Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgVH mutation analysis

Abstract

Lack of immunoglobulin heavy chain genes (IgVH) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgVH mutation status, and its expression in leukemic cells correlates with unmutated IgVH. However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgVH. Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgVH mutation at a cut-off point of 15%. While positive samples (>15% positively) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgVH mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgVH mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgVH mutation status and survival, variations in sample handling and preparation may influence results. We show that IgVH mutation results, unlike ZAP-70 remain correlated with CD38 expression and β-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy. © 2006 International Society for Analytical Cytology.