Correlation of Flow Cytometrically Determined Expression of ZAP-70 Using Two Different Antibodies with IgVH Mutation Status and Cytogenetics in 539 Patients with Chronic Lymphocytic Leukemia.
Authors: Kern Wolfgang , Dicker Frank , Schnittger Susanne , Schoch Claudia , Haferlach Torsten 
Published: April 2020

Abstract
The clinical course of chronic lymphocytic leukemia (CLL) is largely heterogeneous. It may be estimated based on clinical and laboratory findings the most important ones of which include chromosomal aberrations, the IgVH mutation status, and the expression of ZAP-70. The optimum procedure of ZAP-70 assessment including selection of antibody clone and the method of analysis still needs to be established. The validation of ZAP-70 has been performed using the course of the disease as well as using the IgVH mutation status. To further clarify the role of ZAP-70 expression in CLL we prospectively analyzed ZAP-70 expression, IgVH mutation status, and chromosome aberrations in peripheral blood and bone marrow samples of 539 patients with CLL. For the flow cytometric assessment of ZAP-70 expression the antibody clones 1E7.2 (Caltag, n=523) and SBZAP (Immunotech, n=81) have been used. IgVH mutation status has been analyzed in 407 cases and chromosome aberrations in 416 cases applying FISH with probes for del(6q), del(11q), +12, del(13q), del(17p), and t(11;14). ZAP-70 expression has been calculated as percentage of positive cells using normal T-lymphocytes as positive controls as well as using the ratio of mean fluorescence intensity (MFI) for ZAP-70 between T-lymphocytes and leukemic B-lymphocytes. Significant correlations were found between the percentages of ZAP-70 positive cells and IgVH homology (1E7.2, p&lt;0.001, r=0.239; SBZAP, p=0.003, r=0.426). Accordingly, patients with mutated and unmutated IgVH status (98% homology used as cut-off) significantly differed in the percentages of ZAP-70 positive cells (1E7.2, mean 32% vs. 46%, p&lt;0.001; SBZAP, mean 7% vs. 29%, p&lt;0.001). Applying a cut-off level of 20% for positivity of ZAP-70 expression significant correlations (ZAP-70 positive and IgVH unmutated and vice versa) were found for 1E7.2 (p&lt;0.001, rate of concordant cases 59.0%) and SBZAP (p=0.002, 80.0%). Even stronger correlations have been found, however, comparing the MFI ratios for ZAP-70 with the IgVH mutation status: 1E7.2, p&lt;0.001, r=−0.283; SBZAP, p&lt;0.001, r=−0.494. The respective differences between cases with mutated and unmutated IgVH status were: 1E7.2, mean MFI ratio 3.45 vs. 2.67, p=0.001; SBZAP, mean MFI ratio 6.18 vs. 2.86, p&lt;0.001. The ratio of MFI between leukemic and normal B-lymphocytes did not correlate with the IgVH mutation status. No associations of ZAP-70 expression with chromosomal aberrations have been found. The results of the present study
1) confirm the strong correlation between ZAP-70 expression and IgVH mutation status, 2) confirm the independence of ZAP-70 expression from chromosome aberrations, 3) argue in favor of analyzing ZAP-70 expression as MFI ratio between T-lymphocytes and leukemic B-lymphocytes, and 4) suggest that the SBZAP clone has a significant potential in the diagnostic work-up of CLL.
Further studies are warranted to validate the role of the MFI ratio approach and of the SBZAP antibody in the clinical setting.