Apoptosis Gene Expression Profiling in Chronic Lymphocytic Leukemia Using Multiplex Ligation-Dependent Probe Amplification.

Abstract 3457

Poster Board III-345

One of the hallmarks of the clinically heterogeneous disease chronic lymphocytic leukemia (CLL) is defective apoptosis, which is considered to contribute not only to cell accumulation but also to disease progression and resistance to therapy. In an effort to further investigate the ‘apoptotic profile’ in CLL, we have utilized the multiplex ligation-dependent probe amplification (MLPA) method to analyze the expression profile of 35 apoptosis-related genes in 67 CLL cases including 49 peripheral blood, 9 lymph node, 7 bone marrow and 2 spleen samples. CLL cells derived from peripheral blood displayed a different overall expression profile when compared to peripheral blood mononuclear cells (PBMC) from five healthy controls, with several genes including the anti-apoptotic genes BCL2A1 and MCL1 and the pro-apoptotic genes BAX and BMF all being significantly differently expressed (>2 fold difference in median expression). Few differences were detected when comparing peripheral blood CLL samples (25/49 IGHV unmutated/IGHV3-21) to lymph node samples (6/9 unmutated/IGHV3-21) or to bone marrow samples (5/7 unmutated/IGHV3-21), with lower relative expression of BIK and BAX in peripheral blood samples. Furthermore, we correlated the expression profiles of all genes included in the assay with IGHV mutation status, IGHV3-21 gene usage and genomic aberrations, but overall few differences were evident between prognostically different subgroups. An exception to this was the BIK gene which showed significantly higher expression in unmutated/IGHV3-21 compared to mutated CLL. Additionally, MCL1 and BNIP3L displayed higher expression in 17p-deleted cases compared to 13q deletion/no aberration cases. High expression of HRK and BAX indicated a correlation with poor overall survival and shorter time to treatment. However, when performing real-time quantitative PCR on an extended patient cohort (n=112) only prediction of time to treatment could be confirmed for HRK and BAX (p=0.0035 and p=0.024, respectively). Finally, in order to validate our MLPA approach, we compared the gene expression levels of four genes in 19 CLL samples using both MLPA and real-time quantitative PCR, which revealed a significant correlation for all genes tested. In summary, MLPA is a quick and cost-effective method for pathway specific gene expression analysis and the present analysis of 67 CLL cases highlights the rather homogenous apoptosis gene expression profiles among different clinical subsets of the disease.