Abstract 5080: Improved ZAP-70 assay using two clones: Multiple methods of analysis, and a scoring system
Authors: Degheidy Heba A. , Venzon David , Farooqui Mohammed , Abbasi Fatima , Arthur Diane , Wiestner Adrian , Stetler-Stevenson Maryalice , Gerald Marti 
Published: June 2012

Abstract
Background. ZAP-70 expression is a stage independent prognostic marker in CLL. However, inter-laboratory variation is large and there is neither a consensus nor method that has been approved by regulatory authorities.
Methods. Multicolor flow-cytometric expression of ZAP-70 was tested in 45 untreated-CLL patients. Two anti-ZAP70 clones (1E7.2 and SBZAP) were compared. Nine different methods were evaluated: M1 using the isotype control; M2 internal residual T-cell to determine positive; M3, normal donor (ND) T-cell to determine positive; M4 internal T-cell/clone ratio; M5, ND residual T-cell/clone ratio; M6 clone/normal remaining B-cell ratio; M7 clone/ND B- cell ratio; M8 CLL- Z score; M9 modified CLL-Z score. Four methods were selected for designation of a scoring system integrating both clones for ZAP-70 assignment. For a given specimen, each method of analysis was given a score of 1 point if it was positive, and 0 point if it was negative. We considered the cases that show no or one positive method (score 0 or 1) as negative, the cases that showed positivity by two method (score 2) as equivocal, and those with three or four positive methods (score 3 or 4) as positive. This score was calculated for each clone separately and then integrating both clones together. A correlation analysis between IGHV mutational status, FISH, and ZAP-70 score was undertaken.
Results. The correlation coefficients between clones for the four selected statistically significant methods were as follows: M1 0.71, M3 0.72, M7 0.67 and M9 0.64. The two reagents showed agreement using the designed scoring system for 37/45 samples (82%) while 8/45 (18%) showed an equivocal result with one of the two clones. Seven of the eight equivocal samples were resolved using the combined scoring system. For both clones, ZAP-70 expression was significantly correlated with IGHV mutational status with all four methods selected for analysis. The scoring system for a single reagent favored the use of multiple methods of analysis. Furthermore, the combined score indicated a substantial enhancement over the use of a single reagent (p value of 0.0003). Regarding FISH cytogenetics, the absence of ZAP-70 expression correlated with del13q14 (p=0.0049), while the presence of ZAP-70 expression correlated with trisomy 12 (p=0.011) using our designated ZAP-70 score. Conclusions: The use of two independent ZAP-70 reagents increases analytical certitude and the scoring method aids in the resolution of equivocal results. The combined use of two reagents, four methods of analysis, and a scoring system allowed for assignment of ZAP-70 expression in 44/45 samples tested and improved performance of this important prognostic assay. ZAP-70 expression was a good predictor of the IGHV mutational status and 13q14 del/trisomy 12. 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
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2011-5080