A Prospective, Multi Center Phase II Study Evaluating Predictive Factors for Lenalidomide Treatment in Relapse or Refractory Chronic Lymphocytic Leukemia Patients (LE.P.RE.): Preliminary Results about the First 20 Enrolled Patients
Authors: Maffei Rossana , Martinelli Silvia , Debbia Giulia , Rigolin Gian Matteo , Rizzotto Lara , Castelli Ilaria , Bonacorsi Goretta , Bulgarelli Jenny , Fiorcari Stefania , 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 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.