Variability in gene expression patterns of Ewing tumor cell lines differing in EWS-FLI1 fusion type

Type 1 and type 2 EWS-FLI1 fusion products result from variation in breakpo int locations arising from the t(11;22)(q24;q12) recurrent chromosomal tran slocation in Ewing's sarcoma family tumors (EFT). Previously, studies from our institution (updated in the present communication at a median follow-up of more than 6 years) and others suggested a prognostic difference for EFT patients with localized disease depending on the type of EWS-FLI1 fusion p resent in the tumor. It has been suggested that the observed clinical discr epancies result from different transactivation potentials of the various EW S-FLI1 fusion proteins. In an attempt to identify genes whose expression le vels are differentially modulated by structurally different EWS-FLI1 transc ription factors, we have used two related PCR-based subtractive approaches, cDNA representational difference analysis (cDNA-RDA) and linker-capture su btraction (LCS) to compare transcript representations in cDNA pools of type 1 versus type 2 EFT cell lines. About 800 clones obtained by the two appro aches were analyzed by dot blot hybridization to cDNA pools. Eighty-six clo nes showing the highest variability in signal intensities on the dot blots were further hybridized to individual EFT cell line RNAs on Northern blots, and four of them were additionally studied by real-time quantitative PGR ( RTQ-PCR). Although interindividual variations in gene expression patterns i n the range of one- to several-fold were observed, no correlation to specif ic EWS-FLI1 fusion types could be identified. Among the genes differentiall y expressed in individual EFT cell lines are several previously implicated in tumor growth, invasion, and metastasis. Although our data may have revea led candidate genes whose composite expression pattern may be relevant for the biology of individual EFT, they do not support a role of distinct EWS-F LI1 fusion types for EFT prognosis based on different transactivation poten tials.