Treatment of B-CLL Cells with Bortezomib and Rituximab Reduces Cell Viability In Vitro..

Abstract

Introduction: In B-CLL, somatic mutation of IgVH genes defines a group of patients with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Our previous data suggested that BCL-6 mutations identify a subgroup of patients with high risk of progression despite the presence of mutated IgVH gene, but the clinical significance of this molecular alteration remains uncertain. New approaches are now being tested for the treatment of B-CLL. Proteasome inhibitor, Bortezomib (Btz), and monoclonal antibodies specific for surface antigens, Rituximab (Rtx), represent potential therapeutic strategy.Objetives: To study the effects of Btz and Rtx on viability of CLL cells in culture, and to correlate the responses to the mutational status of the samples.Material and Methods: PBMC from 29 B-CLL patients (Binet stage A) were isolated by Ficoll-Hypaque gradient centrifugation. Samples were culture in RPMI supplemented with 20% FCS. Complement rabbit serum was added to the cultures in Rtx treated cells. Cells were exposed to various concentrations of Btz (0.1, 1 and 10 uM), Rtx (10 ug/ml) or both together during 24 hours. Then, cells were stained with Propidium Iodide and subjected to flow cytometry analysis.Results: Btz and Rtx reduce viability of B-CLL cells in a dose and time dependent manner. The kinetic of both drugs were different, whereas Rtx reaches its maximun effect within 3 hours after treatment, Btz do the same after 48 hours. Combined treatment (Btz + Rtx) increases the effect of each one separately. All differences between treatments with increased doses of Btz + Rtx vs Btz alone were statistically significant. In the group of IgVH mutated patients, there were a significant difference in the response to Btz and Rtx in BCL-6 mutated (mut) and unmutated (unmut) groups (n= 10 and 8 respectively), whereas no significant differences were observed in the number of lymphocytes, lymphocyte doubling time, CD38 expression and cytogenetic alterations.Effects of Btz and Rtx on IgVH mutated samples: correlation with BCL-6 mutationsBCL-6Mean viability (%)SDp *Controlunmut82.138.700.42mut86.366.01Btz 0.1 uMunmut49.0510.390.006mut64.2812.16Btz 1 uMunmut39.867.190.040mut53.2713.35Btz 10 uMunmut30.217.410.041mut43.9914.73Rtx 10 ug/mlunmut48.3114.510.050mut34.6212.51Btz 0.1 uM + Rtxunmut32.9614.150.153mut24.5010.01Btz 1 uM + Rtxunmut25.9910.880.315mut19.897.53Btz 10 uM + Rtxunmut19.279.040.491mut16.317.52* U. Mann Whitney testConclusion: Our data show that Btz and Rtx have in vitro activity on B-CLL cells, being used alone or in combination. Interesting, in IgVH mutated cells, Btz and Rtx have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status, but the biological significance of this differential responses remains unclear