These are just a few of the areas discussed by Dr. Cheah and Dr. Billings, with respect to molecular testing for cutaneous soft tissue tumors. Others addressed in their article were clear-cell sarcoma (melanoma of the soft parts), postradiation angiosarcoma, epithelioid hemangioendothelioma, and Ewing sarcoma family of tumors.
Knowledge and identification of the recurrent molecular aberrations in these cutaneous mesenchymal tumors allow for more accurate diagnosis and advancement of understanding about their underlying biology.
BRAF V600E Mutation Detection
The identification of BRAF mutations in the mitogen-activated protein kinase pathway revolutionized the treatment of advanced-stage melanoma, bringing selective small-molecule RAF inhibitors, such as vemurafenib, to the clinical trial stage. In the phase III BRIM-3 trial, vemurafenib was associated with a higher response rate and a significant improvement in survival, compared with dacarbazine.
"The knowledge that melanomas harbor recurring hot spot mutations in the BRAF gene has rapidly brought molecular testing to the clinical stage," wrote Dr. Jonathan L. Curry of the department of pathology at the University of Texas MD Anderson Cancer Center, Houston, and his colleagues.
The cobas 4800 BRAF V600 Mutation Test from Roche, for example, was approved by the FDA as an in vitro diagnostic device to detect mutant BRAF V600E in DNA extracted from a FFPE patient’s sample of melanoma (Sem. Cut. Med. Surg. 2012;31:268-74). The presence of the mutation aids in selecting patients for treatment with vemurafenib.
The authors noted that a number of molecular platforms for BRAF testing have been developed and continue to evolve, offering a more thorough and complex analysis of the genetic components of melanoma.
"The next generation sequencing or massively parallel sequencing will allow sequencing of the entire exon or whole genome. Multiple sequencing molecular platforms are available to examine for BRAF mutations in cutaneous melanoma, and the best technological approach continues to be developed," they wrote.
Among those they described are:
• Sanger Sequencing. Sanger chain–termination sequencing of amplified DNA by PCR was the method used to sequence the human genome, and the Sanger method of sequencing led to the detection of BRAF mutations in cutaneous melanoma. Sensitivity is high (fewer than 5% of tumor cells are necessary in a given specimen), but use in the clinical setting is limited to BRAF testing. Although it remains the gold standard for gene sequencing, the Sanger method has technical and practical limitations. For example, it takes 18-19 hours to perform the test, other tests are more sensitive, and it cannot detect changes in the chromosomal copy number and the translocations.
• Pyrosequencing. Also known as sequencing by synthesis, pyrosequencing is among the platforms more sensitive than Sanger sequencing. The detection ratio of mutant BRAF V600E to wild type is 1:5 for Sanger sequencing, and 1:50 for pyrosequencing. Its clinical application is to detect the presence or absence of known mutations within a specific segment of DNA of a single nucleotide polymorphism.
"Because mutations in melanoma appear to cluster in the BRAF, NRAS, and KIT genes, this molecular platform has been readily incorporated into the mutational analysis of melanoma," the authors explained, noting that pyrosequencing is a rapid and sensitive test for detection of more common BRAF V600E mutations, as well as other variants. It is limited to the length of the DNA template sequenced, and is prone to errors reading through homopolymer sequences.
• Allele-Specific Real-Time PCR. This molecular platform, also known as the amplification-refractory mutation system, enriches known mutations in clinical samples to increase sensitivity of detection, and is particularly useful in FFPE biopsies with low tumor content. It is highly sensitive and is confined to known BRAF mutations that occur in melanomas, but demonstrates greater sensitivity in detecting BRAF V600E mutations in FFPE clinical samples.
• Mass Spectrometry–Based Sequencing (Sequenom). Sequenom uses mass spectrometry to determine the sequence of the FFPE tissue samples of melanoma. The platform allows for simultaneous amplification of multiple genetic hot spots, allowing for analysis of several known mutations in a single clinical sample. In the authors’ experience, it has slightly higher sensitivity than pyrosequencing.
• High Resolution Melting (HRM). Also a highly sensitive method for screening for mutations in clinical samples, high-resolution melting relies on PCR amplification of the DNA template and analysis of the temperature gradient in which the double strands of the PCR products are melted. The strands melt at different temperatures, depending on the sequence of the constituent bases, allowing for detection of the mutant allele in the FFPE tissue sample. An important limitation of this approach is that specific nucleotide alteration is not reported, thus tissues samples that are positive for mutations will require additional sequencing by another method to determine the specific nucleotide alteration.