Activating BRAF and N-Ras mutations in sporadic primary melanomas: an inverse association with allelic loss on chromosome 9 Rajiv Kumar* ,1,2 , Sabrina Angelini 2 and Kari Hemminki 1,2 1 Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, Heidelberg 69210, Germany; 2 Department of Biosciences, Center for Nutrition and Toxicology, Karolinska Institute, Novum, Huddinge 14157, Sweden We searched and report mutations in the BRAF and N-ras genes in 22 out of 35 (63 percent) primary sporadic melanomas. In three melanomas, mutations were con- comitantly present in both genes. In all, 10 out of 12 mutations in the BRAF gene involved the ‘hot spot’ codon 600 (In all communications on mutations in the BRAF gene, the nucleotide and codon numbers have been based on the NCBI gene bank nucleotide sequence NM_004333. However, according to NCBI gene bank sequence with accession number NT_007914, there is a discrepancy of one codon (three nucleotides) in exon 1 in the sequence with accession number NM_004333. The sequence analysis of exon 1 of the BRAF gene in our laboratory has shown that the sequence derived from NT_007914 is correct (Kumar et al., 2003). Due to the correctness of the latter, sequence numbering of codons and nucleotides after exon 1 are changed by þ 1 and þ 3, respectively.), one tandem CT1789-90TC base change represented a novel mutation and another mutation caused a G466R amino- acid change within the glycine-rich loop in the kinase domain. Mutations in the N-ras gene in 11 melanomas were at codon 61 whereas two melanomas carried mutations in codon 12 including a tandem mutation GG4AA. We observed an inverse association between BRAF/N-ras mutations and the frequency of loss of heterozygosity (LOH) on chromosome 9 at 10 different loci. Melanomas with BRAF/N-ras mutations showed a statistically significant decreased frequency of LOH on chromosome 9 compared with cases without mutations (mean fractional allelic loss (FAL) ¼ 0.2970.23 vs 0.7270.33; t-test, P ¼ 0.0001). Difference in the FAL value between tumours with and without BRAF/N-ras mutations on 33 loci on five other chromosomes was not statistically significant (mean FAL 0.1770.19 vs 0.2570.22; t-test, P ¼ 0.24). Melanoma cases with BRAF/N-ras mutations were also associated with lower age at diagnosis than cases without mutations (mean age 80.3877.24 vs 65.77719.79 years; t-test, P ¼ 0.02). Our data suggest that the occurrence of BRAF/N-ras muta- tions compensate the requirement for the allelic loss at chromosome 9, which is one of the key events in melanoma. Oncogene (2003) 22, 9217–9224. doi:10.1038/sj.onc.1206909 Keywords: BRAF; N-ras; melanoma; mutation; FAL; SSCP Introduction Cutaneous malignant melanoma is a potentially fatal neoplasm with complex and heterogeneous etiology (Chin etal., 1998). The sporadic form, which constitutes over 90% of all cases, is linked to sunlight exposure (Gilchrest et al., 1999; Hemminki et al., 2001). Though the mechanism for the casual relationship between sunlight exposure and melanoma is less clear, the epidemiological data are strongly supportive of such an association (Whiteman et al., 2001). The inherited form of melanoma is associated with the melanoma susceptibility locus on chromosome 9p21. Germline mutations in the CDKN2A gene, which encode two cell cycle inhibitors p16 INK4a and p14 ARF , are found in a proportion of melanoma-prone families (Bishop et al., 2002). However, somatic alterations in sporadic mela- noma are heterogeneous. Allelic loss at chromosome 9p21 locus is the most prevalent genetic occurrence in sporadic melanoma, but mutations and other alterations in the CDKN2A gene, though common in melanoma cell lines, are less evident in primary tumours (Kumar etal., 1998b, 1999). The predominant oncogenic changes associated with malignant melanoma have been the activating mutations in the N-ras gene (Castellano and Parmiani, 1999; Saida, 2001). The reported frequency of mutation in the ras genes has varied; the most common mutation detected in melanomas involves codon 61 and these are reported to occur early (van Elsas etal., 1997; Omholt et al., 2002). An activated RAS stimulates a multitude of downstream signalling cascades (Shields et al., 2000). RAF serine/threonine kinases are the key signalling components in the RAS pathway (Kolch, 2000). In melanocytes, BRAF, one of the members of the RAF family, is activated in a cAMP-dependent signalling cascade, as a consequence of a-melanocyte-stimulating Received 30 April 2003; revised 23 June 2003; accepted 26 June 2003 *Correspondence: R Kumar, Division of Molecular Genetic Epide- miology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580 TP3, Heidelberg 69210, Germany; E-mail: rajiv.kumar@cnt.ki.se Oncogene (2003) 22, 9217–9224 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc