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Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications.

There is increasing evidence for the role of chronic antigenic stimulation (CS) in the development of cancer. Clinical data, however, are rare as is the information on outcome. In this study, the occurrence of chronic infections (CI) and autoimmune diseases (AI) in patients with malignant lymphoma at diagnosis was assessed. Of 367 patients [non-Hodgkin's lymphoma (NHL) N = 297, Hodgkin's lymphoma N = 70], 9.8% (N = 36) had a history of chronic antigenic stimulation (4.4% AI, 5.4% CI) at diagnosis. After a median observation time of 74.7 months, 118 patients have died. There were more male patients in this cohort. However, sex ratio among patients with chronic antigenic stimulation was skewed in favor of women (p = 0.018), in particular among lymphoma patients with AI (p = 0.001). NHL patients with autoimmune diseases showed a tendency to develop diffuse large B cell lymphoma [8 of 12 AI + NHL patients (66.7%) vs. 100 of 266 non-CS NHL (37.6%); p = 0.066]. No significant difference in overall survival (OS) between CS and non-CS patients could be observed (median OS after 48 months was: CS 77.7% vs. non-CS 71.8%). In conclusion, chronic antigenic stimulation at diagnosis appears to be associated with a higher prevalence in women, in particular among patients with autoimmune disease. However, no difference in overall survival was observed. This suggests that the presence of chronic inflammatory conditions does not decisively influence the outcome of lymphoma patients.

In chronic lymphocytic leukaemia (CLL), lipoprotein lipase (LPL) mRNA overexpression is an established poor prognostic marker, its function, however, is poorly understood. Measuring extracellular LPL enzymatic activity and protein, we found no difference between levels in CLL patients and those of controls, both before and after heparin treatment in vivo and in vitro . Investigating LPL knock down effects, we determined five potential downstream targets, of which one gene, STXBP3, reportedly is involved in fatty acid metabolism. While possibly reflecting an epigenetic switch towards an incorrect transcriptional program, LPL overexpression by itself does not appear to significantly influence CLL cell survival.

Abstract 3580 The expression of lipoprotein lipase (LPL) in CLL cells is an established mRNA surrogate marker for immunoglobulin heavy chain (IgVH) mutational status. High expression of LPL correlates with poor prognosis. However, the possible functional role of LPL in CLL is still unclear. LPL is normally expressed in muscle cells, adipose tissue and macrophages, transported to the luminal surface of endothelial cells where it is bound heparan sulfate-proteoglycans (HSPG). Heparin competes with HSPG for the binding sites and intravenous injection leads to elevated plasma LPL protein levels and enzymatic activity (“heparin release test”). LPL mRNA levels correlate with intracellular protein expression (Heintel et al. Leukemia. 2005; Mansouri et al. Leuk Res. 2010). Moreover cellular lysates from CLL patients contain elevated LPL enzymatic activity compared to healthy donors (Pallasch et al. Leukemia. 2008.). In this study, we investigated the basal (pre-heparin) LPL protein levels by enzyme-linked immunosorbent assay in the serum of 42 CLL patients, 14 non-CLL patients (lymphoma in remission), and 4 healthy donors (HD): Median pre-heparin LPL protein levels were 40.10 ng/ml (range: 5.66–108.44), 44.11 ng/ml (18.26-84.08), and 68.14 ng/ml (33.28-174.38), respectively. Among CLL patients there were no significant differences between those with high (N=16; median LPL protein in serum: 38.10 ng/ml (8.72-73.49)) and low (N=26; 43.12 ng/ml (5.66-108.44) (p=0.354) LPL mRNA expression. Thirteen patients with known LPL mRNA expression were investigated for LPL protein “release” after heparin injection. Ten and twenty minutes after 50 U/kg heparin injection, the elevation of both parameters, LPL protein amount in serum and enzymatic activity in plasma, was similar to those of HD normal values. In detail, medium serum protein levels in samples with high LPL mRNA (N=5) increased from 16.11 to 214.33 and 332.78 ng/ml and in the samples with low mRNA (N=8) from 13.08 to 219.68 and 386.65 ng/ml, respectively. The corresponding median values of the LPL enzymatic activities in high vs. low expressors were: 7.25/15.52/20.01 and 7.45/19.13/20.57 μ M/ml/h. In addition, release of LPL from peripheral mononuclear cells (PBMC) of CLL patients (N=3) by heparin in vitro was absent. Cell viability and LPL mRNA expression remained unaffected in both in vivo and in vitro samples after heparin addition. In order to assess the impact of LPL on cell survival, CLL cells were cultured (N=3) for up to 72 hours with different doses of purified LPL protein. There was no positive effect on cell survival irrespective of primary LPL mRNA expression or culture conditions (with or without FCS). Since these results point to an intracellular effect of LPL, we aimed to identify downstream targets by knock down with siRNA against LPL in 7 CLL samples and 5 cell lines (hepatocellular carcinoma, cervix carcinoma, colon carcinoma, multiple myeloma and acute monocytic leukemia) with high LPL mRNA expression. Gene expression changes were analyzed by microarrays (GeneChip® Human Gene 1.0 ST Array, Affymetrix). Fifteen genes were up- (N=4) or downregulated (N=11) in at least 3 of 5 cell lines by more than 1.5-fold (e.g. GSTP1, COROC1). Nine genes were at least 1.5-fold downregulated in parallel with LPL in the CLL samples only. These genes belong to various pathways (e.g. cell cycle, signaling in immune system, metabolism of carbohydrates) and seem to be specific for CLL. Cross-validation of individual genes is under way. Our data suggest that (1) neither basal serum LPL protein levels nor heparin-induced LPL release in CLL patients are suitable clinical prognostic markers; (2) Stimulation with external LPL protein does not affect CLL cell survival; (3) siRNA knock-down of LPL induces changes in various functional pathways. We conclude that the key role of LPL expression in high-risk CLL is related to its (intra)cellular expression. Disclosures: No relevant conflicts of interest to declare.

