In Vitro and in Vivo Evidence of an Anti-Angiogenic Effect of Lenalidomide in Chronic Lymphocytic Leukemia
Authors: Maffei Rossana , Bulgarelli Jenny , Rizzotto Lara , Martinelli Silvia , Rigolin Gian Matteo , Fiorcari Stefania , Martinelli Sara , Debbia Giulia , Castelli Ilaria , Bonacorsi Goretta , Zucchini Patrizia , Santachiara Rita , Forconi Francesco , Rossi Davide , Laurenti Luca , Palumbo Giuseppe A , Vallisa Daniele , Cuneo Antonio , Gaidano Gianluca , Luppi Mario , Marasca Roberto 
Published: October 2019

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.