Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423-7).,Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.,We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen.,We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAFV600E mutation, as well as by cloning and sequencing of separated alleles.,Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.,Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells.,Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas.,Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.,Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.
Resistance to BRAF inhibitors is an emerging problem in the melanoma field.,Strategies to prevent and overcome resistance are urgently required.,The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot.,The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture.,BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling.,Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy.,A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance.,Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF-V600E-mutated melanomas.