ZAP-70 expression in B-cell chronic lymphocytic leukemia: evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations.
BACKGROUND
Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV(H)) mutations, although a standardization of the cytometric protocol is still lacking.


METHODS
Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV(H) mutations) by a standard three-color protocol. Identification of ZAP-70(+) cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed.


RESULTS
While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO(+)TNK(+) or ISO(-)TNK(-)), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO(+)TNK(-) profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO(+)TNK(-) cases had a IgV(H) gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL.


CONCLUSIONS
Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV(H) gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed.
Zucchetto
Antonella
A
Clinical and Experimental Hematology Research Unit, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy.
Bomben
Riccardo
R
Bo
Michele Dal
MD
Nanni
Paola
P
Bulian
Pietro
P
Rossi
Francesca Maria
FM
Del Principe
Maria Ilaria
MI
Santini
Simone
S
Del Poeta
Giovanni
G
Degan
Massimo
M
Gattei
Valter
V
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D002138:Calibration; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007694:Killer Cells, Natural / Q000276:immunology*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*
D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human