Expression of ZAP-70 Protein Correlates with Disease Stage in Chronic Lymphocytic Leukemia and is Associated with, but not Generally Restricted to, Non-mutated Ig V_HStatus

Abstract

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.

Quantitative flow cytometry of ZAP‐70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome

“…High ZAP-70 expression was also shown to be an independent poor prognostic factor in CLL, illustrating that it is more than just a surrogate marker for the mutational status in CLL (13). Moreover, Kim et al demonstrated that high expression of ZAP-70 protein correlates with Binet disease stage B or C in CLL and is associated with, but not generally restricted to, non-mutated IgVH status (10).…”

“…Samples considered high by the quantitative method are marked in gray. (4,10,11,13,14,26). On the basis of this data, ZAP-70 has become an important prognostic factor in CLL, and may be utilized for risk stratification and possible risk adapted therapy in CLL.…”

“…Further studies, using Western blot, quantitative RT PCR, or flow cytometry analyses, showed that ZAP-70 level could serve as a surrogate marker for the mutational status in CLL or as a highly significant independent prognostic marker. In CLL, ZAP-70 levels correlate with Binet stage (10), time to progression, time to treatment, and overall survival (4,(11)(12)(13)(14). Unfortunately, the assays described thus far have provided an endless series of technical issues for clinical labo-ratories, as described in the cover story of the December 2004 issue of CAP today (College of American Pathologists) (15).…”

Immunoglobulin Mutational Status Detected through Single-Round Amplification of Partial VH Region Represents a Good Prognostic Marker for Clinical Outcome in Chronic Lymphocytic Leukemia

“…27,28 Nevertheless, ZAP70 expression does not always correlate with the mutational status of Ig genes; in fact it does not correlate with more than 90% of cases. 29 Very recently, Rassenti and colleagues 30 better defined the correlation between Ig mutational status and ZAP70 expression levels. They showed that ZAP70 was highly expressed in only 71% of UM-CLL cases and in 17% of M-CLL cases with an overall concordance of 77%.…”

“…Unfortunately, CD38 expression does not always correlate with the IgV H mutational status [23][24][25] rather it appears to be an independent prognostic marker in B-CLL. 26 In addition, expression of ZAP70 kinase was first recognized to correlate with Ig mutational status and prognosis, 27,28 but recent studies have shown a discordance between ZAP70 and Ig mutational status, 29 although ZAP70 maintains an important prognostic independent value. 30 Here we describe a rapid and simple method to analyze the nucleotide sequence of the variable portion of Ig heavy chain gene using amplicons obtained with degenerate primers commonly used for diagnostic B-cell clonal detection purposes.…”