Results: RNA expression levels showed significant correlation (p-value < 0,01) between the different P53 functional assays with high correlation coefficients (range: 0,7–0,94). Based on combination of FISH-analysis, FASAY and Sanger sequencing the investigated CLL samples could be distinguished into 6 different categories; 1. 17p- with TP53 mutation (n=5), 2. Sole TP53 mutation (n=1), 3. 11q- with ATM mutation (n=1), 4. 11q- in absence of TP53/ATM mutation (n=4), 5. 17p- with both TP53 mutation and ATM mutation (n=1) and 6. No 17p- and 11q- in absence of TP53/ATM mutation (n=3). All p53 functional assays showed absent to minimal induction of expression of respective target genes in samples with a cytogenetic abnormality combined with a TP53/ATM mutation (category 1, 3, 5). In contrast, samples without a cytogenetic abnormality in absence of TP53/ATM mutations (category 6) showed a marked increase in expression of respective target genes in all the assays. The patient with a sole TP53 mutation (category 2) showed a normal DNA damage response in all assays except for FACSp53-p21. Samples with an 11q- in absence of TP53/ATM mutation (category 4) showed high inter-assay variation. The two assays with predefined cut-off values (RT-MLPA and FACSp53-p21) assessed 13 samples equally (9 p53 dysfunctional and 4 p53 functional), except for 2 patients (category 2, 3 respectively). Reproducibility of these assays was tested by blinded sending of 3 samples that have been analyzed previously in this study. Except for p21, all assays showed 100% reproducibility. Discussion: For the first time a comparative side-by-side analysis of different available p53 functional assays was performed, and demonstrated strong correlations between the different assays. All assays could detect samples with expected disturbed and normal DNA-damage responses. To what extent these p53 functional assays could be of clinical relevance especially with respect to chemo-responsiveness, should be further studied in prospective studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3883 Background. MicroRNAs (miRNAs) are known to be involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and affect its clinical course (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012). Moreover, we and others have shown that several miRNAs are down-regulated in the aggressive CLL subtype harboring p53 aberration (Mraz et al. Leukemia, 2009). The role of miRNAs in primary or acquired resistance to therapy in CLL, however, is poorly understood. Aim. In this study, we screened for miRNAs that are induced by fludarabine-mediated apoptosis in vitro, and we suggested that differences in the expression of one of the identified miRNAs (miR-34a) can be used to distinguish patients with impairment of the p53-apoptotic pathway. Results. Ten primary CLL B-cell samples (purity>95% of CD5+19+ cells) were treated in vitro with fludarabine dose (IC50 dose of 3.5 ug/mL, 48hrs), and the expression of 750 miRNAs was subsequently profiled (TaqMan miRNA Cards, ABI). In comparison with untreated control samples, 15 miRNAs were induced by fludarabine (fold change>1.5, SAM FDR Conclusions. Our data provide complex evidence for the use of miR-34a as a marker of fludarabine-resistant disease. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (mutation not accompanied by del17p13), and miR-34a down-regulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease. This can be accomplished without treatment of cells with gamma-irradiation, which was previously used to identify functional impairment of the p53-pathway (Pettitt et al. Blood, 2001; Mous et al. Leukemia, 2009; Lin et al. CCR, 2012). The described assay for absolute quantification of miR-34a encourages further inter-laboratory validation. IGA MZCR NT11218–6/2010, CZ.1.07/2.3.00/20.0045, CZ.1.05/1.1.00/02.0068 Disclosures: No relevant conflicts of interest to declare.

