BRAF is a human gene that encodes for a protein called BRAF. The serine/threonine protein kinase BRAF is a member of the RAF kinase family and plays an important role in the epidermal growth factor receptor (EGFR)-mediated mitogen-activated protein kinase (MAPK) pathway 1. This family consists of 3 kinases, ARAF, CRAF (RAF-1), and BRAF, of which the latter has the highest basal kinase. It catalyzes the phosphorylation of serine and threonine residues in a consensus sequence on target proteins by ATP, yielding ADP and a phosphorylated protein as products 1. Since it is a highly regulated signal transduction kinase, BRAF must first bind RAS-GTP before becoming active as an enzyme 1. Once BRAF is activated, a conserved protein kinase catalytic core phosphorylates protein substrates by promoting the nucleophilic attack of the activated substrate serine or threonine hydroxyl oxygen atom on the γ-phosphate group of ATP through bimolecular nucleophilic substitution. The strength of BRAF, and also its extension to other RAF isoforms (ARAF and CRAF), is to not only activate the MAPK pathway that profoundly affects cell growth, proliferation, and differentiation but also affect other key cellular processes, such as cell migration (through RHO small GTPases), apoptosis (through the regulation of BCL-2), and survival (through the HIPPO pathway) 1. Thus, it is not a surprise that BRAF is found constitutively activated by mutation in 15% of all human known cancer types 2.
BRAF mutations in colorectal cancer
The frequency of BRAF mutations varies widely in human cancers, from more than 80% in melanomas to as little as 0–18% in other tumours 2. In metastatic colorectal cancer (mCRC) BRAF mutations (nearly always V600E) are present in between 8% and 12% and are almost exclusively non-overlapping with RAS mutations 2,3,4.
BRAF is reported to be mutated at several sites; however, the most frequent mutation in BRAF is V600E (1799T-A nucleotide change), characterizing up to 80% of all BRAF mutations 2. This mutation results in amino acid change that confers constitutive kinase activity 2.
BRAF V600E mutated mCRC share peculiar clinical and pathological characteristics: they are more frequent in women than men; are often right-sided; present mucinous histology and microsatellite instability (MSI high). However, BRAF V600E mutation is not associated with the MSI phenotype due to a germline MMR mutation (Lynch syndrome) 5. BRAF mutation showed a higher rate of nodal and peritoneal metastases and a lower rate of lung involvement 3,4. When liver metastases are radically resected, BRAF V600E mutated tumours often relapse early, due to the occurrence of extrahepatic lesions 6. Recently, other mutations have been identified including BRAF 594 or 596 mutated in <1% of mCRC. These mutations identify a rare and unexplored molecular subtype of mCRC with clinical and pathological features different from BRAF V600E mutated 7.
BRAF as a prognostic biomarker in colorectal cancer
BRAF mutation status is consistently associated with poor prognosis in multiple retrospective evaluations. In a cohort of 524 patients, overall survival (OS) for patients with BRAF-mutant colorectal cancer was 10.4 months compared with 34.7 months for BRAF wild-type patients 3. In a multivariate analysis, the hazard ratio (HR) for survival was 10.662 (p < 0.001) 3. This particularly poor prognosis for patients with BRAF-mutant tumours is supported by a number of randomised studies with specific chemotherapy regimens 3,4,8.
Based on this evidence it is possible to conclude that BRAF mutation is a negative prognostic marker in patients with mCRC and that this effect, in contrast to RAS mutations, is not restricted to the outcome of anti-EGFR treatment.
BRAF as a predictive biomarker in colorectal cancer
Regarding standard chemotherapy treatments it has been demonstrated that there is no association between the presence of BRAF mutations and response to chemotherapy with oxaliplatin versus irinotecan 9. Regarding response to standard, targeted therapies, the predictive role towards anti-EGFR agents of V600E activating mutation of BRAF is still debated. In particular, in the meta-analysis that included two second-line trials and two trials involving chemorefractory patients, the lack of a significant efficacy benefit by anti-EGFR monoclonal antibodies (mAbs) over standard chemotherapy alone in patients with BRAF mutated tumours was considered to support the assessment of tumour BRAF mutation status before the initiation of anti-EGFR therapy 10. Conversely, authors of a recent meta-analysis, concluded that there is currently insufficient evidence to definitively consider BRAF mutation a negative predictive biomarker of survival benefit from anti-EGFR mAbs for mCRC 11. The benefit in OS and progression-free survival (PFS) for BRAF mutated tumours treated with anti-EGFR mAbs may be smaller or less likely, but further data are required to clarify this observation. To this extent, studies have not been conclusive maybe due to the low incidence of BRAFV600E mutation and to the intrinsic limitations of retrospective subgroup analyses.
