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
|Method1–4||How it works||Additional information|
|1st- generation sequencing and its different modifications
||Established technology to ‘read the sequence’
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
||Established technology that amplifies a single copy (or a few copies) of a DNA sequence, generating thousands to millions of copies (amplicons).
||There are several methods based on this technology used in clinical practice for BRAF mutation detection|
(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|
|Technology based on the analysis of the temperature gradient in which the double strands of the PCR products are separated or ‘melted.’
||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|