Abstract: Abstract 1782 The aims of LE.P.RE. study include the identification of clinical and biological factors associated with clinical response and toxicity of lenalidomide monotherapy in relapsed/refractory CLL patients enrolled from 9 Italian centers. Lenalidomide treatment schedule starts with 5 mg daily and increases of 5 mg daily every two weeks, up to 25 mg daily or maximum tolerated dose. Therapy is scheduled to be administered for 12 courses (1 course = 4 weeks) unless disease progression or excessive toxicity are observed. Here we show preliminary results about the first 20 enrolled patients up to the 4th treatment course. Patients received a mean daily lenalidomide dose of 8 mg in the 1st course, 10 mg in the 2nd, 14 mg in the 3rd and 4th. Four patients left the study at the 1st course (1 acute renal failure ARF, 1 infection, 1 second neoplasia likely related to CLL, 1 consent withdrawal) and 3 patients at the 3rd (1 Tumor Flare Reaction TFR, 1 infection, 1 thrombocytopenia). The observed toxicities are listed in Table 1. After the 4th course, 13 patients were evaluable for response: 9 partial response (PR), 3 progressive disease (PD) and 1 stable disease (SD) [ORR 69%]. Table 1 Hematological and extra hematological toxicities (n° tot cases=20) grade n° cases grade n° cases Thrombocytopenia 03/04/11 7 TLS 2 1 Neutropenia 03/04/11 14 TFR 1-2 4 Anemia 03/04/11 2 Second Neoplasia 1 Death 1 ARF 02/03/11 3 Infection 03/04/11 3 We compared the levels of several cytokines measured by ELISA in plasma of the 20 patients at baseline and day+8 of therapy. We observed a significant increase of IL2 Receptor (mean 55,31 vs 112,14 ng/ml; p 〈 0,001), IL2 (14,15 vs 17,27 pg/ml; p=0,019), CCL3 (5,21 vs 21,23 pg/ml; p 〈 0,001), CCL4 (24,76 vs 72,99 pg/ml; p=0,003), IL10 (2,65 vs 6,33 pg/ml; p=0,001), IL1b (0,94 vs 2,77 pg/ml; p=0,048), TNFa (35,00 vs 140,59 pg/ml; p 〈 0,001) and IL8 (0,31 vs 3,50 pg/ml; p=0,037) and a decrease of Thrombospondin 1 (693 vs 488 ng/ml; p=0,037). Interestingly, we found that IL1b level decreased from baseline to day+8 in the 4 non responder (PD+SD) patients while increasing in the 9 responder (PR) patients. Moreover, we found that the 5 patients that experienced TFR or tumor lysis syndrome TLS had significantly higher CCL3 level at baseline than the other 15 patients (p=0,025). We also studied peripheral blood cell subsets (T, B, NK, monocyte, dendritic and endothelial cells) in the 20 patients by flow cytometry. From baseline to day+8 we observed a significant increase of the Thelper1/Thelper2 ratio (p 〈 0,001), T cytotoxic1/Tcytotoxic2 ratio (p=0,001) and memory T cells % (p 〈 0,001) as well as a decrease of naïve T cells % (p 〈 0,001) and mean CD69 expression on T cells (p=0,016). Moreover, the expression of CD40 (p=0,001), CD80 (p=0,018), CD86 (p=0,003) and CD95 (p=0,008) were found to be increased on B-CLL cells. Finally, we observed a decrease of endothelial progenitors cells (EPC) (p=0,032) and live circulating endothelial cells (CEC) (p 〈 0,001) and an increase of dead CEC (p 〈 0,001). Interestingly, there was a significant difference in activated CEC (mean 53,57 vs 81,63 CEC %; p=0,031) and resting CEC (46,54 vs 18,37; p=0,031) at baseline between responders and non responders, respectively. Moreover, the patients exhibiting TFR or TLS showed a higher % of CD4+CD3+ cells (p=0,009) and CD4+CD8+ cells (p=0,036) at day+8 than the others. In conclusion: (i) the increase of inflammatory cytokines IL2R, IL2, CCL3, CCL4, IL10, IL1b and TNFa observed from baseline to day+8 suggests that lenalidomide can induce immune activation; (ii) the augmentation of IL2, IL2R and memory T cells and the decrease of naïve T cells noticed from baseline to day+8 indicate that lenalidomide can promote T cell activation; (iii) the shift toward Thelper1 and Tcytotoxic1 phenotypes and the increased expression of co-stimulatory molecules on B-CLL cells observed from baseline to day+8 suggest that lenalidomide can promote an