Correlation of Flow Cytometrically Determined Expression of ZAP-70 Using Two Different Antibodies with IgVH Mutation Status and Cytogenetics in 539 Patients with Chronic Lymphocytic Leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is largely heterogeneous. It may be estimated based on clinical and laboratory findings the most important ones of which include chromosomal aberrations, the IgVH mutation status, and the expression of ZAP-70. The optimum procedure of ZAP-70 assessment including selection of antibody clone and the method of analysis still needs to be established. The validation of ZAP-70 has been performed using the course of the disease as well as using the IgVH mutation status. To further clarify the role of ZAP-70 expression in CLL we prospectively analyzed ZAP-70 expression, IgVH mutation status, and chromosome aberrations in peripheral blood and bone marrow samples of 539 patients with CLL. For the flow cytometric assessment of ZAP-70 expression the antibody clones 1E7.2 (Caltag, n=523) and SBZAP (Immunotech, n=81) have been used. IgVH mutation status has been analyzed in 407 cases and chromosome aberrations in 416 cases applying FISH with probes for del(6q), del(11q), +12, del(13q), del(17p), and t(11;14). ZAP-70 expression has been calculated as percentage of positive cells using normal T-lymphocytes as positive controls as well as using the ratio of mean fluorescence intensity (MFI) for ZAP-70 between T-lymphocytes and leukemic B-lymphocytes. Significant correlations were found between the percentages of ZAP-70 positive cells and IgVH homology (1E7.2, p 1) confirm the strong correlation between ZAP-70 expression and IgVH mutation status, 2) confirm the independence of ZAP-70 expression from chromosome aberrations, 3) argue in favor of analyzing ZAP-70 expression as MFI ratio between T-lymphocytes and leukemic B-lymphocytes, and 4) suggest that the SBZAP clone has a significant potential in the diagnostic work-up of CLL. Further studies are warranted to validate the role of the MFI ratio approach and of the SBZAP antibody in the clinical setting.