Abstract 1770 Background: MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression by inhibition of mRNA translation and/or stability. Numerous miRNAs were described as tumor suppressor genes or oncogenes. In this respect, miR-29 family belongs to the most important miRNAs involved in CLL biology. Three different isoforms of the miR-29 (miR-29a-2, miR-29b-2 and miR-29c) were shown to be down-regulated in aggressive CLL (Calin et al., 2004; Mraz et al., 2009). Moreover, miR-29 was suggested to target the expression of MCL1, CDK6 and TCL1, a critical oncogene in aggressive CLL. Recently, the generation of transgenic mice over-expressing miR-29 in B-cells demonstrated its direct role in CLL pathogenesis. Additionally, mutations in miR-29c and miR-29b-2 have been also detected in CLL patients (Calin et al., 2005); however, their impact and frequency have yet to be elucidated. Aims: In this study, we searched for novel sequence variations in three members of the miR-29 family (miR-29a, miR-29b-2 and miR-29c) using re-sequencing microarray and Sanger sequencing. Methods: The pre-miRNAs (29a, 29b-2, 29c) and 107 other pre-miRNAs were analyzed by a custom re-sequencing microarray (Affymetrix, 50K) in 98 high-risk CLL patients (81.6% of patients had unmutated IgVH; 37.8% had a mutation in the TP53 gene). To cover the whole genomic area of pre-miRNAs, ∼20 nucleotides from both 5' and 3' ends of pri-miRNAs were also analysed. MiRNAs were amplified from genomic DNA isolated from peripheral blood cells (CLL lymphocytes or mononuclear cells) using long range PCR. Amplicons were pooled, fragmented and co-hybridized on the array according to the manufacturer's protocol. Sanger sequencing was used to confirm the presence of the mutations detected by microarray. The genomic regions (455-bp, 940-bp) of pri-miR-29c and pri-miR-29b-2, respectively, were further analyzed by Sanger sequencing in another 192 CLL patients (63.5% with unmutated IgVH; 21.4% with TP53 gene mutation). Results: Using resequencing microarray and confirmatory Sanger sequencing, one heterozygous variation (A/G +22 in 3') was found in the pri-miR-29a in one of 98 CLL patients. Screening of pri-miR-29c (455-bp genomic area) detected two substitutions in 4 of 192 CLL patients. In particular, the known G/A substitution (-31 in 5') was found as heterozygous in two patients. Additionally, novel heterozygous T/A substitution (+137 in 3') was found in two other patients. Screening of 940-bp genomic region of the pri-miR-29b-2 detected insertion (+A) (+107 in 3') in 22 of 192 patients. However, the known G/A substitution (+212 in 3') was not detected in our cohort. Furthermore, 2 novel heterozygous variations in pri-miR-29b-2 (C/G −169 in 5' and A/G − 256 in 5') were found in 5 and 2 patients, respectively. According to our results, pri-miR-29b-2 seems to be the most frequently mutated member of the miR-29 family, since three types of variations were detected in 29 of 192 CLL patients (15.1%). Two substitutions were found in pri-miR-29c in 4 of 192 CLL patients (2.1%) and only one substitution was detected in pri-miR-29a in one of 98 CLL patients (1%). Conclusion: We herein confirm that both known mutations and novel sequence variations are present in three members of the miR-29 family which is already implicated in CLL pathogenesis or progression. Altogether, we have found 6 types of variations in 34 of 290 analyzed CLL patients (11.7%). The insertion (+A) (+107 in 3') in pri-miR-29b-2 was the most common variation detected in our cohort of CLL patients; however, its impact on CLL pathogenesis has to be elucidated. Supported by the grants IGA-MZ-CR NT11218-6/2010, NS10439-3/2009, NS9858-3/2009, MPO-CR-FR-TI2/254 and MSMT-CR-MSM0021622430. Disclosures: Mayer: Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.

Abstract 4600 MicroRNA miR-155 represents a candidate pathogenic factor in chronic lymphocytic leukemia (CLL) and lymphomas (Eis PS, et al. 2005, Fulci V, et al. 2007, Calin GA, et al. 2005, Kluiver J, et al. 2005, Metzler M, et al. 2004, van den Berg A, et al. 2003., Vargova K. et al 2011). In diffuse large B-cell lymphoma (DLBCL) expression of miR-155 was associated with NFkB pathway (Rai et al., 2008). In addition, increased miR-155 levels were associated with more aggressive (Activated B-cell like) DLBCL (Eis et al. 2005). The targets of up-regulated miR-155 apparently include a key hematopoietic transcription factor PU.1. Down-regulation of PU.1 represents an important and critical step for both myeloid as well as lymphoid tumorigenesis. We herein found the increased levels of miR-155 coupled with downregulation of its target PU.1 are frequent events in B cells of a vast majority of CLL patients (N=169). To advance understanding of miR-155 and PU.1 in lymphoid malignancies we studied lymph node biopsies derived from patients with lymphomas. We show that increased miR-155 levels are also found in DLBCL (N=31, P < 0.0001), Hodgkin lymphoma (HL, N=22, P < 0.0001), follicular lymphoma (FL, N=23, P < 0.001), small lymphocytic lymphoma (SLL, N=12, P < 0.01), and marginal zone lymphoma (MZL, N=11, P < 0.01). The miR-155-overexpressing tumors display downregulation of PU.1. In contrast, peripheral T cell lymphoma (T-NHL, N=6) and mantle cell lymphoma (MCL, N=7) expressed miR-155 and PU.1 comparably as control lymph nodes. Our data indicate that expression pattern of miR-155 and its target PU.1 represents a hallmark of some but not all lymphoid malignancies. Interestingly, the miR-155/PU1 levels vary substantially within each diagnosis, therefore, our aim is to further analyze the occurrence of this relationship in different prognostic groups and analyze the clinical outcome as well. (Grants: NT10310-3/2009 & NT11218-6/2010, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507) Disclosures: No relevant conflicts of interest to declare.