active T cell response against leukemic cells; (iv) the alterations in EPC and CEC noticed from baseline to day+8 suggest that lenalidomide may also have an anti angiogenic action. Moreover, our preliminary data seem to show interesting biological differences among CLL patients that respond or do not respond to lenalidomide treatment, which if replicated in additional patients and with increasing time on therapy could give important information for predicting which patients may best respond to therapy or may experience TFR or TLS. Disclosures: Maffei: CELGENE CORPORATION: Research Funding. Off Label Use: Lenalidomide, a thalidomide analogue, is an immunomodulatory drug (IMiD) with antitumoural activity reported in various malignant disorders including multiple myeloma and myelodysplastic syndrome. At preclinical level, lenalidomide has shown to decrease the production of several prosurvival cytokines. This drug is also reported to modulate an effector cell immune response through the activation of T and natural killer cells, inducing apoptosis directly on tumour cells. Currently available data indicate that lenalidomide is active also in heavily pre-treated CLL patients. However, in order to reduce toxicity and to optimize the therapeutic index of lenalidomide treatment in CLL patients, it is necessary to identify features of tumour cells that differ between responder and non responder patients. Hence, we propose a multicenter, phase II study designed in order to identify potential predictive factors correlating with response and toxicity to Lenalidomide treatment in relapsed/refractory CLL patients. Martinelli:CELGENE CORPORATION: Research Funding. Debbia:CELGENE CORPORATION: Research Funding. Rigolin:CELGENE CORPORATION: Research Funding. Rizzotto:CELGENE CORPORATION: Research Funding. Castelli:CELGENE CORPORATION: Research Funding. Bonacorsi:CELGENE CORPORATION: Research Funding. Bulgarelli:CELGENE CORPORATION: Research Funding. Fiorcari:CELGENE CORPORATION: Research Funding. Zucchini:CELGENE CORPORATION: Research Funding. Santachiara:CELGENE CORPORATION: Research Funding. Forconi:CELGENE CORPORATION: Research Funding. Rossi:CELGENE CORPORATION: Research Funding. Laurenti:CELGENE CORPORATION: Research Funding. Palumbo:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Cuneo:CELGENE CORPORATION: Research Funding. Gaidano:CELGENE CORPORATION: Research Funding. Luppi:CELGENE CORPORATION: Research Funding. Marasca:CELGENE CORPORATION: Research Funding.

Abstract: Abstract 1782 The exact mechanism of anti-tumor activity of the immunomodulatory drug lenalidomide remains undefined, but it implies the activation of immune effector cells. Little is known concerning the anti-angiogenic properties of lenalidomide in chronic lymphocytic leukemia (CLL) patients. A total of 27 relapsed/refractory CLL patients were treated within a multicenter Phase II trial. Peripheral blood mononuclear cells and plasma samples were collected before and during treatment. Lenalidomide treatment schedule began with 5 mg daily doses and increased by 5 mg daily every two weeks, up to 25 mg daily or maximum tolerated dose. Several angiogenesis-related cytokines were measured by ELISA in plasma. Circulating endothelial cells (CEC), endothelial progenitor cells (EPC), apoptotic CEC (APO-CEC), and activated CEC (aCEC) were evaluated by flow cytometry. In vitro experiments were performed on CLL cells purified from peripheral blood by using CD19Microbeads. Cells were cultured alone (CLL only) or on a EC monolayer (HC condition). First, CLL cells from 9 patients treated in vitro with up to 10 μmol/L of lenalidomide did not show any direct cytotoxic effect. However, an anti-leukemic effect was observed in vivo in our cohort of heavily pre-treated CLL patients. We found a reduction of white blood cell count (WBC, 1×109/L) from 39.9±8.0 before treatment to 15.2±5.2 and 16.0±5.2 after 4 and 12 months of treatment respectively and a decrement of absolute number of