Resistance to complement activation, cell membrane hypersialylation and relapses in chronic lymphocytic leukemia patients treated with rituximab and chemotherapy.

Anne Bordron,Boutahar Bendaoud,Jacques-Olivier Pers,Caroline Buors,Hussam Saad,Christian Berthou,Florence Dalbies,Yves Renaudineau,Audrey Mohr,Adrian Tempescul,Stéphanie Deshayes,Cristina Bagacean

doi:10.18632/oncotarget.25657

Abstract

The anti-CD20-specific monoclonal antibody rituximab (RTX), in combination with chemotherapy, is commonly used for primary treatment in chronic lymphocytic leukemia (CLL). However, relapses remain important and activation of the complement pathway is one of the mechanisms by which RTX generates the destruction of B cells directly by complement-dependent cytotoxicity (CDC), or indirectly by antibody-dependent cellular phagocytosis. In this study, the RTX capacity to induce CDC was established in 69 untreated CLL patients, this cohort including 34 patients tested before the initiation of RTX-chemotherapy. In vitro CDC-resistance to RTX predicts lower response rates to RTX-chemotherapy and shorter treatment free survival. Furthermore, the predictive value of CDC-resistance was independent from the clinical, cytogenetic and FcγR3A V158F polymorphism status. In contrast, CLL cell resistance to CDC predominates in IGHV unmutated patients and was related to an important α2-6 sialyl transferase activity, which in turn increases cell surface α2-6 hypersialylation. Suspected factors associated with resistance to CDC (CD20, CD55, CD59, factor H, GM1, and sphingomyelin) were not differentially expressed or recruited between the two CLL groups. Altogether, results provide evidence that testing RTX capacity to induce CDC in vitro represents an independent predictive factor of therapeutic effects of RTX, and that α2-6 hypersialylation in CLL cells controls RTX response through the control of the complement pathway. At a time when CLL therapy is moving towards chemo-free treatments, further experiments are required to determine whether performing an initial in vitro assay to appreciate CLL CDC resistance might be useful to select patients.

Highlights

Rituximab (RTX), a monoclonal antibody directed against the B cell molecule CD20, was the first mAb to receive approval to be used in non-Hodgkin’s lymphomas (NHL), and later in chronic lymphocytic leukemia (CLL) [1]
The assay to test complement mediated killing of CLL cells sensitized by the anti-CD20 mAb RTX (10μg/ml) at 37°C was optimized in a preliminary step and, as shown in Figure 1A, complement-dependent cytotoxicity (CDC) induced by RTX was effective after 1h when using the human B cell line Ramos (n=3) but such an effect was delayed to 24h when using CLL cells (n=8) and B cells from healthy controls (n=3)
In order to differentiate CDC-normal from CDC-resistant CLL cells, the 24h time point was selected and a cut-off value of 6% was established by considering the mean of decrease in CLL survival (21.2±7.6%) minus 2 standard deviations of normal B cells from 21 healthy controls (Figure 1C)

Summary

Introduction

Rituximab (RTX), a monoclonal antibody (mAb) directed against the B cell molecule CD20, was the first mAb to receive approval to be used in non-Hodgkin’s lymphomas (NHL), and later in chronic lymphocytic leukemia (CLL) [1]. ADCC is suspected to be the main mechanism of action with regards to RTX therapeutic efficacy and clinical outcomes [15,16,17], several authors suggested that activation of the complement pathway is involved in the clinical response to RTX [18,19,20,21]. To further support the importance of the complement pathway, it was established that RTX therapeutic activity was strongly enhanced in CLL patients resistant to RTX after concurrent administration of fresh frozen plasma [22]. Complement deficiencies are reported in CLL patients and suspected of limiting RTX efficacy in vivo [23]

Methods

Results

Conclusion