While RAF inhibitors such as vemurafenib have produced impressive response rates of ~60–80% in BRAF mutant melanoma patients 12, vemurafenib demonstrated disappointing results in BRAF mutant mCRC patients, producing only a single partial response (overall response rate of ~5%) in 19 evaluable patients 13. The difference in BRAF mutation incidence in these two types of cancer may indicate that BRAF signalling is not similarly required and might be context dependent. Preclinical data suggests that, in mCRC, resistance to BRAF inhibitors may be driven by compensatory feedback reactivation of EGFR and its downstream pathways such as MEK and ERK 14,15. Combination strategies are now under development using combinations of BRAF-mutant inhibitors (dabrafenib, vemurafenib or encorafenib) in combination with MEK and EGFR inhibition, and in some cases conventional cytotoxic therapy 16.
However, in a small subgroup analysis (n = 28) of BRAF mutated mCRC patients, the TRIBE study reported a more favourable outcome in terms of median PFS and OS for FOLFOXIRI-bevacizumab over FOLFIRI-bevacizumab 17.
BRAF testing recommendations in colorectal cancer
BRAF V600E mutation status is determined via PCR amplification and DNA sequence analysis or allele-specific PCR on formalin-fixed, paraffin-embedded tissue from the primary tumour or a metastasis.
Which technique and which algorithm should be used for the analysis of the BRAF status in colorectal cancer?
All methods used to detect BRAF mutations have advantages and disadvantages, and the choice to use one over the other is usually based on current local practices and experience in the different clinical laboratories.
Parameters that should be considered when choosing a suitable companion diagnostic test include sensitivity, specificity, limit of analytical sensitivity and failure rates.
Methods to increase the sensitivity and specificity and to reduce the failure rates include:
- validating the chosen method thoroughly through comparison with 'gold-standard' methods,
- performing macrodissection of specimens to increase the sensitivity of the technique,
- choosing small amplicons for PCR amplification to reduce the failure rate due to DNA degradation,
- ongoing validation of the method through application of best practice and participation in external quality controls 18.
The presence or absence of BRAF alterations should be performed at the time of diagnosis as this represents unique CRC subtype with poor prognosis and limited response to standard-of-care therapies, and in combination with testing for DNA mismatch repair deficiency (dMMR), can assist in the identification of a germline versus somatic cause of dMMR. These results are in according with the National Comprehensive Cancer Network, which “strongly recommends genotyping of tumour tissue in all patients with mCRC for RAS (KRAS exon 2 and non- exon 2; NRAS) and BRAF at diagnosis of stage IV disease 19. In patients with mCRC, BRAF mutation status should be assessed at the same time as RAS mutational status for prognostic assessment (and/or potential selection for clinical trials).
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- Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–954.
- Tran B, Kopetz S, Tie J, et al. Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer. Cancer 2011;117:4623–4632.
- Venderbosch S, Nagtegaal ID, Maughan TS, et al. Mismatch repair status and BRAF mutation status in metastatic colorectal cancer patients: a pooled analysis of the CAIRO, CAIRO2, COIN, and FOCUS studies. Clin Cancer Res 2014;20:5322–5330.
- Domingo E, Niessen RC, Oliveira C, et al. BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes. Oncogene 2005;24:3995–3998.
- Yaeger R, Cercek A, Chou JF, et al. BRAF mutation predicts for poor outcomes after metastasectomy in patients with metastatic colorectal cancer. Cancer 2014;120:2316–2324.
- Cremolini C, Di Bartolomeo M, Amatu A, et al. BRAF codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis. Ann Oncol 2015;26:2092–2097.
- Van Cutsem E, Kohne CH, Lang I, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol 2011;29:2011–2019.
- Richman SD, Seymour MT, Chambers P, et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 2009;27:5931–5937.
- Pietrantonio F, Petrelli F, Coinu A, et al. Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: a meta-analysis. Eur J Cancer 2015;51:587–594.
- Rowland A, Dias MM, Wiese MD, et al. Meta-analysis of BRAF mutation as a predictive biomarker of benefit from anti-EGFR monoclonal antibody therapy for RAS wild-type metastatic colorectal cancer. Br J Cancer 2015;112:1888–1894.
- Sosman JA, Kim KB, Schuchter L, et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med 2012;366:707–714.
- Kopetz S, Desai J, Chan E, et al. Phase II pilot study of vemurafenib in patients with metastatic BRAF-mutated colorectal cancer. J Clin Oncol 2015;33:4032–4038.
- Prahallad A, Sun C, Huang S, et al. Unresponsiveness of colon cancer to BRAF (V600E) inhibition through feedback activation of EGFR. Nature 2012;483:100–103.
- Corcoran RB, Ebi H, Turke AB, et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov 2012;2:227–235.
- Corcoran RB, Atreya CE, Falchook GS, et al. Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600-mutant colorectal cancer. J Clin Oncol 2015;33:4023–4031.
- Cremolini C, Loupakis F, Antoniotti C, et al. FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as first-line treatment of patients with metastatic colorectal cancer: updated overall survival and molecular subgroup analyses of the open-label, phase 3 TRIBE study. Lancet Oncol 2015;16:1306–1315.
- Gonzalez D, Fearfield L, Nathan P, et al. BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel. Br J Dermatol 2013;168:700-7.
- National Comprehensive Cancer Network. NCCN Guidelines: Colon Cancer. Volume 2. Version 3. 2015. Accessed August 12, 2015.