Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy.,BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used.,Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable.,Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples.,Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques.,Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered.,Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples.,Combining different methods excludes that the observed heterogeneity is technique-based.,We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results.,Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.
Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths.,BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60-70% of malignant melanomas.,The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion.,In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK.,These pathways are potential targets for prevention and treatment of melanoma.,In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism.,Treatment of multiple human malignant melanoma cell lines with fisetin (5-20 µM) resulted in inhibition of cell invasion.,BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2.,In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway.,Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion.,Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein).,Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin.,These results indicate that fisetin inhibits melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.