Combined positive/negative selection for highly effective purging of PBPC grafts: towards clinical application in patients with B-CLL

Abstract

Autologous PBPC transplantation is a potentially curative treatment for patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have studied double purging of PBPC using immunomagnetic CD34⁺ cell selection (Isolex 300i) followed by negative depletion with anti-CD19/20/23/37-labeled immunomagnetic beads. In four small-scale experiments using PBPC from patients with CLL or leukemic low-grade lymphoma, double purging resulted in CD34⁺ enrichment from 0.9–4.4% to 95.8–99.4%. Lymphoma cells were always undetectable by FACS and PCR (CDR3 or t(14;18)) after negative depletion. Next, seven heavily contaminated full grafts from five patients with CLL or lymphoplasmocytoid immunocytoma were subjected to the double purging procedure, resulting in a CD34⁺ enrichment from 1.6% (0.7–5.6) to 98.5% (96–99.8). The CD34⁺ yield after double purging was 1.3–6.3 × 10⁶/kg, according to a median recovery of 32%. The overall reduction of lymphoma cells was 5.6 (>4.6–6) log. Although CLL cells were completely absent after purging in five cases as assessed by FACS, all grafts remained PCR positive. The first two patients have been reinfused with double selected products after myeloablative radiochemotherapy and showed prompt and uneventful hematopoietic engraftment. We conclude that without significant loss of CD34⁺ cells, negative depletion adds 2 log of CLL cell depletion to CD34⁺ selection, resulting in an overall purging efficacy of more than 5 log. This combination of positive and negative selection can be successfully applied even to heavily contaminated PBPC grafts.