by Assia Bassarova

Background

BRAF – B-Raf proto-oncogene on chromosome 7 (7q34) is a gene encoding a protein belonging to the RAF family of serine/threonine protein kinases. BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a RAS-regulated serine–threonine kinase and activator of the MAPK signaling cascade. Extracellular signals act via this pathway to regulate cellular growth and proliferation, differentiation, senescence and apoptosis, and survival. Additionally, an increase in protein expression or activity can disturb the Ras–MAPK signalling pathway, which in turn can result in different developmental disorders such as Noonan syndrome (NS), cardio-facio-cutaneous (CFC) syndrome, Costello syndrome, and different types of human cancers. Since 2002, mutations in BRAF have been described in a wide array of benign and malignant neoplasms, including melanoma,benign nevi, colon adenocarcinoma, serrated polyps, thyroid carcinoma, borderline serous ovarian tumors and low-grade serous carcinoma, Langerhans cell histiocytosis, several low-grade glioma subtypes as well as papillary craniopharyngioma, hairy cell leukemia, ameloblastoma, and metanephric adenoma, as well as rare nonsmall cell lung carcinomas, biliary tract carcinomas, head and neck squamous cell carcinomas, gastrointestinal stromal tumors, and plasma cell myelomas.
Approximately 200 BRAF-mutant alleles have been identified in human tumors. So far, almost 30 distinct mutations of BRAF have been functionally characterized. Interestingly, BRAF mutations can be categorized into three classes based on their effect on the activity of BRAF. Recent publications have also shown that oncogenic BRAF gene fusions can also activate BRAF in melanomas and other cancers. In these cases, the BRAF kinase domain is fused with N-terminal partner genes such as SOX10, AGK, SEPT3. The fusions result in an alteration of the BRAF copy number and activity independent of common missense BRAF mutations.

Staining in normal tissues

At present, no data is available on consistent low-level expressing normal tissues/cells. Therefor it is important to secure distinct and an “as strong as possible reaction” for BRAF staining in mutated tumors and still no reaction in negative tissue controls. According to the Human protein atlas, based on RNA expression data and confirmed by immunohistochemical staining of human cerebral cortex shows strong cytoplasmic positivity in neuronal cells B-Raf transcripts are present in high levels in fetal brain and cerebrum. In addition, immunohistochemical studies have localized B-Raf to hippocampal neurons and dendritic spines.

Staining in tumors

• The majority of BRAF mutations occur at amino acid V600, with the V600E mutation being most prevalent. Other mutations as V600K, V600M, V600R, V600D and V600G are less common. The recognition of BRAF V600E mutation as a critical diagnostic, prognostic, and predictive biomarker in many cancers, along with the advent of small-molecule inhibitors of BRAF V600E, prompted interest in developing specific antibody as a potentially faster, less expensive, more widely available methodology to detect the BRAF V600E protein in formalin-fixed and paraffin-embedded (FFPE) tissue.
• The BRAF V600E mutation is detected in approximately 8% of all solid tumours. Mutations in BRAF are present in 40–60% of primary malignant melanomas and roughly half of metastatic melanomas.
• An activating BRAF mutation is the most common genetic event in thyroid carcinoma, found in roughly 45% of papillary thyroid carcinomas.
• Mutations in BRAF occur in approximately 10–15% of colorectal carcinomas.
• Approximately 9 % of lung adenocarcinomas harbor BRAF mutations – 4 % being BRAF V600E and 5 % non-BRAF V600E mutations.
• Among hematologic malignancies, a BRAF V600E mutation is present in virtually all cases of hairy cell leukemia (HCL) and approximately 60% of cases of Langerhans cell histiocytosis (LCH).
• BRAF-V600E mutations are most commonly found in pleomorphic xanthoastrocytoma, ganglioglioma, epithelioid glioblastoma, and gliomas diagnosed at a younger age.

Staining pattern

Weak to strong cytoplasmic staining in tissues with BRAFV600E mutation.

Control tissue

Tumors confirmed with and without BRAF V600E mutation are recommended as positive and negative controls for BRAF. Appendix can also serve as a negative tissue control, where no staining reaction should be seen in the epithelial cells.

Application

• IHC for BRAF V600E mutations can be used as a screening for further molecular analysis. Several publications have shown high concordance between IHC and molecular data indicating overall agreement of 95-99% between the two methods.
• IHC for BRAF V600E is used as predictive biomarker for BRAF inhibitors as vemurafenib and dabrafenib in melanoma.
• BRAF IHC can be used as biomarker to distinguish between hereditary (Lynch) and sporadic MMR deficient colon adenocarcinoma, as BRAF mutations are virtually absent in hereditary colon adenocarcinomas and seen in up 60% of sporadic colon adenocarcinomas.

Selected references

1. Ritterhouse LL, Barletta JA. BRAF V600E mutation-specific antibody: A review. Semin Diagn Pathol. 2015 Sep;32(5):400-8. doi: 10.1053/j.semdp.2015.02.010. Epub 2015 Feb 7. PMID: 25744437.
2. Zaman A, Wu W, Bivona TG. Targeting Oncogenic BRAF: Past, Present, and Future. Cancers (Basel). 2019 Aug 16;11(8):1197. doi: 10.3390/cancers11081197. PMID: 31426419; PMCID: PMC6721448.
3. Stephens RM, Sithanandam G, Copeland TD, et al. 95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site. Mol Cell Biol. 1992 Sep;12(9):3733-42. doi: 10.1128/mcb.12.9.3733-3742.1992. PMID: 1508179; PMCID: PMC360233.
4. Maraka S, Janku F. BRAF alterations in primary brain tumors. Discov Med. 2018 Aug;26(141):51-60. PMID: 30265855.
5. Frisone D, Friedlaender A, Malapelle U, et al. A BRAF new world. Crit Rev Oncol Hematol. 2020 Aug;152:103008. doi: 10.1016/j.critrevonc.2020.103008. Epub 2020 May 26. PMID: 32485528.