Molecular testing for BRAF mutations to inform melanoma treatment decisions: a move toward precision medicine

Abstract

Approximately one-half of advanced (unresectable or metastatic) melanomas harbor a mutation in the BRAF gene, with V600E being the most common mutation. Targeted therapy with BRAF and MEK inhibitors is associated with significant long-term treatment benefit in patients with BRAF V600-mutated melanoma. Therefore, molecular testing for BRAF mutations is a priority in determining the course of therapy. A literature search was performed using MEDLINE/PubMed and scientific congress databases using the terms 'BRAF,' 'mutation,' and 'cancer/tumor.' These results were filtered to include manuscripts that focused on diagnostic tests for determining BRAF mutation status. Numerous BRAF testing methods were identified, including DNA-based companion diagnostic tests and DNA- and protein-based laboratory-developed tests. Herein we review the characteristics of each method and highlight the strengths and weaknesses that should be considered before use and when interpreting results for each patient. Molecular profiling has shown that mutation load increases with melanoma tumor progression and that unique patterns of genetic changes and evolutionary trajectories for different melanoma subtypes can occur. Discordance in the BRAF mutational status between primary and metastatic lesions, as well as intratumoral heterogeneity, is known to occur. Additionally, the development of acquired resistance to combination BRAF and MEK inhibitor therapy is still a formidable obstacle. Therefore, tumor heterogeneity and the development of acquired resistance have important implications for molecular testing and ultimately the treatment of patients with advanced-stage melanoma. Overall, this information may help community oncologists more accurately and effectively interpret results of diagnostic tests within the context of recent data characterizing melanoma tumor progression.

Figure 1

BRAF mutation types in melanoma. Estimated incidence of BRAF mutation frequencies in patients with melanoma is shown.

Figure 2

Genomic evolution of melanoma. Genetic alterations accumulate during melanoma progression, from benign lesions that carry the BRAF V600E mutation to intermediate lesions that have accumulated more genetic mutations to invasive melanoma that has acquired pathogenic mutations that confer the ability to metastasize. As melanoma progresses, distinct evolutionary trajectories for different melanoma subtypes develop.

Figure 3

Processing of lesions for pathological analysis of BRAF V600 mutation. (a) The suspicious lesion is biopsied and prepared for formalin-fixed, paraffin-embedded tissue. The tissue sample is immersed in 10% buffered formalin for 4–6 h, then immersed in a series of increasingly concentrated ethanol to extract water. The final dehydration occurs in xylene, after which the tissue is immersed in a series of increasingly concentrated molten paraffin. Finally, the sample is embedded in a block of paraffin suitable for serial sectioning. Serial sectioning produces ‘ribbons’ of tissue sections that can be directly mounted onto slides, deparaffinized, and then stained. Every other section is stained, typically with hematoxylin and eosin, to identify tumor margins while unstained alternate sections are used for immunohistochemistry (IHC) or to obtain DNA for genetic analyses. In contrast to the process for surgical specimens, fine-needle aspirates are taken and transferred directly from the syringe to a slide, where the material is smeared across its surface, air dried, and fixed. (b) IHC reveals cellular expression of the BRAF kinase in BRAF mutation-positive (dark gray) cells in situ. (c) RT-PCR uses a fluorescent-labeled target-specific probe allowing amplification of the target sequence and real-time quantification of PCR products. EtOH, ethyl alcohol.