ZAP-70 Expression in Acute Lymphoblastic Leukemia: Association with the E2A/PBX1 Rearrangement and with the Pre-B Stage of Differentiation.

The zeta associated protein (ZAP) is a 70-kD molecule associated with the z chain of the CD3 receptorial complex which plays an important role in several signalling pathways following TCR ligation. Historically, this protein was thought to be specific of T and NK cells. A study based on nucleotide arrays on chronic lymphocytic leukemia (CLL) cells revealed the presence of ZAP-70 in CLL samples which carried an unmutated configuration of the IgV_H genes. This finding opened a new area of investigation regarding the presence and putative role of ZAP-70 in B cells. In the present study, we evaluated the expression of ZAP-70 and Syk in acute lymphoblastic leukemia (ALL), using data derived from a series of 128 adult patients analyzed by oligonucleotide arrays and by the quantification of the protein levels. All samples, evaluated for gene expression profiling at the onset of the disease, contained over 90% leukemic cells and were enrolled in the Italian multicenter clinical trial GIMEMA 0496. Of the cases analyzed, 33 had T-ALL and 95 B-lineage ALL: within this latter group, 5 samples carried the E2A/PBX1 rearrangement, 10 the ALL1/AF4 rearrangement, 37 were BCR/ABL+ and 42 samples did not harbor any major molecular abnormality. As expected, ZAP-70 was found highly expressed in T-ALL and a highly significant difference was observed compared to B-lineage ALL (average values 752±268 and 309±105, respectively; p<0.0001). Nonetheless, an increased expression of ZAP-70 was also found in a proportion of B-lineage ALL. When B-lineage ALL samples were stratified according to molecular groups, the highest levels of expression were associated with the E2A/PBX1 abnormality (analyzed by ANOVA; p<0.0001) and, conversely, the lowest levels were detected in the ALL1/AF4+ cases which were all pro-B ALL. When samples were further subclassified according to their stage of differentiation, significantly higher levels of ZAP-70 expression were observed in the pre-B ALL group (analyzed by ANOVA; p<0.0001). Furthermore, when samples were subdivided according to the protein expression of CD20 and Igm, regardless of their molecular abnormalities, there was a trend towards higher levels of ZAP-70 expression in CD20+ samples and significantly higher levels of expression were found in Igm+ samples (p=0.002). The expression of Syk was also studied and found preserved in all cases, with a higher degree of expression in E2A/PBX1+ cases. In order to validate this finding, we evaluated ZAP-70 protein levels on fresh cells by immunocytochemistry on an independent set of 23 newly diagnosed ALL patients (14 adults, 9 children). ZAP-70 was detectable on leukemic cells in 13 cases (56%). Also at the protein level, all pre-B ALL evaluated were ZAP-70+, whereas for the remainders a correlation was found with the expression of CD20. In summary, these results show that ZAP-70 is expressed in a considerable proportion of B-lineage ALL that accounts for about 50% of cases and that the levels of ZAP-70 expression are strongly associated with the E2A/PBX1 rearrangement and with the more mature stages of B-cell differentiation.