ZAP-70 Expression Evaluated by Mean Fluorescence Intensity T/B Ratio Is a More Useful Prognosticator Than Percentage of Positive Cells in Chronic Lymphocytic Leukemia (CLL).
Authors: Rossi Francesca Maria , Del Principe Maria Ilaria , Rossi Davide , Consalvo Maria Irno , Luciano Fabrizio , Zucchetto Antonella , Bulian Pietro , Bomben Riccardo , Benedetti Dania , Dal-Bo Michele , Degan Massimo , Gaidano Gianluca , Del Poeta Giovanni , Gattei Valter 
Published: October 2019

Abstract
Abstract 1257
Poster Board I-279

Introduction
Expression of the T-cells constitutive ZAP-70 protein by CLL cells has been the focus of many studies in the last few years, due to its ability to stratify indolent and more aggressive disease subsets. Although the strength of ZAP-70 as independent negative prognosticator was demonstrated by several studies, concerns about its use derive from a lack of multicentric standardization of flow-cytometric protocols. Analyses in clinical settings are usually performed according to two methods, respectively evaluating the percentage of CLL cells expressing ZAP-70 compared to isotypic control (ISO-method), or to autologous T-cells (T-method). Of note, while the two methods yield concordant results in most patients, a fraction of cases may be discordant as for evaluation of ZAP-70 positivity. Moreover, either method suffers of an operator dependent variability, mainly related to subjectivity in cursor placement to determine the percentage of ZAP-70+ cells. The aim of this study was to compare the ISO- and T-methods with the expression of ZAP-70 evaluated as Mean-Fluorescence-Intensity (MFI) Ratio between gated T and CLL cells (T/B-Ratio-method), and to assess the prognostic significance of the three approaches.

Methods
Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed with BD-DiVa software on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Notably, in the two cohorts, ZAP-70 assessment was accomplished using two different antibody combinations and instrumentations for data acquisition and analysis.

Results
ZAP-70 expression was reviewed in the test set by applying the ISO- and T-methods. Utilizing respectively 11% for ISO-method and 20% of ZAP-70+ cells for T-method, both selected as optimal cut-offs with prognostic relevance by ROC-analysis and maximally selected log-rank statistics, 66 (ISO-method) and 60 (T-method) ZAP-70+ cases were defined. By applying the T/B-Ratio-method, a value of 3.0 was identified as the optimal prognostic cut-point. According to this value, 73 ZAP-70+ cases (i.e. with T/B-Ratio<3.0) were identified in the test set. Univariate analyses resulted in a better separation of ZAP-70+ vs ZAP-70− CLL patients utilizing the T/B-Ratio-method (p=5.6×10−6), compared to T- (p=1.27×10−5) or ISO- (p=0.009) methods. In multivariate analyses with RAI stage, β2microglobulin, IGHV, FISH, CD38 and CD49d, ZAP-70 was selected as independent risk factor, irrespective of the readout employed for evaluation of ZAP-70 expression; however, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio-method was applied (significative hazard ratio=2.72 vs 2.19 with the T-method, or 2.11 with ISO-method). To confirm these findings, we analyzed the 341 cases of the validation set with T-method (cut-off=20%) and T/B-Ratio-method (cut-off=3.0). Analyses yielded 180 (T-method), and 127 (T/B-Ratio-method) ZAP-70+ cases. Univariate analyses also on this cohort resulted in a better separation with T/B-Ratio-method (p=7.77×10−16) than with T-method (p=1.23×10−12). Of note, ROC-analysis and maximally selected log-rank statistics confirmed also in this patient series, the 20% and 3.0 values as optimal cut-offs capable to separate CLL patients into two classes with different treatment probabilities.

Conclusions
We suggest to evaluate ZAP-70 expression in routine settings using the T/B-Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B-Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B-Ratio less than 3.0) from ZAP-70− (T/B-Ratio more/equal than 3.0) cases

Disclosures
No relevant conflicts of interest to declare.