No abstract

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V(H)) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta-associated protein (ZAP)-70 in V(H)-unmutated CLL cases led to its proposal as a surrogate marker for V(H) status. This study investigated the measurement of ZAP-70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP-70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP-70 expression and V(H) status. Three patients (9%) were discordant (two V(H) mutated/ZAP-70 positive, and one V(H) unmutated/ZAP-70 negative). No correlation existed between CD38 and either ZAP-70 expression or V(H) status. Measurement of ZAP-70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.

Samples from 51 chronic lymphocytic leukaemia (CLL) patients (42 typical, nine atypical) were assessed for in vitro response to fludarabine and cladribine (2-CdA) using the flow cytometric terminal deoxynucleotidyl transferase (TdT) assay. No difference was demonstrated between the in vitro response of typical and atypical CLL and previous treatment did not result in a more apoptosis resistant phenotype. The assay could not distinguish those patients who required subsequent treatment from those whose disease remained stable, and universal cross-resistance/sensitivity to the two purine analogues was demonstrated. The assay's potential for use in the rapid assessment of in vivo response to purine analogue therapy in CLL was limited; correctly predicting the clinical outcome of 10/12 patients to treatment but failing to predict progression in two p53 deficient patients. The level of bcl-2 in the clonal lymphocytes did not influence the in vitro, spontaneous or drug-induced, apoptosis.