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e19006 Background: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical course. Identification of prognostic markers is important for proper management of disease. Cytogenetics, IgVH mutation status, TP53 functional status, CD38, ZAP-70 and CD49d expression are accepted prognostic factors in CLL. ζ-chain-associated protein kinase 70 (ZAP-70) is associated with disease progression. However, measurement of ZAP-70 and CD38 expression relies on qualitative flow cytometry. Authors tested hypothesis whether quantitative analysis of markers can improve information about prognosis of CLL. Methods: 217 CLL patients were included; diagnosis was made from peripheral blood by morphology and immunophenotyping by flow cytometry with common antibodies. Expressions of intracellular ZAP-70 and surface CD38 were measured using quantitative fluorescence cytometry. Standardized fluorescent microparticles were used to quantify molecules of equivalent soluble fluorochrome (MESF units). Analysis of rearrangement of the IgVH gene was performed according to the European recommendation. Deletions at 11q22-q23 ( ATM), 17p13 ( TP53), 13q34 ( RB1) loci and trisomy of chromosome 12 were detected by I-FISH. Results: Patients’ ages ranged from 33 to 86 years and included all Rai stages. Expression of ZAP-70 ranged from 546 to 5,955 MESF, CD38 from 1,886 to 31,619 MESF. 48% patients embodied mutated and 52% unmutated IgVH status. In cytogenetics, del(13q) was detected in 59%, trisomy 12 in 13%, del(11q) in 21%, and del(p53) in 8% of patients. IgVH (in %) was plotted against ZAP-70 (in MESF) and patients were divided into high (HR) and low risk (LR) groups by cluster analysis based on Kaplan-Meier overall survival curves. Patients in HR group had significantly poorer prognoses (p = 0.0001) than those in LR group. When patients from HR group were clustered according to CD38 expression, very high risk (VHR) group with high CD38 and significantly worse prognosis (p = 0.035) was identified. Conclusions: We have demonstrated that quantitative ZAP-70 and CD38 expressions cannot replace IgVH analysis, but, contrary to that, addition of quantitative data can further specify prognoses of patients with CLL.

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. Figure Figure

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