Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells

Abstract

Background: ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).

Methods: Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed 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).

Results: The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only.

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.

Figure 1

Flow cytometric analysis of ZAP-70 expression (test set). PB cells of CLL samples were analyzed after staining with anti-CD19-APC, anti-CD3-PE, anti-CD5-PECy7 and AlexaFluor488-conjugated isotype control or anti-ZAP-70 antibodies. Panel A shows the gating strategies used to select lymphocytes in the left plot, CLL cells (CD19+/CD5+/CD3-) or T cells (CD19-/CD5+/CD3+) in middle and right plots, upon gating on lymphocytes. Panel B contains plots showing a representative ZAP-70 negative (upper row) and a representative ZAP-70 positive (lower row) sample, both analyzed according to the three different approaches utilized to evaluated ZAP-70 expression. The ISO- T-, and T/B Ratio-method readouts are shown respectively in the left, middle and right panels. For the ISO-method marker was set to have <1% CLL positive cells with isotypic control. For the T-method, marker was set on the left edge of T cells cluster, to have about 98% of positive cells. For the T/B Ratio-method the ratio was calculated directly from MFI values as separately read from T cell and CLL cell gates defined in panel A.

Figure 2

C index and Kaplan-Meier curves for ZAP-70 evaluation according to ISO-, T- and T/B Ratio-methods (test set). Upper panels in A, B, and C show c index curves applied to ZAP-70 expression values to estimate the optimal cut-off capable to split patients into groups with different time to treatment (TTT) probabilities. X-axes report expression values for ZAP-70, expressed as percent of positive cells (A and B), or T/B ratio values (C); y-axes report the corresponding c index values. For each method, solid line indicates the chosen cut-off value. Lower panels show Kaplan-Meier curves obtained comparing TTT of patients affected by CLL expressing or not ZAP-70, as evaluated according to ISO- (A), T- (B) or T/B Ratio- (C) methods. In all plots, solid lines indicate ZAP-70 negative CLL, while dashed line indicate ZAP-70 positive CLL, according to the three readouts. In (A) Kaplan-Meier curves obtained by dividing CLL patients according to two different cut-offs (11% and 20%) for ZAP-70 evaluation are reported.

Figure 3

Analysis of ZAP-70 concordant and discordant cases among ISO-, T- and T/B Ratio-methods (test set). (A) Venn diagram depicting concordant and discordant cases, as obtained by merging the ZAP-70 positive cases determined by ISO-, T- and T/B Ratio-methods. (B) Kaplan-Meyer curves obtained comparing TTT of patients affected by CLL expressing ZAP-70 according to T/B Ratio-method (73), or expressing ZAP-70 according to either ISO- or T-methods (30).

Figure 4

ZAP-70 expression in the validation set. (A-B) Kaplan-Meier curves obtained comparing TTT of patients affected by CLL expressing ZAP-70 according to T-method (A) or T/B Ratio-method (B). In all plots solid line indicates ZAP-70 negative CLL, while dashed line indicates ZAP-70 positive CLL. (C) Venn diagram depicting concordant and discordant cases, as obtained by merging the ZAP-70 positive cases determined by the two readouts. (D) Kaplan-Meyer curves obtained comparing TTT of patients affected by CLL expressing ZAP-70 according to T/B Ratio-method (127 cases), expressing ZAP-70 according to sole T-method (58 cases), or ZAP-70 negative according to both methods (156 cases).