BRAF mutation analysis
BRAF belongs to a family of serine-threonine protein kinases that includes ARAF, BRAF, and CRAF (RAF1). RAF kinases are central mediators in the MAP kinase signaling cascade and exert their effect predominantly through phosphorylation and activation of MEK. This occurs following the dimerization (hetero- or homo-) of the RAF molecules. As part of the MAP kinase pathway, RAF is involved in many cellular processes, including cell proliferation, differentiation, and transcriptional regulation.
Mutant BRAF has been implicated in the pathogenesis of several cancers, including melanoma, non-small cell lung cancer, colorectal cancer, papillary thyroid cancer, and ovarian cancer.
Somatic mutations in BRAF have been found in ~50% of all malignant melanoma. BRAF mutations are found in all melanoma subtypes but are the most common in melanomas derived from skin without chronic sun-induced damage. In this category of melanoma, BRAF mutations are found in ~59% of samples.
The most prevalent BRAF mutations detected in melanoma are missense mutations, which introduce an amino acid substitution at valine 600. Approximately 80–90% of V600 BRAF mutations are V600E (valine to glutamine) while 5-12% are V600K (valine to lysine), and 5% or less are V600R (valine to arginine) or V600D (valine to aspartic acid). The result of these mutations is enhanced BRAF kinase activity and increased phosphorylation of downstream targets, particularly MEK. In the vast majority of cases, BRAF mutations are non-overlapping with other oncogenic mutations found in melanoma (e.g., NRAS mutations, KIT mutations, etc.).
BRAF inhibitor therapy is associated with clinical benefit in the majority of patients with BRAF V600E-mutated melanoma.
Papillary thyroid cancer
Somatic mutations in BRAF have been found in 40–45% of papillary thyroid cancer. BRAF mutations are also found in anaplastic thyroid cancer (30–40%) and poorly differentiated tumors (20–40%).
The most prevalent BRAF mutations detected in thyroid cancers are missense mutations which introduce an amino acid substitution at valine 600. The vast majority (98%) of BRAF mutations are V600E (valine to glutamic acid). The result of these mutations is enhanced BRAF kinase activity and increased phosphorylation of downstream targets, particularly MEK.
Approximately 8–15% of colorectal cancer (CRC) tumors harbor BRAF mutations. The presence of BRAF mutation is significantly associated with right-sided colon cancers and is associated with decreased overall survival.
FFPET specimens are processed and genomic DNA isolated using the cobas® DNA Sample Preparation Kit, a generic manual specimen preparation based on nucleic acid binding to glass fibers. A deparaffinized 5 μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration to be added to the amplification/detection mixture. The target DNA is then amplified and detected on the cobas z 480 Analyzer using the amplification and detection reagents provided in the cobas® 4800 BRAF V600 Mutation Test kit.
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