Since the identification of activating mutations of BRAF in melanoma, the technology for detection has improved dramatically. Standard mutational testing for BRAF in tumor tissue typically utilizes techniques such as bidirectional direct fluorescent sequencing and allele-specific polymerase chain reaction which are commercially available and offer high specificity. The sensitivity of these assays, however, is limited in that they are only able to detect the mutation if the tumor cells constitute >5–10% of the specimen submitted for genetic analysis [16
]. While this degree of sensitivity is typically sufficient to detect the presence of the BRAFV600E
mutation in a homogenous tumor nodule, this is likely not sensitive enough to detect a few tumor cells in the background of a high percentage of stromal or lymphatic elements, infiltrating lymphocytes, or peripheral blood cells.
One concern regarding the utilization of mutation detection techniques with enhanced sensitivity is that a positive test might actually reflect the detection of a small subset of mutant cells. While this might have interesting scientific consequences, the clinical relevance of a tumor containing a small amount of mutant BRAF cells is none, as these patients would not be expected to benefit from BRAF inhibitors. This concern is warranted, as tumor heterogeneity has been described in primary melanomas [18
]. In addition, while BRAF mutations are seen in the great majority of melanocytic nevi, vertical growth phase melanomas, and metastatic melanoma, they are rarely detected in radial growth phase melanomas (10%), which is thought to be the initial malignant lesion prior to a frankly invasive lesion [19
]. This suggests that BRAF mutation may actually be an acquired event in early melanoma that leads to clonal expansion and tumor progression. Such polyclonality has not been seen in individual metastatic tumors nor when tumors across multiple sites from individual patients are sampled [18
]. Nevertheless, the application of enhanced sensitivity mutational analysis may not be just testing tumor samples but detecting small numbers of representative tumor cells in a background of nonmalignant cells such as in lymph nodes and peripheral blood.
More advanced techniques and assays have been developed which either provide increased sensitivity or obviate the need for increased sensitivity. These next generation tests allow for more accurate testing on samples which contain only a small amount of tumor, as well as for the detection mutations in various peripheral blood components (i.e., lymphocytes, mononuclear cells, plasma, serum). The utility of many of these tests have been explored in samples from melanoma patients with varying results.
Amplification refractory mutation systems (ARMSs) are a recently described, allele-specific technique which has enhanced sensitivity (able to detect mutation sample containing 1% mutant cells) compared to standard DNA sequencing of formalin fixed paraffin-embedded (FFPE) tissues [21
]. Another approach which greatly enhances sensitivity for mutation detection is the utilization of assays which selectively amplify mutant DNA/RNA in a sample. Using a combination of allele-specific primers and locked nucleic acid primers, the detection of 10 melanoma cells in 1
mL of blood has been described [22
]. A third approach to increase the sensitivity of mutation detection is reported to be able to detect one mutant cell in a thousand nonmutant cells, taking advantage of a unique restriction enzyme site in the wild-type alleles which allows for the digestion of the wild-type alleles and thus the enrichment of the mutant alleles [23
]. Finally, the incorporation of COLD-PCR leads to a near doubling of sensitivity in the detection of BRAF mutation from FFPE tissue when using standard sequencing and pyrosequencing [24
In addition to new technologies (ARMS) and modifications to routine techniques which lead to a greater sensitivity of mutation detection, the application of standard assays on previously untested samples is also changing how we approach BRAF testing. BRAF analysis on free DNA in the serum and plasma has been reported as has the detection of BRAF mutations from isolated, circulating tumor cells (CTCs) [25
]. While CTC, serum and plasma BRAF analysis appears possible, it is yet to be determined whether there will be routine clinical use for one or more of these assays or if this will remain as only an experimental approach.
While the role of standard and experimental molecular diagnostics is being utilized to identify specific mutations of interest (i.e., BRAFV600E
), both in tissue or blood, it also may be worthwhile to test for other mutations and anomalies as these may indicate sensitivity to a particular treatment. For example, Sequenom MassARRAY technology is being used to query larger panels of oncogenic mutations, using a primer extension reaction followed by mass spectrometry to detect the products and identify mutations with potential clinical consequences [31
]. Array comparative genome hybridization (aCGH) offers the opportunity to examine the entire genome for copy number changes, including both amplifications and deletions that may confer sensitivity to a targeted therapy [33
]. However, all these technologies are obviously limited in that they can only identify known, preselected anomalies. Whole genome analysis (WGA) has the potential to not only consolidate all or most of these modalities and tests to a single technology platform but also to identify additional genetic changes outside the design parameters of these other assays [34
]. WGA also offers the opportunity to uncover previously unknown (perhaps patient-specific) mutations in melanoma genomes and to explore whether particular profiles of mutations or polymorphisms may be predictive of benefit from a particular therapy (i.e., BRAF inhibitors, HD IL-2) [35
]. Still, the clinical utility of these “Next Generation” tests in the care of patients with melanoma is completely unknown.