Abstract 3115 Background. Rituximab significantly improves the outcome of patients with B-cell non-Hodgkin lymphomas, but it does not completely eradicate residual B-cell populations in the bone marrow (BM) and lymph nodes (LN). The architecture and gene expression profile of LN stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with R-CHOP (Lenz et al. 2008). Interestingly, multiple studies have demonstrated the importance of B-cells adhesion and pro-survival signaling mediated by integrin alfa-4-beta-1 (VLA-4/CD49d), which is constitutively expressed on malignant B-cells. Although several studies have demonstrated that adhesion to stromal cell cultures or ligand coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance (CAM-DR); Dalton, 2002), a similar mechanism of resistance to rituximab has not, to our knowledge, been described. Hypothesis. In this study we tested the hypothesis that interactions with the microenvironment protect malignant B-cells from rituximab induced apoptosis, and that targeting these interactions with natalizumab, a humanized monoclonal anti-alfa-4 antibody approved for treatment of multiple sclerosis and Crohn's disease, can overcome this protection. Methods. Lymphoma B-cell lines were cultured on either a plastic surface, confluent HS-5 cells or a fibronectin (alfa-4-beta-1 ligand) coated surface. HS-5 is an immortalized human bone marrow stromal cell line and a well characterized model for a component of the BM microenvironment (Roecklein and Torok-Storb, 1995). Cells were treated with rituximab (10 ug/mL; Genentech), natalizumab (10ug/mL; Biogen IDEC), control IgG (10ug/mL, Sigma-Aldrich). Cell viability was measured by flow cytometry using Annexin V-FITC, propidium iodide in malignant B-cell population (anti-CD19 antibody, BD PharMingen). Results. We studied ten CD20-positive B-cell lymphoma cell lines and the CD20-positive MEC-1 cell line (derived from prolymfocytoid transformation of CLL) for rituximab induced apoptosis. The four most rituximab sensitive cell lines were used for further experiments (Karpas-422, Raji, DOHH2, SUDHL-4). Cell lines were co-cultured for 24hrs with confluent HS-5 stromal cells and subsequently treated for 24hrs with rituximab. The percentage of apoptotic cells was significantly lower (17-23% vs. 30–42%, p We next examined if natalizumab could disrupt cell adhesion to fibronectin and overcome the stromal cell-mediated resistance to rituximab. Natalizumab reduced the number of lymphoma cells that adhered to fibronectin by 75–95% (p Conclusion. We have shown that human bone marrow stromal cells (HS-5) protect lymphoma B cells from rituximab induced apoptosis suggesting existence of stromal cell adhesion-mediated antibody resistance (CAM-AR) analogous to CAM-DR. Targeting integrin alfa-4-beta-1 with natalizumab partially overcomes this cell adhesion-mediated resistance to rituximab. Research supported by P50CA97274-8 LYMPHOMA SPORE, Genentech, MSMT-MSM0021622430 and IGAMZCR NT11218-3/2010. Disclosures: Zent: Genentech: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Abstract 1231 Poster Board I-253 Background Chromosomal abnormalities are present in the majority of CLL cases (>80%) and recently, a novel deletion of 22q11 was described in CLL patients. This locus encodes lambda immunoglobulin light chain (IgL) subgenes and several protein-coding genes, which distinguishes IgL from kappa Ig or heavy Ig locus. Gunn et al. (2008) previously characterized the minimally deleted region of 22q11 in CLL as 0,34Mb (containing PRAME, ZNF280A, ZNF280B, GGTLC2) and described the PRAME as a candidate gene with prognostic impact in CLL. Interestingly, the IgL variable segment (V2-8) located at 22q11 includes an annotated miR gene miR-650. According to the performed BLAST search also other subgenes of V2 family include homologues for miR-650, which overlay the leader exon of the V2 subgenes. This could be of particular importance because the expression of miRNAs was previously shown to be associated with known prognostic markers in CLL: 13q deletion, IgVH status (Calin, 2002, 2005) and p53 deletion/mutation (Mraz, 2009). Moreover, miRNA pathways are involved in the regulation of immune system functions including IgG production and V(D)J recombination (Vigorito, 2007; Koralov, 2008). Aim We analyzed genomes of 40 CLL patients using array-CGH (CGH Microarray 4×44 K, Agilent) and FISH (del13q14, del17p13, del11q23, +12), and in particular focused on detailed characterization of 22q11 deletion. An expression analyses (RT-qPCR, TaqMan miRNA Assay, ABI) was performed for protein-coding genes and miR-650 located at 22q11. Results Using array-CGH additional aberrations to FISH were found in the majority of samples and among them, the deletion of 22q11 locus was observed most often (ranging in size up to ∼ 0,77 Mb). Altogether, 40% of patients (16/40) had detectable deletion in this locus and according to our finding, all observed deletions were a consequence of a VJ rearrangement event, which lead to an elimination of specific genes located between VJ elements. These rearrangements were characterized in detail by PCR detection (BIOMED-2 protocol) and sequencing. In our cohort, deletions of 22q11 containing PRAME gene constituted