ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia

Objective: The mutational status of the immunoglobulin (Ig) V-H gene in B-cell chronic lymphocytic leukaemia (B-CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP-70 expression with IgV(H) mutational status in B-CLL by quantifying ZAP-70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) assay for the detection of ZAP-70 expression in a group of patients whose mutational status and cytogenetics had been determined previously. Methods: RQ-PCR was used to analyse ZAP-70 expression from 42 B-CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ-PCR was performed using Taqman PCR. Results: Twenty-five patients (60%) had mutated and 17 (40%) had unmutated IgV(H) genes; 94% (16/17) of patients with unmutated IgV(H) gene were ZAP-70 positive as assessed by RQ-PCR and 92% (23/25) of patients with mutated IgV(H) gene were ZAP-70 negative. In three patients, ZAP-70 expression and IgV(H) mutational status were discordant. Conclusion: This paper describes an RQ-PCR assay for the detection of ZAP-70 expression and confirms that IgV(H) unmutated CLL cells have a high expression of ZAP-70 in comparison with IgV(H) mutated CLL. This robust method acts as a surrogate marker for IgV(H) mutational status albeit with < 100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.