Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano (PN), Italy.
J Transl Med. 2010 Mar 8;8:23. doi: 10.1186/1479-5876-8-23.
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).
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).
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.
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.
ZAP-70 是慢性淋巴细胞白血病（CLL）的独立预后不良标志物。通常，通过流式细胞术方案检测其表达，其中以相对于同型对照（ISO 法）或残留的 ZAP-70 阳性 T 细胞（T 法）确定 ZAP-70 阳性 CLL 细胞的百分比。然而，这些方法除了在应用中存在固有主观性之外，在某些情况下可能会得出不一致的结果。本研究旨在评估这些方法与另一种方法的预后意义，后者将 ZAP-70 表达评估为门控 T 细胞和 CLL 细胞之间的平均荧光强度比（T/B 比值法）。
回顾性分析了 173 例患者（测试集）的三种读数中 ZAP-70 测定的流式细胞仪文件，所有患者均具有完整的临床和生物学预后评估以及治疗时间（TTT）。然后在来自不同机构的 341 例独立病例的验证集中验证了这些发现。
在测试集中，ZAP-70 表达的最佳预后截断值为 11%（ISO 法）或 20%的阳性细胞（T 法），以及 3.0（T/B 比值法）；这些截断值分别产生了 66、60 和 73 个 ZAP-70+病例。与 T 或 ISO 方法相比，利用 T/B 比值法可更好地分离 ZAP-70+与 ZAP-70-CLL 患者的单变量分析。在包括 CLL 主要临床和生物学预后标志物的多变量分析中，当应用 T/B-Ratio 方法时，ZAP-70 的预后影响似乎更强。这些发现在验证集中得到了证实，其中 T-（截断值=20%）或 T/B 比值-（截断值=3.0）方法评估的 ZAP-70 表达分别产生了 180 或 127 个 ZAP-70+病例。与 ZAP-70-CLL 相比，与仅通过 T 方法分类为 ZAP-70+的病例相比，T/B 比值方法的 ZAP-70+患者 TTT 更短。
鉴于这种方法的操作员和实验室独立性，我们建议在常规环境中使用 T/B 比值法评估 ZAP-70 表达。我们提出 3.0 T/B 比值值作为区分 ZAP-70+（T/B 比值小于 3.0）和 ZAP-70-（T/B 比值大于或等于 3.0）病例的最佳截断值。