only ∼44% (n=7) and 56% patients (n=9) harbored smaller deletions not involving PRAME. To analyze the gene expression, we performed a Real-Time PCR analyses for previously mentioned protein-coding genes and miR-650. The Real-Time PCR assay for miR-650 reported higher expression of miR-650 in CLL cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=5) compared to samples utilizing different V lambda family or expressing kappa Ig (n=36). It remains to be elucidated how and whether all the V2 subgenes give rise to a mature and functional miRNA. The overlap between miR-650 and the leader exon of IgL gene could provide a unique mechanism for coordinated IgL and miRNA expression. To assess the function of miR-650 we performed a microarray (Human OneArray, Phalanx) expression profiling 24/48 hours post transfection of CLL cells with a short RNA mimicing miR-650 (miRIDIAN Mimic, Dharmacon). The transfection led to a significant down-regulation of dozens of mRNAs that could be considered as potential targets for miR-650. Interestingly, the predicted targets for miR-650 include early B cell factor (EBF3; target with highest total score – TargetScan) and VPREB1 (pre B lymphocyte gene 1) whose expression is important for immature phenotype of pre-B lymphocytes prior to light chain recombination. The coupled activation of miR-650 and lambda Ig expression following productive rearrangement of IgL locus could potentially affect the expression of VPREB1/EBF3. Conclusion Deletion of 22q11 is frequently detected in CLL patients and we suggest that it is caused by physiological process of lambda immunoglobulin variable locus rearrangement. This leads to the elimination of protein-coding genes located between VJ elements. Interestingly, the V2 family of variable segments for lambda light chain at 22q11 includes an annotated miR gene miR-650 and its homologues. The productive lambda locus rearrangement utilizing V2 family could be also coupled with activation of miR-650 expression with consequent relevance for B cell biology. This work is in progress and in further analyses we expect complex characterization of miR-650 functions in B cells and CLL. Supported by IGA MZ CR NR9293-3/2007 and IGA MZ CR NS10439-3/2009. Disclosures Mayer: BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.

Abstract 4645 Background Graft-versus-host disease (GvHD) is the life-threatening complication of allogeneic hematopoietic stem cells transplantation (allo-HSCT). GvHD is mediated by an immune reaction of donor T lymphocytes against recipient's tissues/cells. Acute GvHD (aGvHD) appearing within the first 100 days post transplantation is the most frequent cause of recipient's death and characterization of biomarkers for early prediction of aGvHD or resistance to corticoid treatment could be of great clinical relevance. Biomarker panels for aGvHD are currently extensively studied by proteomic and gene expression based approaches but so far very few markers were described and validated (Kaiser et al., 2004; Baron et al., 2007; Weissinger et al., 2007; Paczesny et al., 2009). Aim In this study, we performed microarray gene expression analysis (whole genome Human OneArray, Phalanx) of 43 leukemia patients who received allo-HSCT. Mononuclear cells isolated from peripheral blood samples (Ficoll-Paque) were collected at i) 14 days before transplantation, ii) 20 and iii) 30 days after transplantation and iv) at the time of aGvHD manifestation. We also performed gene-expression analysis for corticoid-resistant vs. corticoid-sensitive aGvHD cases. Results The SAM supervised analysis of samples collected at day +20 post transplantation revealed set of differentially expressed genes between groups of patients that developed aGvHD vs. aGvHD-free recipients. Among others, genes CASP1 (encoding caspase 1, protein implicated in apoptosis), HLA-DRA (member of MHC class II family) and LILRA3 (leukocyte immunoglobulin-like receptor, subfamily A member 3) showed the highest difference in expression. Gene expression with regard to corticoid response was analyzed at the time of first aGvHD manifestation. The SAM supervised analysis of gene expression between patients with corticoid-sensitive aGvHD (n=10) or aGvHD resistant to corticoid treatment (n=4) revealed a set of significantly differentially expressed genes including NR4A2 (nuclear receptor subfamily 4; member of the steroid-thyroid hormone-retinoid receptor superfamily), DEDD2 (death effector domain containing 2), TREM1 (triggering receptor expressed on myeloid cells 1), TPK1 (thiamin pyrophosphokinase 1) and HBEGF (heparin-binding EGF-like growth factor). Conclusion Oligonucleotide microarrays proved to be a useful tool for expression studies of hematological malignancies and our work shows that they may help to identify markers for early diagnosis/treatment of aGvHD. The limited patients' cohort and their heterogeneity complicate such studies. Our future effort will be focused on experimental group extension, cohort uniformity and verification of the obtained data. This work is supported by the grant NS9683-4/2008 provided by the IGA MH of the Czech Republic, and MSM0021622430 provided by MEYS of Czech Republic Disclosures: No relevant conflicts of interest to declare.

