title: Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method.
abstract: 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.
authors.affiliation: Laboratoire d'Hématologie, CHU Dupuytren, Faculté de Médecine de Limoges, Université de Limoges, Centre National de Recherche Scientifique, UMR CNRS 6101, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gachard
authors.forename: Aurélie
authors.initials: A
authors.lastname: Salviat
authors.forename: Catherine
authors.initials: C
authors.lastname: Boutet
authors.forename: Christine
authors.initials: C
authors.lastname: Arnoulet
authors.forename: Françoise
authors.initials: F
authors.lastname: Durrieu
authors.forename: Bernard
authors.initials: B
authors.lastname: Lenormand
authors.forename: Stéphane
authors.initials: S
authors.lastname: Leprêtre
authors.forename: Sylviane
authors.initials: S
authors.lastname: Olschwang
authors.forename: Fabrice
authors.initials: F
authors.lastname: Jardin
authors.forename: Marina
authors.initials: M
authors.lastname: Lafage-Pochitaloff
authors.forename: Dominique
authors.initials: D
authors.lastname: Penther
authors.forename: Danielle
authors.initials: D
authors.lastname: Sainty
authors.forename: Liliane
authors.initials: L
authors.lastname: Reminieras
authors.forename: Jean
authors.initials: J
authors.lastname: Feuillard
authors.forename: Marie C
authors.initials: MC
authors.lastname: Béné
mesh_terms: 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
chemical_list: 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