Division of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, Food & Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Cytometry B Clin Cytom. 2011 Sep;80(5):309-17. doi: 10.1002/cyto.b.20593. Epub 2011 Apr 6.
In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation.
Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken.
There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation.
In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.
在一项配套的方法学研究中，我们比较了两种抗 ZAP-70 克隆（1E7.2 AF488 和 SBZAP PE）和四种选定的分析方法。需要进行临床相关性验证。
对 45 例未经治疗的 CLL 患者进行 ZAP-70、CD38、CD69、CD26、CD49d 和 CD27 的多色流式细胞术评估。设计了四种 ZAP-70 表达分析方法和评分系统。对 ZAP-70 评分、免疫球蛋白重链可变区（IGHV）突变状态、荧光原位杂交（FISH）以及这些生物标志物之间的相关性进行了分析。
ZAP-70 表达与 IGHV 突变状态之间存在很强的相关性。使用单个试剂的评分系统（P = 0.0006 或 0.0002）有利于使用多种分析方法。联合评分基本相当（P = 0.0003）。与 del13q14（P = 0.017）和 trisomy12（P = 0.011）也存在相关性。当使用 1E7.2 AF488 和 SBZAP PE 时，均观察到 CD38 和 ZAP-70 评分之间存在相关性，且当使用≥20%或≥7%截断值时具有相关性。两种试剂均观察到 CD49d 表达与 ZAP-70 表达之间存在正相关。CD26 与 ZAP-70 表达存在相关性，但依赖于分析方法。CD69 和 CD27 与 ZAP-70 表达之间无统计学显著相关性。
在我们的研究人群中，ZAP-70 表达是 IGHV 突变状态的更好预测指标。相关性分析证实，使用单个试剂或两种试剂的四种分析方法的组合优于使用单一分析方法。CD38、CD49d 和 CD26 的常规使用需要标准化。
