GENE EXPRESSION PROFILING IN B-CELL CHRONIC LYMPHOCYTIC LEUKAEMIA REVEALS DIFFERENTIAL EXPRESSION LEVELS OF EDAG AND ANGIOPOIETIN-2 GENES BETWEEN UNMUTATED AND MUTATED PATIENTS

B-cell chronic lymphocytic leukemia (B-CLL) shows a high heterogeneity in the clinical course, spanning from aggressive to completely indolent behaviour. To identify the correspondent gene expression variability, we selected 29 well-defined, newly diagnosed B-CLL patients and we hybridized them on ink-jet printed microarrays comprising about 20,000 oligonucleotide probes. The mutational status of IgVH genes was examined in all 29 CLL cases. Using the classical homology cut off value of 98%, 14 patients (48.3%) had mutated VH genes (M-CLL group) and 15 (51.7%) had unmutated VH sequences (UM-CLL group). As previously reported, different immunoglobulin gene se ments revealed a biased usage related to the mutational immunoglobulin VH gene and poor prognosis was associated with the unmutated subset. Firstly, using 4 different unsupervised clustering algorithms to analyze gene expression data, we identified two robust CLL clusters. Altered expression of genes mainly involved in protein biosynthesis and oxidative phosphorylation differentiated the two main CLL subgroups. These B-CLL biological clusters were not associated with Ig mutational status. Furthermore, we applied another unsupervised algorithm after the exclusion of the genes characterizing the previously identified CLL subsets identifying two new CLL clusters, showing a clear association to the Ig mutational status. In order to identify how the immunoglobulin status could affect the levels of specific genes, we applied supervised clustering based on differences in the IgVH mutation status. Differentially regulated genes were revealed at a significant level of 0.01, yielding 128 genes. Of note, we observed in unmutated CLL cases a down-regulation of genes belonging to B-cell- and antibody-mediated immunity and immunity/defence categories. Moreover, genes involved in Wnt signalling, integrin signalling, inflammation mediated by chemokine and cytokine signalling pathways as well as in T cell activation were found down-regulated in unmutated CLLs. The analysis identified EDAG as the best discriminating gene between M- and UMCLLs. Interestingly, EDAG regulates the proliferation and differentiation of hematopoietic cells and apoptosis resistance through the activation of NF-k B. Again, we found the high expression in UM-CLL of angiopoietin 2 (ANGPT2), inducer of angiogenesis. We evaluated the mRNA levels of EDAG and ANGPT2 in a large cohort of both unpurified and CD19+ purified CLL samples as well as in some cases of LMA, LLA, LMC and in normal PBMCs and CD19+ selected cells, using Real-Time PCR. Then, we analyzed the correlation between EDAG and ANGPT2 mRNA levels as well as their association with immunoglobulin mutational status, haematological parameters and clinical outcome of patients. PO-060