In-Vitro Treatment of Chronic Lymphocytic Leukemia (CLL) Cells with Fludarabine, Etoposide and Alemtuzumab (Campath-1H) Reveals Different Rates and Mechanisms of Cell Death.
Authors: Winkler Dirk , Schneider Christof , Nitsch Daniela , Habermann Annett , Doehner Hartmut , Stilgenbauer Stephan 
Published: October 2019

Abstract
Chemotherapeutic agents such as fludarabine, etoposide and the monoclonal anti-CD52 antibody alemtuzumab induce cell death and clinical responses in CLL. However, the mechanisms by which these processes occur are not well understood. In an effort to gain better insight into these mechanisms CLL cells from 21 patients were collected and individually treated with fludarabine 500μM (n=19) and etoposide 60 μM (n=19) for 24 and 48 hours respectively, and 24 hours with alemtuzumab 10 μg/ml ± cross-linking F(ab’)2 fragments. Each sample treated with alemtuzumab was also cultured with and without serum as a source of complement. Enrichment for B-cells was also done in 4 cases using negative selection with anti-CD2 and anti-CD14 magnetic beads. Of 18 cases investigated 10 were unmutated VH and 6 of 19 had del11q and/or del17p. A FACS analysis with double staining for Annexin V/7AAD was used to measure rates of cell death and caspase-3 activation. Results: treatment with fludarabine and etoposide induced apoptosis in all cases. However, rates of apoptosis decreased in cells from patients with genetically high-risk CLL, and these cells showed higher caspase-3 activation in response to fludarabine. Response to alemtuzumab was significantly dependent on presence of serum in the culture: 8% Annexin-V/7AAD-positive cells in serum-free cultures vs 53% in cultures with serum. However, addition of F(ab’)2 fragments increased the percentage of Annexin-V/7AAD-positive cells even in serum-free cultures: 61% with serum vs 25% without serum. Response to alemtuzumab was found to be independent of the genetic subgroup of the case. Notably, treatment with alemtuzumab in serum cultures did not produce cells that stained Annexin-positive/7AAD-negative, a typical feature of early apoptosis, whereas treatment with fludarabine, etoposide and alemtuzumab in serum-free medium resulted in a significant number of Annexin-positive/7AAD-negative cells. This was also observed in CD19+ purified cultures. In the presence of serum alemtuzumab did not induce caspase-3 activation, neither did the addition of F(ab’)2 fragments. However, in 5 of 20 serum-free cell cultures, all of had unmutated VH, active caspase-3 was clearly detectable after alemtuzumab treatment, and caspase-3 activity was further up-regulated when F(ab’)2 fragments were also added.
Summary: in CLL cells mechanism and rate of cell death dramatically differed depending on in-vitro treatment with fludarabine, etoposide and alemtuzumab, and differed between genetic subgroups. CLL cells from high-risk patients were more capable of caspase-3 activation when treated with fludarabine or alemtuzumab. Alemtuzumab killed CLL cells effectively and independently of serum as a source of complement, but the mechanism of response was different when serum was added. In serum-free CLL cultures, alemtuzumab induced apoptosis with activation of caspase-3, and addition of cross-linking F(ab’)2 fragments increased the rate apoptosis, whereas in the presence of serum treatment with alemtuzumab induced no typical features of apoptosis, even in B-cell enriched cultures. These findings favor CDC rather than apoptosis or ADCC as the major cell kill mechanism activated by in vivo alemtuzumab.
mean % of cells AnnexinV+/7AAD+ caspase-3 activation etoposide (48 hrs) fludarabine (48 hrs) fludarabine (48 hrs) IgVH unmutated 33% 24% 32% IgVH mutated 74% 38% 20% del 11q/del 17p 39% 25% 37% del 13q/normal karyotype 61% 32% 21%