We found all cases to show at least some
We found all cases to show at least some IGF-1R expression by immunohistochemistry. This is consistent with its ubiquity at the mRNA or protein level in ES/PNET cell lines or clinical samples as shown by others.12, 14 Evidence that IGF-1R expression is required for EWS-FLI1 transformation of mouse fibroblasts suggests that in vivo, this fusion protein may have a similar requirement for a IGF-1R-positive precursor cell or for induction of the IGF-1R pathway. Although essentially all cases showed at least some IGF-1R expression, its level could be critical for maintaining cell proliferation in ES/PNET, because only cases with >40% of 3-Bromopyruvic acid clinical expressing IGF-1R had a significant Ki-67 immunoreactivity (>10%; see Figure 3). The differences in extent of IGF-1R immunoreactivity between type 1 and non-type 1 EWS-FLI1 groups, although statistically significant (P = 0.015), were relatively small (65% vs. 82%). Nonetheless, there is experimental evidence that small differences in IGF-1R expression level per cell can effectively modulate cell proliferation. We have recently reported in a partially overlapping series that tumors with EWS-ERG fusions have a similar clinical presentation and behavior as those with EWS-FLI1 chimeric genes. It may thus be unexpected that there was a difference in proliferative index, as assessed by Ki-67 immunoreactivity, between these two groups. No differences in IGF-1R expression were seen between EWS-FLI1 and EWS-ERG cases, suggesting that the observed difference in proliferative rate could be due to differential activation of other target pathways. The extent to which activation of downstream targets may differ quantitatively or qualitatively between EWS-FLI1 and EWS-ERG is unknown. Indeed, outside their DNA-binding domains, FLI1 and ERG show major sequence differences which could be significant in the protein-protein interactions involved in the regulation of certain promoters. It is therefore not unreasonable to consider the possibility that EWS-FLI1 and EWS-ERG may deregulate some partly non-overlapping pathways to generate clinical phenotypes which are so far indistinguishable. Because both EWS-FLI1 and EWS-ERG fusion proteins have been shown to inhibit apoptosis in model systems, we were interested in comparing relative numbers of apoptotic cells in these pretreatment clinical samples. However, the numbers of apoptotic cells shown by the TUNEL technique were small and there were no significant differences among the fusion types. Other determinants of proliferation rate may include secondary genetic alterations in cell cycle regulators. In two series of ES/PNET which partially overlapped with the present study group, we have found evidence of p53 alterations in 11% and INK4A deletion in 17%.40, 41 Similar percentages have been reported by other groups.42, 43, 44, 45, 46, 47, 48 These genetic alterations are relatively uncommon but appear prognostically significant.40, 41, 48 Because of their low prevalence, however, alterations in cell cycle regulators are unlikely to account for the significant differences in Ki-67 values between groups in the present study, but may explain some of the variability in proliferative rate within these groups. Establishing whether the distribution of these secondary genetic alterations in cell cycle regulators is random in relation to EWS-ETS fusion type will require a much larger systematic multiparameter study. In summary, the most common type of EWS-FLI1 fusion transcript, type 1, is associated with a favorable prognosis6, 7 and appears to encode a functionally weaker transactivator compared to other fusion types. The present study indicates that these differences are paralleled by similar differences in proliferative rate, perhaps mediated in part by the putative differential regulation of the IGF-1R pathway by these alternative forms of EWS-FLI1. Further elucidation of the target genes of EWS-FLI1 should clarify this intriguing aspect of the biology of ES/PNET.