Comparison of chronic lymphocytic leukemia patients expressing high or low level of ZAP70 mRNA: new prognostic factors differences in microRNA expression and interaction with the microenvironment

Stamatopoulos B, Haibe-Kains B, Equeter C, Debruyn C, Meuleman M, Hanosset D, Sorree A, Bron D, Martiat M, Lagneaux L 

Zeta-associated protein 70 (ZAP70) is a prognostic factor in B chronic lymphocytic leukemia (B-CLL) and a powerful surrogate marker for the immunoglobulin VH gene (IgVH) status. In this study, we used gene expression profile to identify genes associated with clinical disparity in CLL patients expressing high versus low ZAP70 mRNA measured by quantitative real time PCR (qPCR). Using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array, two groups of 7 patients selected for either high and low ZAP70 mRNA expression in purified CD19+ cells were compared. Selected genes were verified by qPCR in an extended patient cohort (n=85) with a median follow-up of 74 months. 27 genes were differentially expressed with a FDR<10% and several genes were significant predictors of treatment-free (TFS) and/or overall (OS) survival. Of these, ITGA4, PDE8A, and FCRL genes were selected for further analysis. PDE8A and FCRL family genes were downregulated in ZAP70 positive patients and can predict TFS and OS. ITGA4 mRNA was upregulated in ZAP70 positive patients and can significantly predict OS. Moreover this gene is known to be implicated in adhesion to fibronectin and in the migration. Therefore, we investigated and demonstrated the adhesion/migration capacities of ZAP70 positive cells into a stromal microenvironment or in response to conditioned medium. Finally, in order to complete this RNA study, we investigated the differential expression of some microRNA between ZAP70 positive and negative patients and found that miR-29c and miR-223 had an individual prognostic power. In conclusion, this study identifies new prognostic factors (genes and microRNA) and shows clearly the better adhesion and migratory capacities of ZAP70 positive cells in their microenvironment explaining their better survival and the aggressiveness of the disease.