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.

Igβ(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity

“…In the analysis of 29 patients who were fully evaluated and underwent a recently optimized immunohistochemical analysis of ZAP70 expression [32], we noted a positive correlation between clinical severity and ZAP70 protein expression and a less prominent correlation between clinical severity and hypomutation at the expressed IgV H locus (Fig. 7).…”

“…This procedure and its clinical application is described in detail elsewhere [32]. Briefly, peripheral blood mononuclear cells (PBMCs) isolated from 10 mls of heparinized whole blood using Ficoll-Hypaque (Amersham Biosciences, Buckinghamshire, England) were fixed, centrifuged, and submitted for parafin cell-block preparation.…”

“…An immunohistochemical assay of ZAP70 protein expression in peripheral lymphocytes was developed for this purpose. The details of this analysis and its validation are separately reported [32]. The level of ZAP70 expression, measured by this immunohistochemical analysis of PBMCs, was considered to be positive if unequivocal staining was observed in >20% of the B-cells in a sample [32].…”

“…The details of this analysis and its validation are separately reported [32]. The level of ZAP70 expression, measured by this immunohistochemical analysis of PBMCs, was considered to be positive if unequivocal staining was observed in >20% of the B-cells in a sample [32]. Of note, although >20% was the designated threshold for positivity, this was typically an essentially ''all-or-none'' phenomenon.…”

Customized Oligonucleotide Array-Based Comparative Genomic Hybridization as a Clinical Assay for Genomic Profiling of Chronic Lymphocytic Leukemia

“…2,12,15,16,38,39 However, levels of ZAP70 expression do not always correlate with mutational status, and it has proven difficult to standardize reagents and methodologies for its detection. 40 Laboratory studies to detect genetic aberrations in CLL thus have been easier to validate and standardize 41,42 and provide correlations with other biomarkers, such as the strong association of del ATM/11q22.3 cases with unmutated/ZAP70ϩ status. 21,43,44 We present a model for routine application of aCGH methodology in the clinical laboratory.…”