1. ¹Human Tumor Immunobiology Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy
2. ²Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy
3. ³Drug Resistance and Experimental Therapeutic Section, Department of Drug Research and Evaluation Pharmacogenetic, Istituto Superiore di Sanità, Milano, Rome, Italy
4. ⁴Unit of Immunotherapy of Human Tumors, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy

Correspondence: Dr M Sensi, Human Tumor Immunobiology Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, via Venezian 1, Milano 20133 Italy. E-mail: marialuisa.sensi@istitutotumori.mi.it

Received 15 August 2005; Revised 29 November 2005; Accepted 5 December 2005; Published online 6 February 2006.

Abstract

Activating 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.