BACKGROUND: Despite the recent update to the AJCC staging criteria for melanoma, this system is still unable to identify truly high-risk stage I tumour subsets. OBJECTIVE: To determine clinical utility of combined epidermal AMBRA1/Loricrin (AMLo) expression as a prognostic biomarker for AJCC stage I cutaneous melanoma. METHODS: Peri-tumoural AMBRA1 expression was evaluated in a retrospective discovery cohort of 76 AJCC I melanomas. Multivariate analysis of AMLo expression was subsequently correlated with clinical outcomes up to 12-years in two independent powered, retrospective validation and qualification cohorts comprising of 379 AJCC I melanomas. RESULTS: Decreased AMBRA1 expression in the epidermis overlying primary melanomas in a discovery cohort of 76 AJCC I tumours was associated with 81.5% 7-year DFS versus 100% survival with maintained AMBRA1; P<0.081. Following automated IHC protocol development for semi-quantitative analysis of AMLo further analysis was undertaken in validation (n=218) and qualification cohorts (n=161) of AJCC I melanomas. Combined cohort analysis revealed a DFS of 98.3% in the AMLo low-risk group (n=239) versus 85.45% in the AMLo high-risk cohort (n=140, P<0.001). Sub-cohort, multivariate analysis revealed the AMLo hazard ratio of 4.04 ((95% CI 1.69-9.66) P = 0.002), is a stronger predictor of DFS than Breslow depth (multivariate analysis 2.97 (95% CI 0.93-9.56) P = 0.068) in AJCC stage IB patients. CONCLUSIONS: Loss of AMLo expression in the epidermis overlying primary AJCC stage I melanomas identifies high risk tumour subsets independently of Breslow depth and represents a major paradigm shift in future prognostic assessment and stratification of primary melanomas. This article is protected by copyright. All rights reserved.