SAGE Analysis Demonstrates Increased Expression of TOSO Contributing to Fas Mediated Resistance in CLL.

The molecular pathogenesis of CLL remains unclear; therefore we sought to identify new genes involved in the pathogenesis or mechanisms of resistance in CLL and other B cell malignancies. To this end we performed SAGE in CLL cells and compared results with those from healthy B cells (nCD19+). Gene expression profiles were obtained by SAGE and individual gene expression was measured by real time PCR. Analysis revealed only 27 genes with significant (p< 0.001) differences between mutated and unmutated subtypes of CLL cells. In contrast to the high level of similarity between the CLL subtypes, comparison of the complete CLL SAGE libraries versus nCD19, revealed that 56 genes were over represented and 50 genes were down regulated in CLL (fold change higher than 20x, p<0.001 as a cut-off). A gene ontology analysis using the top 1,000 transcripts revealed some genes aberrantly expressed in CLL not previously reported. One of these is TOSO (regulator of Fas-induced apoptosis) of interest since CLL cells are resistant to Fas mediated killing. Real-time RT-PCR analysis of 78 cases of CLL and 12 nCD19+ confirmed that TOSO mRNA was over expressed (4.4 fold) in CLL (p<0.0001), and its expression was positively correlated with BCL-2 expression. There was no correlation between TOSO expression and prognostic factors, including IgVH mutational status, cytogenetics, evolution or previous treatment. In addition, using tissue microarray of 1mm cores arranged in triplicate followed by indirect immunohistochemistry we evaluated TOSO protein distribution and intensity in 44 CLL lymph nodes and 5 reactive tonsils and appendix. Scoring was performed by two histopathologists independently and demonstrated that more than 50% of CLL cases express TOSO strongly (2+, 3+) with a range of 10% to 95% of positive tumour cells (p=0.0057, Chi-Square test), whereas among control samples analyzed all were negative except the mantle cells from one tonsil and one appendix were scored as weakly positive (1+); the remainder were negative. Flow cytometry analysis in 12 CLL cases and 5 healthy subjects demonstrated a 5.6 fold increase of TOSO in peripheral CLL cells (p=0.013). SAGE results confirmed that both CLL subtypes share a similar expression profile and highlighted a novel gene, TOSO. This protein plays a functional role upstream of Fas in the extrinsic apoptosis pathway and ongoing studies are assessing the mechanisms whereby TOSO mediates resistance of CLL cells to anti-Fas treatment and ways to reverse this.