ZAP-70 and Flow Cytometry

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[495] Correlation of Flow Cytometrically Determined Expression of ZAP-

70 Using Two Different Antibodies with IgVH Mutation Status and

Cytogenetics in 539 Patients with Chronic Lymphocytic Leukemia.

Session Type: Oral Session

Wolfgang Kern, Dicker, ne Schnittger, Schoch,

Torsten Haferlach MLL, Munich Leukemia Laboratory, Munich, Germany

The clinical course of chronic lymphocytic leukemia (CLL) is largely

heterogeneous. It may be estimated based on clinical and laboratory

findings the most important ones of which include chromosomal

aberrations, the IgVH mutation status, and the expression of ZAP-70.

The optimum procedure of ZAP-70 assessment including selection of

antibody clone and the method of analysis still needs to be

established.

The validation of ZAP-70 has been performed using the course of the

disease as well as using the IgVH mutation status. To further clarify

the role of ZAP-70 expression in CLL we prospectively analyzed ZAP-70

expression, IgVH mutation status, and chromosome aberrations in

peripheral blood and bone marrow samples of 539 patients with CLL.

For the flow cytometric assessment of ZAP-70 expression the antibody

clones 1E7.2 (Caltag, n=523) and SBZAP (Immunotech, n=81) have been

used. IgVH mutation status has been analyzed in 407 cases and

chromosome aberrations in 416 cases applying FISH with probes for del

(6q), del(11q), +12, del(13q), del(17p), and t(11;14).

ZAP-70 expression has been calculated as percentage of positive cells

using normal T-lymphocytes as positive controls as well as using the

ratio of mean fluorescence intensity (MFI) for ZAP-70 between T-

lymphocytes and leukemic B-lymphocytes.

Significant correlations were found between the percentages of ZAP-70

positive cells and IgVH homology (1E7.2, p<0.001, r=0.239; SBZAP,

p=0.003, r=0.426). Accordingly, patients with mutated and unmutated

IgVH status (98% homology used as cut-off) significantly differed in

the percentages of ZAP-70 positive cells (1E7.2, mean 32% vs. 46%,

p<0.001; SBZAP, mean 7% vs. 29%, p<0.001). Applying a cut-off level

of 20% for positivity of ZAP-70 expression significant correlations

(ZAP-70 positive and IgVH unmutated and vice versa) were found for

1E7.2 (p<0.001, rate of concordant cases 59.0%) and SBZAP (p=0.002,

80.0%). Even stronger correlations have been found, however,

comparing the MFI ratios for ZAP-70 with the IgVH mutation status:

1E7.2, p<0.001, r=-0.283; SBZAP, p<0.001, r=-0.494. The respective

differences between cases with mutated and unmutated IgVH status

were: 1E7.2, mean MFI ratio 3.45 vs. 2.67, p=0.001; SBZAP, mean MFI

ratio 6.18 vs. 2.86, p<0.001. The ratio of MFI between leukemic and

normal B-lymphocytes did not correlate with the IgVH mutation status.

No associations of ZAP-70 expression with chromosomal aberrations

have been found.

The results of the present study 1) confirm the strong correlation

between ZAP-70 expression and IgVH mutation status, 2) confirm the

independence of ZAP-70 expression from chromosome aberrations, 3)

argue in favor of analyzing ZAP-70 expression as MFI ratio between T-

lymphocytes and leukemic B-lymphocytes, and 4) suggest that the SBZAP

clone has a significant potential in the diagnostic work-up of CLL.

Further studies are warranted to validate the role of the MFI ratio

approach and of the SBZAP antibody in the clinical setting.