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

Introduction: In B-CLL, somatic mutation of IgV_H genes defines a group of patients with favorable prognosis, whereas the absence of IgV_H 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 IgV_H 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 IgV_H 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 mutations

Conclusion: Our data show that Btz and Rtx have in vitro activity on B-CLL cells, being used alone or in combination. Interesting, in IgV_H 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