Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma

BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS 'double mutants' may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAF(V600E) and the activating NRAS(Q61R) mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAF(V600E), but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRAS(Q61R)/wild-type BRAF). When compared to BRAF(V600E) clones, NRAS(Q61R) clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRAS(Q61R) or BRAF(V600E) is associated with distinct in vitro and in vivo growth properties of neoplastic cells.