Apoptosis Taqman Low-Density Array: Analysis of Programmed Cell Death Patway in CLL.

The clinical course of CLL is highly heterogeneous: some patients progress rapidly thus requiring early chemotherapy whereas others exhibit a stable disease over years. Gene expression studies have identified a relatively small number of genes that are differentially expressed between these subsets. Resistance to programmed cell death seems to be one of the preferential pathways of neoplastic B cells to survive and to develop resistance to therapy. We investigated by MicroFluidic Card™ technology patients affected by untreated CLL cells for alterations of agonist and antagonist apoptosis genes. Our aim was to shed light on programmed cell death pathway in this particular subset of patients.

Methods

34 CLL and 30 normal controls were evaluated. Highly purified (>90%) B-CLL cells were obtained from all the patients after magnetic cell separation (CD19+ microbeads, Miltenyi Biotech). 92 human apoptosis-related genes as well as 4 reference genes were analyzed in duplicate by MicroFluidic Card method based on TaqMan™ technology (Applied Biosystems). Raw data from the analysis were converted to relative gene expression quantity (RQ) by GeneNorm software. RQs were median normalized, log2 transformed, and variations among patients were investigated by both unsupervised (hierarchical clustering) and supervised methods. Selection of significant genes between normal and neoplastic samples was accomplished by the following criteria:

• a ratio between the averages of the two groups ≥ 2 or ≤ 0.5 and

• a p value of Welch T test ≤0.01.

Results

Unsupervised hierarchical clustering (dChip software) with the complete gene dataset revealed the homogeneity of normal samples (Euclidean distance, average linkage method p=0.0012). 14 were differently regulated in CLL, in particular 7 genes (CASP8AP2, TNFRSF4, TNFSF14, BCL2, CD40LG, CDKN2A and ZAP-70) were up regulated and 7 (CASP10, BIRC5, LTB, BCL2A1, TNFSF10, TNFRSF8 and BID) were down modulated. ZAP-70+ and ZAP-70- groups showed 5 genes differentially expressed (p≤ 0.01, BIK, LTBR, TNFSF11, TNFRSF1A and BCL2L2), with all the targets up regulated in the ZAP-70 subset. When the chromosome status was investigated, ANOVA test revealed 5 genes differentially regulated (p< 0.05, TNFRSF1B, BIRC6, BCL2, BCL2L2 and TNFRSF10D) between the groups (11q-, 13q- and negative sets). Hierarchical cluster revealed the formation of specific sample groups for specimens with β2microglobulin increase (>2,2 mg/l) and for those with mutated IgV_H(Euclidean distance, p=0.0074 and p=0.0006, respectively).

Conclusions

Indolent CLL is poorly characterized in its molecular aspects. The comprehensive profiling of gene expression in CLL can provide a molecular framework for understanding the pathophysiology of this disease. Significant alterations of the apoptotic pathway at both the extrinsic and the intrinsic levels among asymptomatic CLL patients were found. The system uses only 2 ng of sample and small volumes of reagent, and the precaptured primers and probes avoided labor-intensive pipetting steps. This procedure could be an useful method to identify targets associated with clinical outcome.