Abstract

Ig mutation status discriminates aggressive from more indolent chronic lymphocytic leukemia (CLL). Several gene expression studies distinguished CLL from other leukemia but failed to discriminate these two types of patients. Nevertheless, differentially expressed genes including zap70 were identified and further validated as an important prognostic factor. Zap70 is a normal component from the T cell receptor pathway, and may have a critical role in the B cell receptor (BCR) signaling pathway in CLL. We questioned whether zap70 might alter response to BCR signaling in CLL cells and explain the difference in biologic characteristics of this disease. To examine this we crosslinked the BCR in CLL cells from 12 patients and B cells from 6 healthy donors. We examined the response to BCR stimulation on cell proliferation, apoptosis and gene expression profile. Here we analyzed the temporal gene expression after BCR crosslinking at different time points, in addition to the basal level of gene expression. Based on the results of a pilot study examining multiple different time points, four time points were analyzed in all cases ranging from 60 min to 390 min and gene expression of stimulated (S) and unstimulated (US) CLL and control cells analyzed for a total of 170 HU133 plus2.0 DNA-chips. We defined the expression data from the logarithmic ratio log(S/US) for each time point. In keeping with previous findings, the basal gene expression distinguished CLL samples from healthy B-cells, but did not distinguish mutated from unmutated patients. We defined the linear combination first for the initial time point and then for all four time points to score for expression over time. This temporal gene profile of BCR engagement was based on filtering of 2850 genes that now distinguish healthy B-cells from CLL-cells, but is now capable also of distinguishing a specific BCR signature of a group of patients. Of note, this distinguished patients based less upon Ig gene mutation status but on zap70 protein level, with a mean protein expression difference of 9 fold between these two groups. This more aggressive pattern reveals changes in gene expression in important genetic pathways associated with BCR crosslinking, including JUN and NFkB. Stimulating a crucial pathway for CLL biology identified temporal gene expression pathways linked with the aggressiveness of this leukemia. This suggests that a fuller understanding of the biologic differences in leukemia cells may be identified by a more dynamic approach to gene profiling by examination of response to specific stimuli, rather than examination at the basal level. Based upon these results, ongoing studies examine the biologic impact of gene silencing of zap70 using RNAi on BCR stimulation in CLL cells.

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