[CANCER RESEARCH 63, 3955–3957, July 15, 2003] Analysis of BRAF and N-RAS Mutations in Metastatic Melanoma Tissues 1 Alexis Gorden, Iman Osman, Weiming Gai, Dan He, Weiqing Huang, Anne Davidson, Alan N. Houghton, Klaus Busam, and David Polsky 2 Ronald O. Perelman Department of Dermatology [A. G., I. O., W. G., D. H., D. P.], Kaplan Comprehensive Cancer Center [I. O.], New York University School of Medicine, New York, New York 10016; Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 [W. H., A. D.]; and Memorial Sloan-Kettering Cancer Center, New York, New York 10024 [A. N. H., K. B.] ABSTRACT We examined mutations in BRAF exons 11 and 15 and N-RAS exons 2 and 3, in 77 metastatic melanoma cases and 11 melanoma cell lines. Significant differences in the mutation rates observed at different meta- static sites could not be detected. The most frequent mutation, the V599E amino acid substitution in BRAF exon 15, was observed in 31 of 77 (40%) tissues and 5 of 11 (45%) cell lines. Tandem base-pair substitutions encoding V599R and V599K amino acid changes were observed in two cases. Novel findings with respect to melanoma include a cell line possess- ing a 2 base-pair substitution in BRAF exon 11 and a case harboring mutations in both BRAF exon 11 and N-RAS exon 3. Our data show that BRAF mutation is common in melanoma metastases, regardless of their site, that mutations include both exons 11 and 15, and suggest that anti-RAS/RAF strategies may be effective in metastatic melanoma patients. INTRODUCTION Novel treatment modalities are clearly needed to improve the dismal outcome of metastatic melanoma patients (1–3). In the treat- ment of solid tumors, strategies to target specific oncogenes are becoming increasingly important. Efficacy has been demonstrated for trastuzumab (Herceptin) in the treatment of metastatic breast cancer patients with HER-2 overexpression (4), and ST1571 (Gleevec), a tyrosine kinase inhibitor that targets the product of the BCR-ABL translocation found in chronic myeloid leukemia, as well as the constitutively active KIT receptor found in gastrointestinal stromal tumors (5). Activation of the RAS/RAF/MAPK 3 pathway is a critical component of tumor cell proliferation and survival in general (6, 7) and specifically in melanoma (8 –10). Until recently, mutational stud- ies of the RAS/RAF/MAPK pathway in melanoma tissues were lim- ited primarily to RAS genes, detecting mutation rates of N-RAS in 5–33% of cases (11–13). BRAF, a member of the RAF family of serine-threonine kinases, activates the MAPK cascade when it is bound to activated RAS proteins (6). Recent studies reported frequent BRAF mutation in melanoma cell lines and primary cultures (14, 15). Limited attention has been given to metastatic melanoma clinical specimens (16), which would be more representative of the specific patient population to be treated by anti-RAS/RAF targeted therapy. In this study, we focused on examining the frequency of BRAF and N-RAS mutations in a large cohort of metastatic melanoma tissues to determine whether patients with this stage of disease could be con- sidered for clinical trials using anti-RAS/RAF-based strategies. MATERIALS AND METHODS Tumor Specimens and Cell Lines. Seventy-seven frozen metastatic mel- anoma samples and corresponding normal tissues were retrieved from the Memorial Sloan-Kettering Cancer Center Tumor Bank. Pathology information, excluding patient-identifying information, was reviewed to determine the anatomical site of metastatic tumor. Fifty of 77 lesions (65%) were from lymph nodes or soft tissue sites; 27 of 77 (35%) were from visceral sites. The Institutional Review Boards of both Memorial Sloan-Kettering Cancer Center and New York University approved the conduct of this study. Human meta- static melanoma cell lines (SK-MEL 19, 29, 85 ,94, 100, 103, 147, 173, 187, 192, and 197) were maintained in culture using DMEM with 10% FCS and 1% penicillin/streptomycin (BioWhittaker). Mutation Detection. DNA was extracted from tumor tissues and cell lines using the QIAgen Tissue Kit (Qiagen, Valencia, CA). BRAF exons 11 and 15, and N-RAS exons 2 and 3, were amplified by PCR using QIAgen HotStar Taq. The primer sequences for BRAF exons 11 and 15 have been published (14); the primer sequences and annealing temperatures used for analysis of N-RAS were: Exon 2: Ta = 63°C F: 5'-CTGGTTTCCAACAGGTTCTTGC; R: 5'-CTACCACTGGGCCTCACCT Exon 3: Ta = 53°C F: 5'-CATACTGGATACAGCTGGAC; R: 5'-TGACTTGCTATTATTGATGG, or, Ta = 58°C F: 5'-GGTGAAACCTGTTTGTTGGA; R: 5'-ATACACAGAGGAAGCCTTCG PCR products were subjected to direct sequencing using an ABI PRISM 310 Genetic Analyzer. The resulting traces were analyzed using SeqScape software (Applied Biosystems, Foster City, CA) to identify mutations. Mutations were confirmed by repeat PCR and sequencing using a different primer. For one case containing a 2-bp substitution in BRAF exon 15, PCR products from four separate reactions were cloned into Topo TA (Invitrogen, Carlsbad, CA), and 17 colonies were sequenced. RESULTS Overall, mutations in BRAF exons 11 and 15, and N-RAS exons 2 and 3, were detected in 36 of 77 (47%) tissues and 8 of 11 (73%) cell lines (Table 1). Among metastatic sites, mutations were seen in 9 of 16 (56%) visceral sites, 14 of 28 (50%) lymph node metastases, 5 of 11 (45%) lung metastases, and 8 of 22 (36%) soft tissue metastases. There was no statistically significant difference between these fre- quencies ( 2 , P = 0.65). Importantly, no germ-line mutations were identified in normal tissue samples obtained from the patients whose tumors sustained BRAF or N-RAS mutations (n = 36; Table 1). The most frequent mutation was the T1796A substitution, resulting in the V599E amino acid change in BRAF exon 15. This mutation was detected in 31 of 77 (40%) tissues and 5 of 11 (45%) cell lines. Tandem bp substitutions, GT1795-96AG and GT1795-96AA, which encode V599R and V599K amino acid changes, respectively, were detected in two other melanoma tissues (Fig. 1A). Cloning and se- quencing of PCR products from the GT/AG tandem mutation con- firmed that this case did not possess a compound heterozygous mu- tation. Both of these tandem mutations occurred in lung metastases. In the cell lines, all five mutations were the T1796A substitution (Table 1). Among specimens that contained an exon 15 mutation, no BRAF exon 11 or N-RAS mutations were observed. Received 12/23/02; accepted 5/8/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grant K08 AR02129 (to D. P.). This was also supported in part by the use of facilities at the Manhattan Veterans Affairs Medical Center. 2 To whom requests for reprints should be addressed, at Department of Dermatology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. Phone: (212) 686-7500, extension 3927; Fax: (212) 951-3214; E-mail: david.polsky@med.nyu.edu. 3 The abbreviation used is: MAPK, mitogen-activated protein kinase. 3955 on July 22, 2015. © 2003 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from