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


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.
Gachard
Nathalie
N
Laboratoire d'Hématologie, CHU Dupuytren, Faculté de Médecine de Limoges, Université de Limoges, Centre National de Recherche Scientifique, UMR CNRS 6101, France.
Salviat
Aurélie
A
Boutet
Catherine
C
Arnoulet
Christine
C
Durrieu
Françoise
F
Lenormand
Bernard
B
Leprêtre
Stéphane
S
Olschwang
Sylviane
S
Jardin
Fabrice
F
Lafage-Pochitaloff
Marina
M
Penther
Dominique
D
Sainty
Danielle
D
Reminieras
Liliane
L
Feuillard
Jean
J
Béné
Marie C
MC
D051997:ADP-ribosyl Cyclase 1 / Q000096:biosynthesis / Q000235:genetics; D014408:Biomarkers, Tumor / Q000096:biosynthesis* / Q000235:genetics; D001782:Blood Donors; D002872:Chromosome Deletion; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics; D005260:Female; D005434:Flow Cytometry / Q000592:standards; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D011379:Prognosis; D014314:Trisomy; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000096:biosynthesis* / Q000235:genetics
D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1