Lenalidomide and Dexamethasone Combination in Patients with Chronic Lymphocytic Leukemia (CLL) Relapsing or Resistant to Treatment (LENDEX-LLC-09): A Gene Expression Profiling Study

Background. Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which immune evasion of tumoral cells, as well as, an impaired CD4 and CD8 T-cell function have been described. Immunomodulatory drugs, such as lenalidomide, alone or in combination with other treatments are promising strategies for those patients with refractory disease. The combination of lenalidomide with dexamethasone has been investigated in multiple myeloma and has revealed as a highly efficient treatment. Nonetheless, the efficacy and mechanisms of action of this combination in CLL have not been elucidated.

Aim. To assess the effect of lenalidomide and dexamethasone combination in gene expression of CLL B cells, as well as CD4+ and CD8+ T cells from CLL patients enrolled in LENDEX-LLC-09 trial.

Methods. Four patients included in the LENDEX-LLC-09 trial (NCT01246557) were studied (2M/2F, med age 72). All presented with advanced CLL (2 B and 2 C Binet stages), and were previously treated by a minimum of two chemo-immunotherapy regimens. Peripheral blood samples were taken at the recruitment and the 7th day of the first cycle of lenalidomide (2.5mg/day) and dexamethasone (20mg/day, 4 days). Total RNA was extracted from CLL B cells (CD5+ CD19+) and T cells (CD4+ or CD8+) positively selected by immunomagnetic methods (Miltenyi Biotec). Good quality RNA (RIN>7) was hybridized to Human Gene 2.0 ST array (Affymetrix). Differences between gene expression of pretreated and treated samples were assessed for each cell type using linear models for microarrays. Genes with a |logFC|>1 were considered as potentially relevant. Functional analysis was performed using Ingenuity Pathway Analysis (IPA).

Results and discussion. The major effect in the gene expression due to treatment was observed in CD4+ T cells, which presented 290 up-regulated genes and 103 down-regulated. CLL cells showed up-regulation of 189 and down-regulation of 53 genes, while increase and decrease in the expression of 112 and 37 genes, respectively, were found in CD8+ T cells. Globally, the most important involved networks were related to cell-to-cell signaling, cellular growth and proliferation, cell death and survival, as well as inflammatory response and immune cell trafficking. Regarding CLL B cells, TNF-α was the most up-regulated gene, as previously described in lenalidomide treated B cells. Contrarily, we did not observe significant differences in genes involved in the immunologic synapse, as CD80, CD86, CD200, PD-L1, CD276 and CD270, which have been reported as key regulators in lenalidomide mechanism of action. Of note, a general increase of genes associated with binding to cells (CD68, CTLA4, ADAM28, ITGAX, LY96) was detected. In contrast to previous studies that demonstrated a growth arrest and induction of apoptosis by lenalidomide or dexamethasone in monotherapy (Baptista et al, 2012; Fecteau et al, 2014), a global inhibition of the apoptosis (up-regulation of BTK and CD79B and inhibition of SMAD7, among others) were observed when both drugs were combined. Considering CD8+ T cells gene expression, an up-regulation of genes involved in leukocyte activation and cell-to-cell binding was detected. The most remarkable changes were found in TNF-α and IFN-γ induction, as well as in ADAM28, LY96 and CD68. In contrast to CD8+ T cells, an inhibition of CD4+ T cell proliferation was observed after the combined treatment (up-regulation of VSIG4, LILRB4 and down-regulation of ICOS). This observation suggests that dexamethasone administration inhibits the CD4+ activation promoted by lenalidomide, as has been described in multiple myeloma (Hsu et al, 2011). Regarding response to treatment, two patients initially presented a complete response with positive minimal residual disease. However, all patients finally progressed after treatment and one died due to disease progression. No significant differences in gene expression patterns were found among patients.

Conclusions. Our results suggest that lenalidomide and dexamethasone combination leads to an anti-tumoral activity displayed by an activation of CD8+ T cells against the tumor, rather than an increase of apoptosis in CLL cells. More studies are needed to confirm these preliminary findings of the combined effect of lenalidomide and dexamethasone in refractory CLL patients.

Acknowledgments. This work was funded by Celgene, and supported by PI11/1621, 14SGR585 and Fundació LaCaixa.