Single-agent lenalidomide in the treatment of previously untreated chronic lymphocytic leukemia

Abstract

Purpose: Lenalidomide is an oral immunomodulatory drug with multiple effects on the immune system and tumor cell microenvironment leading to inhibition of malignant cell growth. Based on encouraging reports of lenalidomide in relapsed and refractory chronic lymphocytic leukemia (CLL), we investigated the first-line use of single-agent lenalidomide in CLL.

Patients and methods: Using a starting dose of lenalidomide 10 mg/d for 21 days of a 28-day cycle and weekly 5-mg dose escalations to a target of 25 mg, we encountered severe toxicities (tumor lysis, fatal sepsis) in the first two patients enrolled. The study was halted and the protocol amended to a more conservative regimen: starting dose of lenalidomide 2.5 mg with monthly escalations to a target dose of 10 mg, and extended tumor lysis prophylaxis and monitoring. Gene expression profiles from patient samples before and after 7 days of lenalidomide were performed.

Results: Twenty-five patients were enrolled on the amended protocol. No further tumor lysis events were reported. Tumor flare was common (88%) but mild. Grade 3 to 4 neutropenia occurred in 72% of patients, with only five episodes of febrile neutropenia. The overall response rate was 56% (no complete responses). Although rapid peripheral lymphocyte reductions were observed, rebound lymphocytoses during the week off-therapy were common. Lenalidomide-induced molecular changes enriched for cytoskeletal and immune-related genes were identified.

Conclusion: Lenalidomide is clinically active as first-line CLL therapy and is well-tolerated if a conservative approach with slow dose escalation is used. A lenalidomide-induced molecular signature provides insights into its immunomodulatory mechanisms of action in CLL.

Fig 1.

Proportion of cycles affected by grade 3 to 4 cytopenias. Of a total of 470 cycles administered to all patients, 14.7% were complicated by grade 3 to 4 neutropenia, 3.2% grade 3 to 4 thrombocytopenia, and 2.3% grade 3 to 4 anemia. For each grade 3 to 4 cytopenia (represented by each bar), the proportion occurring at each dose level is depicted by the different colors. The majority of grade 3 to 4 cytopenias occurred at low doses 2.5 to 5 mg daily. ANC, absolute neutrophil count.

Fig 2.

Cyclic rebound of peripheral lymphocytosis. Although significant reductions in absolute peripheral lymphocyte counts can be seen by day 15 of each cycle, a rebound increase in lymphocytes can be seen in some patients before start of next cycle. This figure demonstrates this rebound in eight representative patients.

Fig 3.

Kaplan-Meier survival curves. (A) Progression-free survival in months. (B) Overall survival in months.

Fig 4.

Lenalidomide-induced molecular changes. Molecular responses were evaluated on days 1 (before initiation of therapy) and 8 (after lenalidomide dosing) for cycles one and two. A volcano plot identified 42 probe sets with a greater than two-fold difference in expression at day 8 with a P lower than .05 using Benjamini and Hochberg false detection rate multiple test correction. The heatmap shows the change in gene expression (red three-fold up, and blue three-fold down) of genes at day 8 compared to day 1 in 22 patients (pooling the data from cycle one and two for each patient). Twenty-six probe sets were upregulated and sixteen probe sets were downregulated. In general, we observed that the same direction of lenalidomide-induced changes in gene expression (down or upregulation) was found in the majority of patients.

Fig A1.

Lenalidomide enhances tumor necrosis factor alpha (TNF-α) production in activated chronic lymphocytic leukemia (CLL) cells. Pretreatment-derived CLL cells were cocultured on bone marrow stroma in the presence of 0.5 μmol/L lenalidomide or stimulated with 100 U/mL interferon-alfa (IFN-α) and 50 U/mL recombinant human-interleukin-2 ± 0.5 μmol/L lenalidomide for 24 to 96. The cells were then analyzed for cell surface expression of CD19, and intracellular TNF-α Brefeldin A (10 μg/mL) was added to the last 6 hours of culture to allow for intracellular accumulation of protein. Shown is a representative experiment demonstrating higher than threefold induction of TNF-α in interleukin-2/IFN-α–induced cells that are costimulated with lenalidomide for 96 hours.

Fig A2.

Lenalidomide induces cell surface expression of lymphocyte activation gene-3 (LAG3) in chronic lymphocytic leukemia (CLL). Dot blots from three representative patients show LAG3 expression increase on CD19 cells from day 8 compared to day 1 of cycle 2. Cells were thawed and labeled with goat anti-LAG-3 (R&D Systems, Minneapolis, MN) or isotype control, anti-CD19-fluorescein isothiocyanate (Beckman Coulter, Mississauga, Ontario, Canada), 7AAD (Sigman, Oakville, Ontario, Canada) followed by phycoerythrin-conjugated antigoat secondary antibody. Flow cytometry was performed using a FACSCaliber flow cytometer (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Treestar, Ashland, OR).

Fig A3.

Lenalidomide increases cell surface expression of tropomyosin 2 in chronic lymphocytic leukemia (CLL). Immunofluorescence confirms increased expression of tropomyosin in day 2 (C2D8) treated CD19+ cells compared to day 1 (C2D1) of cycle two in a representative patient experiment. Increased expression on day 8 compared with day 1 detected by immunofluorescence was observed in five of eight samples tested. Cytospin slides of fresh purified CD19⁺ cells were permeabilized with 0.5% saponin (Sigma, Oakville, Ontario, Canada) fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and stained with a 1:200 dilution of antitropomyosin B (Santa Cruz Biotechnology, Santa Cruz, CA), then 1:500 dilution of secondary conjugated to Alexa Fluor 488 (Invitrogen, Burlington, Ontario, Canada), following which 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Confocal slices of 2 μmol/L were collected. The slides were again washed and DAPI was applied. Slides were visualized on a Zeiss LSM510 microscope using a 40× objective (Carl Zeiss, Thornwood, NY).