ZAP70 Expression Assessed by Immunohistochemistry on Peripheral Blood: A Simple Prognostic Assay for Patients With Chronic Lymphocytic Leukemia

Abstract

The somatic hypermutational (SHM) status of the immunoglobulin heavy-chain variable (IgVH) gene is a powerful prognostic factor in patients with chronic lymphocytic leukemia (CLL). However, IgVH SHM analysis is not well-suited to routine use in the clinical diagnostic laboratory. ZAP70 expression is a potential surrogate for the absence of SHM. Given the current problems with the standardization of ZAP70 assessment by flow cytometry, we sought an alternative approach, using immunohistochemistry (IHC). The utility of IHC is largely restricted to tissues, precluding its routine application to most patients with CLL who are typically diagnosed based upon peripheral blood (PB) findings. Accordingly, we developed an IHC assay that can be performed on PB. Enriched PB mononuclear cells from 29 patients with CLL were analyzed for ZAP70 expression by IHC on paraffin-embedded cell blocks, using standard techniques. IgVH SHM analysis was performed on all cases, and clinical features recorded. Seventeen specimens (59%) were negative for ZAP70 expression and 12 (41%) were positive for ZAP70 expression. SHM was evident in 20 specimens (69%), and absent in 9 (31%). Seventy-six percent of the specimens (22/29) displayed "concordant" ZAP70 and SHM results, in that 15 (52%) were SHM-positive/ZAP70 negative, whereas 7 (24%) were SHM-negative/ZAP70 positive. ZAP70 expression in this small cohort correlated with poor clinical outcome. Importantly, IHC analysis of ZAP70 in PB is a simple, reliable, robust assay that may have a valuable role in the routine clinical laboratory assessment of patients with CLL.