A Novel Multiparameter Flow Cytometric Cytotoxicity Assay Detects p53 Deletion as the Major Cause of In Vitro Resistance to Fludarabine in B-Cell Chronic Lymphocytic Leukemia.

Abstract

Individual patients with B-CLL demonstrate variable responses to standard induction and salvage therapeutic regimens. It would be highly desirable to develop a predictable and reproducible laboratory diagnostic strategy that guides the selection of appropriate drugs and/or regimens based on the drug sensitivity and resistance profiles of leukemic cells for individual patients. As a first step towards this goal, a study was designed to investigate the differences of in vitro drug sensitivity profiles of leukemic cells with different cytogenetic abnormalities from CLL patients. CLL cells from 43 patients were incubated in vitro with four commonly used chemotherapeutic agents (fludarabine, chlorambucil, cladribine or prednisolone) individually or in combination. Multiparameter flow cytometry was utilized to determine the decrease in leukemic cell viability after drug exposure. Both fresh and cryopreserved samples were assessed and were found to be equivalent for assay, regardless of the percentage of B-CLL cells or the degree of spontaneous apoptosis. The highest in vitro resistance to fludarabine, was seen in all seven cases of B-CLL cells with deletions of p53, a cytogenetic abnormality associated with poor clinical outcome. Interestingly, in vitro response to chlorambucil and prednisolone was seen some CLL cases with p53 deletion and correlated with clinical response to these drugs. In CLL cases without p53 deletion, a marked variability in vitro drug sensitivity CLL cells was observed but no significant difference was detected among cases with normal cytogenetics (n=13), ATM deletion (n=4), trisomy 12 (n=3), or 13q deletion (n=7). Our findings provide direct evidence of cellular resistance to fludarabine in CLL associated with p53 deletion, confirming prior clinical observations. In vitro drug sensitivity assay may prove useful in guiding choices for therapy for CLL patients based on the drug sensitivity profile of leukemic cells in individuals.