In Vivo Purging with Rituximab Prior to Collection of Stem Cells for Autologous Transplantation in Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) cells express the CD20 antigen, and monoclonal antibodies against CD20 have resulted in remissions. We hypothezised that the anti-CD20 antibody rituximab (Rituxan™) may be useful in reducing the number of contaminating CLL cells in stem cell collections for use in autologous transplantation. A pilot study in 5 patients was designed using rituximab 375 mg/m² as an in vivo purging step following cyclophosphamide 4 gm/m² and granulocyte colony-stimulating factor/granulocyte-macrophage colony-stimulating factor (G-CSF/GM-CSF) mobilization therapy for patients with advanced-stage CLL undergoing autologous stem cell transplantation. Eligible patients had 0-30% marrow involvement prior to mobilization. A single pre-rituximab leukapheresis product was obtained after the white blood cells (WBC) reached 800/mm³ to serve as a control but was not reinfused. Rituximab was administered the following day and subsequent leukaphereses were commenced 48 h later to reach a total of >2 × 10⁶ CD34⁺ cells/kg. Dual-color flow cytometry CD5/CD19 and consensus PCR using primers to the joining region and FR3 of the variable region of the immunoglobulin heavy chain (IgH) were used to evaluate the degree of contaminating CLL cells in the leukapheresis product and to monitor disease status post transplant. All 5 patients were informative for the consensus PCR assay. Four of 5 patients mobilized >2 × 10⁶ CD34⁺ cells/kg and proceeded to cyclophosphamide 120 mg/kg and total body irradiation (6 × 200 cGy) with stem cell rescue. All leukaphereses products were positive by PCR for the IgH rearrangement and 4/5 contained CD5/CD19 dual-positive cells. Comparing the pre- and post-rituximab leukapheresis products, a reduction in the percentage of CD5⁺/CD19⁺ cells was seen in 4/5 patients. All patients engrafted at a median of 13.5 days to ANC > 500/mm³ and 11 days to platelets>20,000/mm³. No regimen-related mortality was seen. Although 2 patients tested positive on PCR for the IgH rearrangement early after transplant, all patients had absence of the IgH gene rearrangement at 1 year and no CD5/CD19 dual-positive cells were could be detected in the bone marrow. This includes 1 heavily pretreated patient who received stem cells containing up to 30% CD5⁺/CD19⁺ cells. We conclude that purging with Rituximab 48 h prior to stem cell collection was able to reduce significantly (but not eliminate) the percentage of CLL cells in the leukaphereses. However, despite the infusion of CD5⁺/CD19⁺ cells in the stem cell coions, patients were able to obtain durable complete molecular remissions, implying that the PCR-positive cells in the leukaphereses may not have long-term clonogenic potential. The results also support the recommendation to test if rituximab should be part of a maintenance regimen after transplant to prevent disease recurrence in high-risk patients.