Prognostic Stratification of Patients with Chronic Lymphocytic Leukemia Based on Quantitative Flow Cytometry Evaluation of ZAP-70 Versus Homology of IgVH Plot.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the adult population. It has a heterogeneous behavior and variable prognosis. While some patients experience indolent disease requiring no therapy for many years, others demonstrate a more aggressive type unresponsive to therapy. An accurate prognostic stratification is essential for optimizing the therapeutic strategy. Many prognostic factors are presently known, but their relationships and significance are often not clearly understood. Several markers have been identified, including IgVH mutation status and ZAP-70. Mutation of IgVH (less than 98% homology with the germline sequence) is correlated with better prognosis. Expression of ZAP-70, which is a cytoplasmic ζ-associated tyrosine-kinase essential for T-cell receptor signal transduction, is associated with more rapid disease progression and shorter survival. However, its detection by flow cytometry has many technical difficulties, resulting in high interlaboratory variability. The expression of intracellular ZAP-70 in 217 patients with diagnosis of B-CLL was determined using a new approach: quantitative flow cytometry on CD5⁺19⁺ tumor cells. Other laboratory and clinical parameters were evaluated, including gender, age, type and number of therapies, CD38 expression, cytogenetics and mutation status of IgVH. The expressions of ZAP-70 and CD38 were measured in molecules of equivalent soluble fluorochrome (MESF units). The results were correlated with mutation status of IgVH. Patients were divided into two groups by cluster analysis in a plot of IgVH homology versus ZAP-70 MESF (left panel). Overall survival curves for these groups are shown (right panel). It could follow that patients above the diagonal (triangles, group B) in the IgHV versus MESF plot have significantly poorer prognoses (p=0.0001) than do patients below this diagonal (circles, group A). Expression of CD38 above 15000 MESF showed worse prognosis for patients in the group B (p=0.035). Only three of 61 patients with chromosomal aberrations associated with poor prognosis (del11q and del17p) were found in the group A, but all of them had del13q present, as well. The authors introduced a new approach for evaluating ZAP-70 expression in B-CLL cells using quantitative flow cytometry that is easily standardized and not burdened with technical problems. Plotting IgVH homology (%) versus ZAP-70 quantitative expression (MESF) could clearly divide patients according to their prognoses.

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