(P 17) METASTASIS-SPECIFIC GENE EXPRESSION PATTERNS IN EWING SARCOMA

Objective: Prognosis for Ewing Sarcoma (EwS) patients with disseminated disease or at relapse remains poor. In most cases, the site of relapse corresponds to the site metastases. Though it is speculated that the transient reduction of oncogene expression promotes dissemination, the mechanisms of metastasis in EwS remain elusive. Therefore, we aimed to identify differentially expressed genes specific for metastases.

Methods: We re-evaluated published microarray datasets on gene expression levels of primary, metastatic and recurrent tumor samples, healthy tissue samples and cell lines. We checked whether the resulting hits were directly regulated by EWSR1-FLI1 by published RNA-Seq data and qRT-PCR after EWSR1-FLI1 knockdown. Furthermore, the 27 highest expressed hits were tested in qRT-PCRs for four EwS cell lines and two human mesenchymal stem cell (hMSC) lines to provide a basis for in-vitro studies to reverse-engineer metastatic mechanisms.

Results: Our data analysis revealed 72 upregulated genes in EwS metastases. One of the top hits was the cancer testis antigen PRAME. Interestingly, most upregulated genes (68%) were also highly expressed in EwS cell lines. These results were confirmed by qRT-PCRs for the top hits in four EwS cell lines, so far. While 36 upregulated genes were downregulated, only one gene (HOXB13) was upregulated upon EWSR1-FLI1 knockdown in an RNA-Seq dataset.

Conclusion: Most of the upregulated genes we found in EwS metastasis data have already been associated with migration, invasion or aggressiveness in other cancer entities. We are currently analyzing whether PRAME expression is associated with severity of the disease in histological patient samples. Strikingly, only HOXB13 seemed to be negatively regulated by the fusion oncogene, rendering it a putative mediator in metastasis. We will evaluate our findings by analyzing the impact of manipulating the expression levels of some of the top hits in vitro and in vivo.