Prognostic Role of BRAF^V600E Cellular Localization in Melanoma.

Abstract

Approximately half of cutaneous melanoma tissues harbor BRAF^V600E mutations, resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Nuclear-cytoplasmic transport machinery is dysregulated in neoplastic cells and alters the key regulatory proteins that can lead to tumor progression and drug resistance. The significance of nuclear localization of BRAF^V600E has not been fully understood. We examined the clinical significance of intracellular localization of BRAF^V600E in cutaneous melanoma. Immunohistochemical analysis of BRAF^V600E was performed on formalin-fixed, paraffin-embedded specimens of cutaneous melanoma (n = 91). Staining intensity was graded in a blinded manner. Correlations to clinical factors were analyzed by Fisher's exact test and 2-tailed t-test. Localization of BRAF^V600E was determined in melanoma cells, and we investigated their resistance to BRAF^V600E-specific inhibitor according to nuclear localization in both in vitro and in vivo models. We included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAF^V600E. Nuclear BRAF^V600E was observed in 30% (27 of 91). Overall, BRAF^V600E expression correlated with TNM stage (p = 0.011), mitotic activity (p = 0.010), and ulceration (p = 0.045). Nuclear BRAF^V600E expression correlated with overall clinical stage (p < 0.001), tumor size (p < 0.001), regional lymph node (p < 0.017), depth of invasion (p = 0.005), Clark level (p < 0.001), mitotic activity (p < 0.001), ulceration (p < 0.001), and margin status (p = 0.017). On a cellular level, BRAF^V600E was identified in the nucleus, and its translocation was serum dependent. Our in vitro and in vivo data revealed sequestration of BRAF^V600E in the cytosol-sensitized resistant cells to vemurafenib; nuclear retention of BRAF^V600E was associated with aggressiveness and drug resistance. Nuclear localization of BRAF^V600E is associated with melanoma aggressiveness. Further multi-institutional studies are warranted to confirm the clinical relevance of nuclear localization of BRAF^V600E.