Beschreibung:
Abstract Background & Objectives: Relapse of Ewing sarcoma (ES) can occur months or years after initial remission, and salvage therapy for relapsed disease is usually ineffective. There is great need to develop biomarkers that can predict which patients are at risk for relapse, so that therapy and post-therapy evaluation can be adjusted accordingly. In the current study we sought to identify prognostic gene expression signatures in patients diagnosed with ES. Methods: Specimens were obtained from the Children's Oncology Group (COG) Biorepository. All samples were prospectively acquired from patients treated on clinical trials INT-0154 and AEWS0031. Criteria for inclusion included confirmation of localized ES, registration on a clinical trial and availability of frozen tumor. Total RNA was processed for whole genome expression profiling using Affymetrix GeneChip Human Exon 1.0 ST arrays. Affymetrix Power Tools (APT) were used to generate normalized gene-level signal intensity estimates from raw CEL file data. Differentially expressed genes with false discovery rate (FDR) of < 0.2 and absolute fold-change > 1.3 were considered to be significantly associated with outcome (survivor vs. non-survivor, relapse vs. no-relapse). Differentially expressed genes were used to create prognostic gene signatures. An independent group of ES samples from the Euro-Ewing tumor Biorepository in Muenster, Germany was similarly analyzed as a validation cohort. Results: Frozen tumor tissue was available from 254 COG patients. Following RNA and sample QC, Affymetrix CEL files were successfully generated from 56 unique patients with localized disease for whom outcome data were available. Analysis of processed gene expression data led to exclusion of 10 cases from further analysis due to issues of batch effect and outlier status. The demographic, event-free (EFS), and overall (OS) characteristics of the remaining 46 cases were shown to be representative of the INT-0154 and AEWS0031 studies as a whole. Unsupervised clustering of transcript-level data failed to demonstrate segregation of the 46 tumors into groups based on relapse or survival status. Supervised analysis of survivors vs. non-survivors identified a small number of differentially expressed genes and several statistically significant gene signatures, but none of these candidate classifiers could be internally validated. Interestingly, gene set enrichment analysis (GSEA) reproducibly revealed that integrin and chemokine receptor pathways were significantly over-represented as discriminators of EFS and OS (FDR<0.25; nominal p<0.01). However, the significance of these pathways was limited to tumor samples that contained at least 30% stromal cell contamination. Analysis of an independent cohort of 39 tumors from the Euro-Ewing biorepository (all with <30% stromal content) failed to identify prognostic genes, and no pathways reached statistical significance upon GSEA analysis. Conclusions: Robust prognostic gene signatures that pass internal and external cross-validation were not identified in this study. These findings demonstrate the profound degree of molecular heterogeneity in ES and suggest that the heterogeneous nature of these tumors is likely to preclude identification of prognostic signatures that will be clinically useful for all cases. Whether prognostic gene signatures will be useful for differentiating among subsets of ES cases is unknown and will require larger cohort analyses. Nevertheless, these studies support recent laboratory-based discoveries implicating cell adhesion and chemokine receptor signaling in ES aggression. Further investigation of these pathways in the context of tumor cell:tumor stroma interactions is warranted. Citation Format: Samuel L. Volchenboum, Jorge Andrade, Donald A. Barkauskas, Mark Krailo, Richard Sposto, Andreas Ranft, Jenny Potratz, Uta Dirksen, Timothy J. Triche, Elizabeth Lawlor. Do prognostic gene signatures exist in Ewing sarcoma? A report from the Children's Oncology Group. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A72.