Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA

Abstract

Background: Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained.

Design and methods: In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression.

Results: Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70(+) patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70(+) patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70(+) cells had better adhesion/migration capacities and only ZAP70(+) patient cells responded to microenvironment contact by CXCR4 downregulation.

Conclusions: We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70(+) cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.

Figure 1.

Disease progression and underlying gene expression in chronic lymphocytic leukemia patients with high versus low ZAP70 expression. ZAP70 expression was expressed as fold change of ZAP70 in the Namalwa cell line and normalized with cyclophilin expression. Patients expressing ZAP70 more than 114 fold were deemed “+” (n=41) while patients expressing ZAP70 with less than 114-fold were deemed “−” (n=44). (A) Treatment-free survival as a function of ZAP70 measured by qPCR. (B) Overall Survival as a function of ZAP70 measured by qPCR plotted using Kaplan-Meier analysis and compared using the log-rank test. (C) Multidimensional scaling of samples using the ZAP70 gene expression signature (n=39, FDR<10%). Red samples represent ZAP70^high and black samples represent ZAP70^low patients. (D) Hierarchical clustering of 39 probe sets discriminated between ZAP70^high and ZAP70^low chronic lymphocytic leukemia samples. Color scale indicates units of standard deviation from the mean expression of each gene.

Figure 2.

Migration of chronic lymphocytic leukemia cells in response to SDF1α and MSC conditioned medium and modulation of ZAP70, CXCR4 and CD69 in response to the stromal microenvironment. (A) Results are expressed as the mean of migration index in response to SDF1α or MSC conditioned medium (CM). (B) ZAP70 measured by FC in the upper and lower migration chamber. Chronic lymphocytic leukemia cells from ZAP70− (C) and + (D) patients were co-plated either with stromal cells, with stromal cells separated by a 0.45 μm transwell not allowing contact between the two cell types, or alone. After 4 h of incubation, ZAP70, CXCR4 and CD69 were measured by FC on adherent and non-adherent cells. Furthermore, ZAP70 was measured on chronic lymphocytic leukemia cells cultured alone and in transwell conditions.