ZAP-70 intron1 DNA methylation status: Determination by pyrosequencing in B chronic lymphocytic leukemia

ZAP-70 expression is a strong prognostic indicator in chronic lymphocytic leukemia. However, ZAP-70 quanti. cation by flow cytometry lacks sufficient standardization. Based upon the correlation between ZAP-70 expression and its gene methylation status, we have developed a quantitative pyrosequencing assay for the determination of ZAP-70 methylation adapted for routine use. Methylation in four CpG pairs (C-223, C-243, C-254, and C-267) in the first intron of ZAP-70 is associated with repression of ZAP-70. Moreover, it correlates with CD38 expression (n = 111, p<.0001), IgHv mutation status (n = 106, p<.0001), time to treatment (p<.0001), and overall survival (p=.0014). Pyrosequencing of ZAP-70 provides a good alternative to flow cytometry. (C) 2009 Elsevier Ltd. All rights reserved.