Abstract 10: High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer.

Abstract At time of diagnosis 25% of all bladder cancers are muscle invasive and require cystectomy, radiotherapy, or chemotherapy. Metastasis is the principal event leading to death of individuals with cancer, and several attempts have been made to identify prognostic biomarkers for metastatic disease. The aim of this study was to compare gene-expression profiles from 10 primary bladder tumors with 12 matched lymph node metastases and eight primary tumors without metastasis to identify markers associated with metastatic disease. All 30 tumors were laser micro-dissected and mRNA expression was measured using Affymetrix U133 +2 GeneChips. MicroRNA expression was measured using TaqMan® MicroRNA Assays from Applied Biosystems. We compared our data to gene expression data from a previously published study with 69 primary bladder tumors to identify potential markers for metastasis (GEO ID: 200031684), and Gene Set Enrichment Analysis (GSEA) was used to test if previously published gene signatures were enriched in our data. We identified 12 genes significantly up-regulated at least two fold in primary tumors with metastasis compared to non-metastatic tumors in both patient cohorts. Of the 12 genes, GEM (GTP binding protein overexpressed in skeletal muscle), EDNRA (Endothelin receptor type A), and PXDN (peroxidasin homolog) were also significantly up regulated in the corresponding lymph node metastases. Validation at the protein level by immunohistochemistry using tissue microarrays containing 417 core biopsies from both primary tumors and lymph node metastasis showed that high expression of GEM (P = 0.032) and EDNRA (P = 0.037) were significantly associated with decreased cancer-specific survival. High EDRNA expression was significantly associated with decreased cancer-specific survival (P = 0.039) while GEM showed no significance (P = 0.15) in multivariate Cox regression analysis. Furthermore, 94% of the lymph nodes showed similar expression as in the primary tumors for EDNRA and 71% for GEM. PXDN expression was not significantly associated with outcome (P = 0.52). GSEA analysis showed that gene-sets involved in DNA double-strand repair was significantly enriched in tumors without metastasis (P = 0.002). Furthermore, a previously reported gene-set identified from comparison of metastases and primary tumors were significantly enriched in the primary tumors with metastasis (P = 0.03). In addition, we found 29 microRNAs to be down regulated in the lymph nodes compared to the primary tumors, and only one was up-regulated. Interestingly, miR-143 and miR-145 were both significantly down regulated in the lymph nodes. They are known to be down-regulated in bladder cancer, and this further down-regulation in the metastases supports the reported tumor suppressor function of these molecules. Additional studies are needed to validate the functions and clinical relevance of GEM and EDNRA. Citation Format: Jens R. Laurberg, Jørgen Jensen, Troels Schepeler, Michael Borre, Torben Ørntoft, Lars Dyrskjøt. High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 10. doi:10.1158/1538-7445.AM2013-10 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.