Amy K. Church

Bone marrow stromal cells protect lymphoma B-cells from rituximab-induced apoptosis and targeting integrin α-4-β-1 (VLA-4) with natalizumab can overcome this resistance.

Rituximab improves the outcome of patients with non‐Hodgkin lymphoma, but does not completely eradicate residual B‐cell populations in the microenvironment of the bone marrow and lymph nodes. Adhesion to stromal cells can protect B‐cells from apoptosis induced by chemotherapy drugs [(cell adhesion‐mediated drug resistance (CAM‐DR)]. A similar mechanism of resistance to rituximab has not, to our knowledge, been described. We tested the hypothesis that the microenvironment protects malignant B‐cells from rituximab‐induced apoptosis, and that blocking these interactions with natalizumab, an antibody targeting VLA‐4 (integrin alfa‐4‐beta‐1/CD49d), can overcome this protection. VLA‐4 is an adhesion molecule constitutively expressed on malignant B‐cells and is important for pro‐survival signalling in the bone marrow and lymph node microenvironment. The human bone marrow stromal cell line HS‐5 was shown to strongly protect B‐cell lymphoma cells from rituximab cytotoxicity, suggesting the existence of a stromal cell adhesion‐mediated antibody resistance (CAM‐AR) mechanism analogous to CAM‐DR. Natalizumab decreased B‐lymphocyte adherence to fibronectin by 75–95% and partially overcame stromal protection against rituximab and cytotoxic drugs. These pre‐clinical findings suggest that the addition of stromal adhesion‐disruptive drugs to rituximab‐containing therapy could improve treatment efficacy.

BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma

Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.

Induced Resistance to Ofatumumab-Mediated Cell Clearance Mechanisms, Including Complement-Dependent Cytotoxicity, in Chronic Lymphocytic Leukemia

Ofatumumab (OFA), a human CD20-targeting mAb, kills B lymphocytes using the innate immune system including complement-dependent cytotoxicity (CDC). The efficacy of OFA in patients with chronic lymphocytic leukemia (CLL) is limited by drug resistance, which is not well characterized. To better understand mechanisms of resistance, we prospectively studied CLL cells isolated from blood samples collected before and after in vivo exposure to the initial dose of OFA therapy in 25 patients undergoing their first treatment for progressive CLL. As previously reported, OFA therapy rapidly decreased the absolute lymphocyte count, CD20 expression by CLL cells, and serum complement levels. We now show that after administration of the first dose of OFA, there was a modest rebound in the absolute lymphocyte count and serum complement levels, but substantial ongoing loss of CD20 expression by CLL cells. These post-OFA treatment CLL cells were highly resistant to OFA-mediated CDC but retained sensitivity to alemtuzumab-mediated CDC in vitro. Posttherapy serum OFA levels correlated inversely with both the amount of pretreatment circulating cell-bound CD20 and with the decrease in this value following treatment. In vitro OFA-mediated CDC did not predict clinical responses, and the patients with first-dose reactions to OFA did not have markers of increased complement activation in vivo. We propose that optimal efficacy of CD20- targeted therapy for CLL requires determining an mAb dose size and frequency that optimizes CLL killing without exceeding the capacity of the cytotoxic mechanisms and thus minimizes loss of CD20 expression in the surviving CLL cells.

Complement dependent cytotoxicity in chronic lymphocytic leukemia: ofatumumab enhances alemtuzumab complement dependent cytotoxicity and reveals cells resistant to activated complement

Abstract Complement dependent cytotoxicity (CDC) is an important mechanism of action for monoclonal antibodies (mAbs) used in the treatment of chronic lymphocytic leukemia (CLL). We hypothesized that alemtuzumab (ALM) mediated CDC would be increased by the addition of ofatumumab (OFA). CLL cells from 21 previously untreated patients with progressive disease were tested in vitro for mAb binding, complement activation and CDC. The subpopulation of CDC resistant CLL cells was examined for levels of C3b and C5b-9 binding, and expression of complement regulatory proteins. OFA significantly increased complement activation and CDC in ALM-treated CLL cells, suggesting that combining ALM and OFA could improve clinical outcome in patients with CLL. Approximately 10% of CLL cells were resistant to CDC because of lower levels of complement activation or decreased cytotoxicity of activated complement. Improvement of clinical responses will require determining the mechanisms of CDC resistance and developing methods to overcome this problem.

Anti-CD20 monoclonal antibody-dependent phagocytosis of chronic lymphocytic leukaemia cells by autologous macrophages.

Unconjugated monoclonal antibodies (mAbs) are an important component of effective combination therapies for chronic lymphocytic leukaemia (CLL). Antibody‐dependent phagocytosis (ADP) is a major mediator of mAb cytotoxicity, but there is limited knowledge of the determinants of ADP efficacy. We used macrophages derived in vitro from autologous circulating monocytes to test the effects of mAb structure and concentration, target : effector cell ratio, duration of co‐incubation and CLL cell CD20 expression on ADP. Next‐generation anti‐CD20 mAbs (ofatumumab, ublituximab, obinutuzumab, ocaratuzumab) were significantly more effective at inducing ADP compared to rituximab, but none were as effective as the anti‐CD52 mAb alemtuzumab. Ofatumumab (10 μg/ml) used as a representative next‐generation anti‐CD20 mAb achieved an ADP plateau at 3 h co‐incubation with a target : effector ratio of 10 : 1 (mean = 2·1 CLL cells/macrophage, range = 1·5–3·5). At 0·156 μg/ml (the lowest concentration tested) ofatumumab ADP was significantly higher than alemtuzumab. However, ofatumumab‐induced ADP did not increase significantly at higher mAb concentrations. We show that anti‐CD20 mAb ADP efficacy is determined by the mAb characteristics, target : effector ratio and incubation time. We suggest that preclinical evaluation of anti‐CD20 mAbs to understand the determinants of ADP could be useful in designing future combination therapies for CLL.