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Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method

¹ Laboratoire d’Hématologie, CHU Dupuytren, Faculté de Médecine de Limoges, Université de Limoges, Centre National de la Recherche Scientifique, UMR CNRS 6101;
² Laboratoire d’Hématologie, Centre Hospitalo-Universitaire Charles Nicolle, Rouen, France;
³ Département de Biopathologie, Institut Paoli Calmettes, Marseille;
⁴ Laboratoire d’Hématologie, CLCC Bergonié, Bordeaux;
⁵ Département d’Hématologie Clinique, CLCC Henri Becquerel, Rouen;
⁶ INSERM U599, Université de la Méditerranée, Marseille;
⁷ Laboratoire de Génétique Oncologique, CLCC Henri Becquerel, Rouen;
⁸ Service d’Hématologie Clinique, CHU Dupuytren, Faculté de Médecine de Limoges, Université de Limoges;
⁹ Laboratoire d’Immunologie du CHU, Faculté de Médecine de Nancy, Vandoeuvre-lès-Nancy, France

Correspondence: Marie Christine Béné, Laboratoire d’Immunologie du CHU Faculté de Médecine de Nancy, BP 184, 54500 Vandoeuvre les Nancy, France. E-mail: bene{at}medecine.uhp-nancy.fr

Background: Flow cytometry allows specific assessment of the expression of ZAP-70, a promising new prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL), but suffers from a lack of multicenter standardization.

Design and Methods: An optimized method for direct detection of ZAP-70 in flow cytometry was tested in a multicenter fashion. Adapted for frozen cells, this method includes a normalization step by addition of B cells from a pool of peripheral blood mononuclear cells collected from normal donors. ZAP-70 expression levels were assessed for 153 patients with typical B-cell chronic lymphocytic leukemia chronic lymphocytic leukemia. Results were expressed as the ratio of ZAP-70 mean fluorescence intensity between B-CLL cells and normal B cells.

Results: The statistically optimized cut-off of ZAP-70 positivity was a ratio of 1.4. Concordance between ZAP-70 and CD38 expression was 67%. Concordance between the mutational status of IgVH genes and ZAP-70 or CD38 expression was 87% and 65%, respectively. ZAP-70 was significantly expressed in 28%, 54% and 61% of patients with Binet stages A, B and C B-cell chronic lymphocytic leukemia, respectively (p=0.008). The absence of ZAP-70 expression was associated with isolated del(13q14), a cytogenetic abnormality with a good prognosis, while most patients with the del(17p13) poor prognosis cytogenetic marker expressed ZAP-70 (p<10^–5). ZAP-70 expression was not related to the other poor prognosis cytogenetic abnormality del(11q22.3) nor to trisomy 12.

Conclusions: This new technique provides highly reliable results well correlated with the mutational status of IgVH genes, CD38 expression, Binet stage and cytogenetic abnormalities. This robust discriminative technique appears of particular interest for routine diagnosis and assessment of ZAP-70 expression in large, prospective, multicenter therapeutic trials.

Key words: B-cell chronic lymphocytic leukemia, ZAP-70, CD38, prognosis.