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BRAF mutation testing methods

There are several molecular platforms and methods available to analyse the BRAF mutation status of melanoma tissue, such as first-generation (Sanger) sequencing, polymerase chain reaction (PCR), mass spectrometry base sequencing, next generation sequencing (NGS), high-resolution melting (HRM) analysis, pyrosequencing, and immunohistochemistry.^1,2 Each of these methods has different technical and practical advantages and certain limitations, which makes some methods better suited for particular settings and to the workflow in individual laboratories.¹ One important consideration when selecting the testing methodology is its ability to detect not just the V600E mutation but also other less-common mutations at this location (e.g. V600K, V600E, V600D).

It is essential that the chosen method and test procedure is validated based on appropriate requirements of local regulatory and professional bodies and the laboratory undergoes continued quality assessment and control to assure best performance.

Sole reliance on a single assay is not always appropriate – a false-negative BRAF mutation result means that a patient who could potentially benefit from targeted BRAF-inhibitor therapy has been missed. Therefore, use of alternative second-line testing for tumour samples that are negative on the first assay can sometimes be a rational approach.³

Method^1–4	How it works	Additional information
1^st generation sequencing and its different modifications Sanger chain-termination sequencing Pyrosequencing Mass spectrometry based sequencing	Established technology to ‘read the sequence’ Determining the order of the deoxynucleotide bases (dNTPs): Adenine, Guanine, Cytosine and Thymine, in analysed DNA and therefore identify mutations in genes of interest such as BRAF It is able to generate and read sequence in a gene or genes of interest (e.g. genotyping)	There are several methods based on this technology used in clinical practice for BRAF mutation detection Sanger sequencing was once considered the ‘gold standard’ method for detection of DNA mutations and is often still used as a reference to which new methods are compared
Real-time PCR (polymerase chain reaction) and its different modifications Taqman PCR ARMS (amplification refractory mutation specific system)	Established technology that amplifies a single copy (or a few copies) of a DNA sequence, generating thousands to millions of copies (amplicons). Allows for detection and measurement of the initial amount of DNA present in the sample	There are several methods based on this technology used in clinical practice for BRAF mutation detection
NGS (next generation sequencing)	Emerging sequencing technology which is able to generate and read very large amounts of sequence (e.g. whole genome sequencing – 'genome-typing'). It is also referred to as massive parallel sequencing	There are a number of cancer-related gene target panels which are currently being evaluated/used in clinical practice for detecting BRAF mutations among others; for example Ion Torrent™ NGS Ion AmpliSeq™ Panels⁵
HRM (high-resolution melting)	Technology based on the analysis of the temperature gradient in which the double strands of the PCR products are separated or ‘melted’. Different DNA molecules (based on whether they harbour given mutation or not) have different melting curve patterns	This technology is more often used in the research environment and to compare the new tests with a reference method
IHC (immunohistochemistry)	Detects tumor cells harbouring a specific antigen by cytoplasmic staining of cells containing a mutation-specific monoclonal antibody²	Considered rapid, inexpensive technique, however specificity may be lower than other methods²

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REFERENCES:

Curry JL, et al. Molecular Platforms Utilized to Detect BRAF V600E Mutation in Melanoma. Semin Cutan Med Surg. 2012;31(4):267–73.
Ihle MA, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations. BMC Cancer. 2014;14:13.
Colomba E, et al. Detection of BRAF p.V600E Mutations in Melanomas Comparison of Four Methods Argues for Sequential Use of Immunohistochemistry and Pyrosequencing. J Mol Diagn. 2013;15(1):94–100.
Anderson S, et al. Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed Paraffin-Embedded Tissue Specimens of Malignant Melanoma. Arch Pathol Lab Med. 2012;136(11):1385–91.
Thermo Fisher Scientific. Ion Torrent™ NGS Ion AmpliSeq™ Panels. http://www.thermofisher.com/uk/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-select-targets/ampliseq-target-selection.html. Accessed August 28, 2019.

Disclaimer: This is an international website on the role of mutated BRAF in melanoma and BRAF testing and is intended for healthcare professionals outside the US. The information on this site is not country-specific and may contain information that is outside the approved indications in the country in which you are located.

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