Thalidomide Alone and in Combination with Fludarabine Exerts Distinct Molecular and Antileukemic Effects in B-Cell Chronic Lymphocytic Leukemia.
Authors: Giannopoulos Krzysztof , Dmoszynska Anna , Kowal Malgorzata , Wasik-Szczepanek Ewa , Wlasiuk Paulina , Bojarska-Junak Agnieszka , Rolinski Jacek , Stilgenbauer Stephan , Bullinger Lars 
Published: October 2019

Abstract
Background: By targeting the tumor microenvironment and inhibiting angiogenesis chronic lymphocytic leukemia (CLL) might be more effectively treated. Recently, thalidomide (THAL) has been shown a promising immunomodulatory compound to achieve this goal. Therefore THAL based treatment might represent a novel therapeutic strategy for patients (pts) with CLL, and addition of fludarabine (FLU) might enhance anti-leukemic effect of THAL. We aimed to treat CLL pts with THAL and FLU and monitor effects of THAL treatment in vivo.
Patients and methods: Thirty eight pts (females=23, males=15) were enrolled (median age 67, range 43–75 years). Eighteen pts received THAL+FLU as first line and 20 as second or third line therapy. Pts received THAL (100mg po/day) starting at day 0 (d0). FLU was added for 5 consecutive days every 28 days (25 mg/m2 iv/day) starting at day 7 (d7) for up to 6 cycles. Control group consisted of 20 healthy volunteers (HVs) matched by age. To evaluate the influence of THAL on gene expression profile (GEP) 13 paired d0 and d7 samples were analyzed using U133plus2.0 microarrays. TNF plasma level were measured using a high sensitivity ELISA test on d0, d7 and d12. Tregs were identified as CD4+CD25highFOXP3+ by FACS analysis on d0, d7 and d12. Clinical response was evaluated in pts who completed at least 3 cycles of THAL + FLU therapy according to NCI criteria.
Results: Paired supervised analysis of GEP data based on sample comparison before and during THAL treatment revealed a THAL induced signature comprising 66 differentially expressed genes (p<0.001). Gene set enrichment analyses showed 19 deregulated pathways, including the apoptosis and FAS signaling pathway involving expression changes of FAS, FADD, caspase 8 and caspase 3 (p<0.005). Furthermore, we observed a significant association with CD40L signaling which has been previously reported to play a role in immunomodulatory drug action. The frequency of Tregs was significantly higher in CLL pts when compared to HVs (12.1 vs 1.9%, P<0.001). After 7 days of THAL therapy significant reduction of Tregs and leukemic lumphocytes was observed. The addition of FLU did not result in further descreasing of Tregs. In contrast leukemic cells decreased significantly on d12 comparing to d0 and d7. THAL treatment resulted in a mean reduction TNF plasma level of 47% within the first 7 days, while addition of FLU did not result in a further reduction. All pts receiving THAL+FLU as first line therapy responded (4CR+14PR), while in pts who were previously treated response rate was about 44% and no CR was noted. Four pts were excluded due to serious infectious complications (grade 3–4) resulting in one death (all pts. were heavily pretreated). Six pts progressed during therapy. The most common side effects were: a flare reaction observed during the first cycle in 9 pts (24%), neutropenia (24%), thrombocytopenia (8%) and AIHA (10%).
Conclusions: Our study provides novel biological insights into the molecular effects of THAL, which might act (i) by enhancing apoptosis of CLL cells, and (ii) by reducing Tregs thereby enabling T-cell dependent tumor rejection. Furthermore, combined THAL+FLU therapy demonstrated efficacy in CLL pts not only as a first line treatment, but also as salvage therapy with acceptable safety profile.