Abstract 2371 Poster Board II-348 Chronic lymphocytic leukemia (CLL) is a clonal expansion of B cells which is characterized by a defect in apoptosis and is associated with the co-expression of CD19, CD5 and CD23. Lumiliximab is a monoclonal antibody against CD23 which has been shown to exert a promising therapeutic effect in CLL in vivo and to induce apoptosis in CD23+ lymphoma cells in vitro. The aim of this study was to investigate the direct effect of lumiliximab on cell viability and to explore its molecular mechanism of action in primary CLL cells. PBMC from twenty CLL patients were used in this study. Nine patients had previous therapy and 11 were untreated. Nine patients had unmutated and 11 had mutated IgVH genes. The mean percentage of CD19+/CD5+/CD23+ cells was 80% (range 53-98%). PBMC were exposed to various concentrations of lumiliximab (1-50 μg/mL) for various durations (1-15 days). The long term incubation with lumiliximab was performed in a microenvironment co-culture model using primary human stromal cells which prevent spontaneous apoptosis of CLL cells. Cell viability was assessed by flow cytometric analysis using annexin V/PI staining and by MTT assays. The results showed that single exposure to lumiliximab had a minimal effect on cell viability (< 1 fold increase in apoptosis rate compared to untreated cells and to the isotype control antibody). Cross-linking with goat anti-human IgG was more effective in inducing cell death. Interestingly, repeated exposure to lumiliximab without cross-linking had a significant pro-apoptotic effect selectively in the CD19+/CD5+ cells. Western blotting analysis demonstrated a significant biological response to the single and repeated exposure to lumiliximab in spite of the moderate pro-apoptotic effect. Lumiliximab induced a significant decrease in Bcl-2, Mcl-1 and Hsp70 protein expression. In addition, it resulted in a significant decrease in the phosphorylation of Akt1 at serine residue 473 and dephosphorylation (activation) of the tumor suppressor PTEN at serine residue 380 suggesting the involvement of the PI3-K/Akt/PTEN cascade in priming CLL cells to undergo apoptosis by lumiliximab. Pre-incubation of CLL cells with lumiliximab enhanced the pro-apoptotic effect of PI3-K inhibitor LY292004 and the CK2 inhibitor apigenin. Consistent with previous observations, pre-exposure of CLL cells to lumiliximab augmented the cytotoxic effect of Fludarabine. The in vitro response to lumiliximab did not appear to be influenced by IgVH mutation status, cytogenetics or by previous therapy. To gain further insight into the downstream targets of lumiliximab in CLL, microarray analysis and pathway exploration was performed. The data revealed that lumiliximab regulates several sets of genes which are involved in chemokine signaling, cytokine/cytokine receptor interaction, oxidative stress, PI3-K and integrin signaling, complement cascade and Toll-like receptor signaling. Single exposure to lumiliximab led to down-regulation of several genes including LY9, GPR183, RGS1, HSPA6, and INPP5F and up-regulation of FN1, FAIM3 and LOX. Repeated exposure to Lumiliximab led to a significant down-regulation of TXNIP, CTSB, SODS, IFI44, IRF7, TNFSF13B, MS4A7, CCL4, CCL7, CCL8, cathepsin B, MARKS, ADAMS and FCER1G and up-regulation of SPP1, C10orf10, ANGPTL6, RBBP4 and GSTA4. In conclusion, these data demonstrate that repeated exposure to Lumiliximab is effective in priming CLL cells to undergo apoptosis through inactivation of the PI3-K/Akt pathway and render the cells more sensitive to cytotoxic compounds. The data also provide further evidence of a promising therapeutic role for lumiliximab in CLL and a rationale for lumiliximab-based drug combinations to improve treatment of this incurable disease. Disclosures: Shehata: Biogen Idec: Research Funding. Hughes:Biogen Idec: Employment. Maclaren:Biogen Idec: Employment. Jaeger:Biogen Idec: Research Funding.