lymphocytes from 25.6±6.4 before treatment to 7.9±2.7 and 5.4±2.4 after 4 and 12 months of treatment (p 〈 0.05, Wilcoxon test). To evaluate whether lenalidomide exerts its anti-CLL effect disrupting leukemic cross-talk with endothelial cells, we co-cultured CLL cells (n=9) on EC layer in presence or absence of increasing doses of lenalidomide. All CLL cells cultured on EC layer displayed a significant survival advantage (mean viability, 16% vs. 47% in CLL alone and HC) that was reduced by lenalidomide treatment (28%) (p 〈 0.001). We then evaluated the effect of lenalidomide treatment on circulating endothelial cells in treated CLL patients (n=27). We found that the absolute number of CEC and EPC decreased from 8.2±1.5/μL and 4.7±1.0/μL pre-therapy to 2.1±0.6/μL and 1.0±0.5/μL after 4 months respectively (p=0.002 for CEC and p=0.01 for EPC). Moreover, the percentage of APO-CEC increased from 51% to 84% of the total CEC (p 〈 0.0001). No differences were found in the aCEC subset during treatment. Moreover, we examined the effect of lenalidomide treatment on a panel of angiogenesis-related factors. The plasma levels of vascular endothelial growth factor (VEGF) and thrombospondin-1 (THBS1) showed a significant decrease by month 4 compared to pre-treatment levels (VEGF, 77±13 to 43±8 pg/mL, p=0.014; THBS1, 662±80 to 416±75 ng/mL, p=0.01). Angiogenin plasma levels increased after 4 months of treatment from 441±16 ng/mL to 460±21 ng/mL (p=0.025). No significant differences were detected in plasma levels of basic fibroblast growth factor (bFGF,) and Angiopoietin-1 (Ang1). Angiopoietin-2 (Ang2) showed an increase during the first phase of treatment from 5183 to 8126 pg/mL (p=0.001). Furthermore, patients were grouped under the clinical classification of stable/progressive disease (not responders, NR n=6) and partial response (responders, R n=10). There was an Increased percentage of aCEC detected in NR patients (54% vs. 74% in R and NR at baseline respectively, p=0.039). We also found that VEGF plasma levels significantly decreased during treatment exclusively in the R subset (83±26 pg/mL at baseline to 26±6 pg/mL after 4 months, p=0.007). Moreover, bFGF plasma levels showed a strong decrease in R patients from 168±105 pg/mL at baseline to 41±11 and 19±8 pg/mL after one week and 4 months of treatment (p=0.017 and p=0.005 respectively), whereas increased levels were measured in NR patients from 120±58 pg/mL at baseline to 426±195 and 234±147 pg/mL. Our study provides evidence that the mechanism of action of lenalidomide in CLL patients is mediated not only through the activation of immune effector cells, but also through the modulation of angiogenesis-related factors and disruption of CLL cross-talk with endothelial cells. Of interest, we identified plasmatic factors (VEGF and bFGF) that are correlated with clinical response to lenalidomide. Disclosures: Off Label Use: Lenalidomide, a thalidomide analogue, is an immunomodulatory drug (IMiD) with antitumoural activity reported in various malignant disorders including multiple myeloma and myelodysplastic syndrome (MDS). At preclinical level, lenalidomide has shown to decrease the production of several prosurvival cytokines. This drug is also reported to modulate an effector cell immune response through the activation of T and natural killer (NK) cells, inducing apoptosis directly on tumour cells. Currently available data indicate that Lenalidomide is active also in heavily pre-treated CLL patients with unfavourable prognostic factors. However, in order to optimize the therapeutic index of Lenalidomide treatment in CLL patients, it will be necessary to identify features of tumour cells that differ between responder and non responder patients. Hence, we reported results from a multicenter, phase II study designed in order to identify potential predictive factors correlating with response to Lenalidomide treatment in relapsed/refractory CLL. Marasca:CELGENE CORPORATION: Honoraria, Research Funding.