Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia.
BACKGROUND
Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers.


METHODS
We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19(+) cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS).


RESULTS
The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [chi(2) (1) = 50.95; P <0.0001], and the value of Cramer's V statistic (0.72) indicated a very strong relation. This method also demonstrated sensitivity, specificity, and positive and negative predictive values of 87.8%, 85.7%, 87.5%, and 86%, respectively. ZAP70 expression was significantly associated with OS (P = 0.0021) and TFS (P <0.0001). ZAP70(+) patients had significantly shorter median TFS (24 months) than ZAP70(-) patients (157 months) (P <0.0001). Moreover, qPCR-measured ZAP70 expression has greater prognostic power than IGHV mutational status and the other prognostic markers tested.


CONCLUSIONS
ZAP70 mRNA quantification via qPCR is a strong surrogate marker of IGHV mutational status and a powerful prognostic factor.
Stamatopoulos
Basile
B
Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels, Belgium. bstamato@ulb.ac.be
Meuleman
Nathalie
N
Haibe-Kains
Benjamin
B
Duvillier
Hughes
H
Massy
Martine
M
Martiat
Philippe
P
Bron
Dominique
D
Lagneaux
Laurence
L
D051997:ADP-ribosyl Cyclase 1 / Q000097:blood; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D018941:Antigens, CD19 / Q000097:blood; D001402:B-Lymphocytes / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000097:blood*; D015331:Cohort Studies; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis*; D008071:Lipoprotein Lipase / Q000097:blood; D008297:Male; D008562:Membrane Glycoproteins / Q000097:blood; D008875:Middle Aged; D009154:Mutation; D011237:Predictive Value of Tests; D011379:Prognosis; D016016:Proportional Hazards Models; D012333:RNA, Messenger / Q000097:blood*; D012372:ROC Curve; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000097:blood*
D018941:Antigens, CD19; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D008071:Lipoprotein Lipase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1