Mutational status of immunoglobulin genes coding for heavy chain variable region (IgVH) stratifies patients with B-cell chronic lymphocytic leukemia (CLL) into two distinct prognostic groups. Expression level of specific genes such as LPL, ZAP-70, ADAM29 or CLLU1 has previously been shown to distinguish these two subgroups, although their exact role in CLL pathogenesis remains unclear. In our study we performed microarray and Real-time PCR analyses of gene expression in B-CLL cells with different IgVH mutational status in an attempt to further identify potential discriminating genes. Heparinised peripheral blood samples were collected from 92 previously untreated B-CLL patients (40 IgVH unmutated vs. 52 IgVH mutated) with a median age of 62 years (ranging from 40 to 81 years). B-cells were separated using cocktails of selected bispecific antibodies directed against cell surface antigens of hematopoietic cells and glycophorin A (RosetteSep® B Cell Enrichment Kit, StemCell Technologies). Great attention was paid to T-cell depletion using Human CD3 Depletion Cocktail. Samples with lower percentage of B-CLL cells were further processed by immunomagnetic separation (EasySep® FITC Selection Cocktail, anti-CD5FITC) and only samples with B-CLL cell content higher than 95 % (assessed using flow cytometry) were used for the gene expression analysis. Microarray gene expression analysis of 20 B-CLL patients (10 with mutated and 10 with unmutated IgVH) was performed using Agilent Human 1A Arrays and obtained data were statistically analysed using SAM (Significance Analysis of Microarrays). Among approx. 50 diferentially expressed genes, high expression of LPL and LAG3 (Lymphocyte- Activation Gene 3) was the most prominently associated with unmutated IgVH subtype. The mRNA levels of selected genes (LAG3, LPL, ZAP-70, AICDA, BCL2, CLLU1, TCF7, SEPT10, ADAM29) were further tested on a cohort of 92 B-CLL patients using quantitative RT-PCR (TaqMan® Gene Expression Assay, Applied Biosystems). Among them, LAG3, LPL and ZAP-70 reached the best statistical significance (p-value < 0.001; Wilcoxon’s rank-sum test). LAG3 expression level can predict unmutated IgVH with 100% sensitivity and could be used as a novel potential CLL prognostic marker. LAG3 plays role in T-cell dependent B-cell activation during affinity maturation and its over-expression in cells with unmutated IgVH may be a consequence of improper signalling in this process. Combination of expression data of three genes LAG3, LPL and ZAP-70 reliably assigned 95% of CLL samples into a correct IgVH subtype and seems to be better predictor of IgVH status than either of these genes itself. Moreover, we have analysed the correlation between expression of those three IgVH surrogate markers and necessity of therapeutical intervention. The patients manifesting high expression of LAG3, LPL and ZAP-70 required therapy significantly earlier than patients with low expression of those genes as calculated by Kaplan-Meier estimator (median time to the first therapy was 797 days in patients with high expression of surrogate markers, median was not reached in patients with low expression of surrogate markers). Differences of time to the first therapy between patients with differential expression of three IgVH surrogate markers (p=4.4*10−8, log-rank test) and differences between patients with different IgVH status (p=7.5*10−8, log-rank test) showed high significance. These results suggest that IgVH status could be precisely assessed using expression analysis of three genes LAG3, LPL and ZAP-70. The expression data of those surrogate markers provide even better statistical concordance with time to requirement of the first therapy than IgVH mutational status itself and possibly could be used in the diagnostics.