Search

Background: ZAP-70, after being considered as a potential surrogate for VH mutational status, has seen its own prognostic value emerge. We aimed at standardizing a simple, fast, and reproducible flow cytometry method., Methods: AntiZAP-70 antibody 2F3.2 was used with indirect labeling and secondary anti-IgG2a antibody. The reference values for the expression of the results were determined on 45 normal blood samples. ZAP-70 protein expression was investigated in 192 CLL samples. The indirect technique was compared with FITC-conjugated 2F3.2 clone, and with clone 1E7.2-FITC, -PE or -AlexaFluor 488., Results: Using FITC or PE-conjugated antibodies, 2F3.2 and 1E7.2 clones allowed a much less adequate discrimination between positive and negative cells and discordant cases were most likely true negative cases. Using the AlexaFluor 488 conjugated 1E7.2 clone, the discordant cases were mostly negative with the conjugated antibody and positive with the 2F3.2 clone but Western blotting or RNA microarray confirmed discordant cases were false negative with the conjugated antibody. Subsequently, recommendations were used by 13 centers participating in an interlaboratory quality control protocol. The use of MFI ratio appeared to be more reliable., Conclusions: Results suggested that slight differences in the procedure had little impact on the interpretation in characteristic cases; however, careful interpretation was required for values close to threshold., ((c) 2006 International Society for Analytical Cytology.)

Purpose: Rai's and Binet's staging systems have contributed significantly to the identification of major prognostic groups in chronic lymphocytic leukemia (CLL), though they fail to accurately predict disease progression at the individual level. Biologic factors, such as the mutational status of the immunoglobulin heavy-chain variable genes (VH, cytogenetics, CD38 expression, and some serum markers, have recently improved prognostic assessment in CLL. In this study, we analyzed the prognostic value of VH mutational status within the different stages of Binet's classification in 145 patients., Patients and Methods: Our series consisted of 83 VH mutated (MT) and 62 VH unmutated (UM) patients. MT cases predominated within Binet's stage A (70%), whereas UM cases predominated among stages B and C (62%)., Results: Median overall survival (OS) was 84 months for UM patients and was not achieved for the MT group (70% 12-year survival, P <.0001). Concerning Binet's stage A, both median OS and progression-free survival were significantly shorter for UM patients when compared with those of MT patients (97 months v not achieved, P =.0017; and 42 v 156 months, P <.0001), which compared favorably with the classical A' and A" substaging. The VH mutational profile could also segregate stage B and C patients into two groups with different survival patterns (median OS, 78 v 120 months for UM and MT patients, respectively; P =.002)., Conclusion: The significant survival differences observed between the VH mutational groups, among stage A and stage B and C patients, indicate that Binet's classification and VH genes are independent prognostic variables and are most likely complementary.