Abstract
Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs.
Highlights
Melanoma is a highly aggressive malignancy and accounts for the majority of all skin cancer-related deaths [1]
To identify frequently occurring mutations to be included in the melanoma-specific mutation panel, 43 stage III melanoma cell lines were screened with the Sequenom OncoCarta panel
BRAF and NRAS mutations were mutually exclusive, with mutations occurring in approximately 60% (25 of 43) and 20% (9 of 43) of cell lines, respectively
Summary
Melanoma is a highly aggressive malignancy and accounts for the majority of all skin cancer-related deaths [1]. The high mortality rate for advanced stage metastatic melanoma is largely due to the ineffectiveness of currently approved systemic treatment strategies [2]. Use of traditional chemotherapeutic approaches has shown poor response rates; less than 10% of patients demonstrate a clinical response to standard treatment with dacarbazine and less than 5% patients survive. Authors' Affiliations: 1Queensland Institute of Medical Research, Oncogenomics Laboratory; 2Faculty of Science and Technology, Queensland University of Technology; 3Sequenom Inc. Asia Pacific; 4Queensland Institute of Medical Research, Cancer Immunotherapy Laboratory; 5Morris Tower, Brisbane, Queensland; 6University of Sydney at Westmead Millennium Institute, Westmead; 7Melanoma Institute Australia (formerly the Sydney Melanoma Unit), North Sydney; 8Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown; and 9Discipline of Pathology, The University of Sydney, New South Wales, Australia. Novel effective moleculartargeted therapies for the treatment of metastatic melanoma have emerged
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