ZAP-70 expression in B-cell chronic lymphocytic leukemia: Evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV_H mutations

Background: Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV_H) mutations, although a standardization of the cytometric protocol is still lacking. Methods: Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV_H mutations) by a standard three-color protocol. Identification of ZAP-70⁺ cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed. Results: While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO⁺TNK⁺ or ISO ^-TNK^-), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO⁺TNK^- profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO⁺TNK^- cases had a IgV_H gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL. Conclusion: Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV_H gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed.