e21037 Background: Staging of cutaneous melanoma (CM), as defined by the American Joint Committee on Cancer (AJCC), includes determination of the sentinel lymph node (SLN) status as a major prognostic factor. We have previously identified and validated a gene expression risk score (GRS) in primary CM and adjacent stroma, consisting of 7 protective genes and 1 risk-associated gene, which predicts patient survival, independently of AJCC stage. This external validation study evaluated single and combined performance of GRS and SLN in predicting relapse-free survival (RFS). Methods: The study cohort included 203 formalin-fixed, paraffin-embedded (FFPE) -primary CMs (AJCC stages IA-IIIC, n=31 RFS events). Tissue samples and clinical data were provided by two centers, and gene expression was determined externally using real-time RT-PCR (double-blind study). External prospective data analysis followed a pre-specified protocol. Results: Integration Discrimination Improvement (IDI) analysis demonstrated non-inferiority of GRS vs. SLN status in predicting RFS (IDI estimate=-0.8%, p=0.040). In univariate Cox regression analysis, GRS and SLN status predicted RFS (GRS: p=0.011, HR=2.44 [1.2-4.95]; SLN: p=0.038, HR=2.12 [1.03-4.38]). Multivariate Cox regression demonstrated independent contribution of prognostic information by both parameters (GRS: p=0.015, HR=2.40 [1.18-4.89]; SLN: p=0.046, HR=2.11 [1.02-4.37]. IDI analysis demonstrated that combining GRS and SLN status improved prognostic performance, as compared to SLN status alone (IDI estimate=3.2%, p=0.044). When complementing SLN status with GRS (SLN positive or high GRS), sensitivity of relapse detection increased from 38.7% for SLN alone to 67.7%. Conclusions: GRS is a non-invasive predictor of RFS, complementary to SLN analysis. Combining GRS and SLN status significantly improves accuracy of melanoma prognosis and, therefore, may guide follow-up care as well as treatment decisions regarding new adjuvant therapies, e.g. of SLN positive patients with high GRS. [Table: see text]