Williamson et al interrogated the fusion gene status of appr
Williamson et al.  interrogated the fusion gene status of 210 appropriately reviewed and annotated RMS specimens divided into three groups: ERMS (n=77); fusion gene-positive ARMS (n=94); and fusion gene-negative ARMS (n=39), the latter comprising a group of tumor samples that appeared histologically to be ARMS, but did not have an identifiable PAX-FOXO1 translocation. The authors demonstrated that ERMS and fusion gene-negative ARMS arise in the same locations and have a comparable frequency of metastases — distinct from fusion gene–positive ARMS. Most importantly, they had indistinguishable outcomes with therapies that were not stratified for histologic subtype. In ES samples we find a low IGF2 gene expression compared to control (P<0.0001). EWS–FLI1-negative samples showed a higher IGF2 expression than EWS–FLI1-positive samples (p=0.0231). Steigen et al. (2009) investigated 14 ES samples by immunohistochemistry. In 27% of the samples, they detected a high expression of the IGF2 protein. However, in comparison to other tumors, such as SS, the IGF2 expression level in the ES samples was low. Signaling of the Hedgehog pathway is a necessary component of embryonic development. IHH gene initiates Hedgehog pathway signaling by binding to the transmembrane PTCH1 receptor protein. In the unbound state, PTCH1 inhibits the G-protein coupled receptor smoothened (SMO). The zinc finger transcription factor GLI1, a potent oncogene and a key effector of normal and aberrant SHH pathway signaling, functions solely as a transcriptional activator . In skeletal muscle, the Hedgehog signaling pathway regulates self-renewal versus differentiation fates of muscle stem Natural Product Library receptor and thus controls the balance of proliferation versus differentiation. This embryonic signaling pathway is responsible for normal skeletal muscle differentiation may therefore play a critical role in RMS tumorigenesis by stabilizing muscle precursor cells . In RMS samples, compared to control, the IHH gene showed a high expression in ERMS samples (p=0.0104) and ARMS samples (p=0.0334). Moreover, the ARMS samples showed a higher expression than the ERMS samples (p=0.0095). Increased expression of the IHH gene may suggest that Hedgehog pathway could be activated in these tumors. Hedgehog pathway participates in the development, acting in processes such as proliferation and suppression of cell differentiation. Thus, activation of Hedgehog pathway may lead to tumor development. IHH gene is essential to skeletal muscle development in vertebrates . In Ihh−/−embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, Bren-Mattison et al.  manipulated the Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh−/−mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by retroviral infection of chicken embryo hindlimbs restores muscle mass . The increased expression of IHH gene, as observed in our samples, may have contributed to the tumorigenesis by acting in the opposite way to that observed by the authors mentioned above. An increased expression would increase the cell division, causing imbalance of the focal skeletal muscle and therefore acting on tumor development. ES samples showed higher IHH, PTCH1 and GLI1 gene expressions than control (P<0.0001; p=0.0173 and p=0.0113, respectively). EWS–FLI negative samples showed a higher IHH gene expression than control (p=0.0016). Moreover, EWS–FLI1-positive samples showed a higher GLI1 gene expression than control (p=0.0267). Our results corroborate a recent finding from the literature. Joo et al.  performed qPCR to quantify relative expression the IHH, PTCH1 and GLI1 genes, in 12 ES samples positive of EWS–FLI1 rearrangement. Although, PTCH1 and GLI1 genes showed high expression levels, IHH gene showed a low expression. They performed a reduction of the EWS–FLI1 rearrangement expression in ES cell lines and observed a decrease of IHH gene expression. The differences in IHH gene expression levels observed in both studies could be explained by the number of samples, 60, in our study and 12 on study of Joo et al. . Moreover, they used only EWS–FLI1 positive samples. In our study, we also used EWS–FLI1-negative samples.