Definition of BRAF
BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a serine/threonine protein kinase that plays a critical role in the RAS-RAF-MEK-ERK mitogen activated protein kinase (MAPK) cell signalling pathway. Activation of this pathway transfers extracellular signals through the cell via a cascade of phosphorylation events, leading to altered gene expression, cell growth, survival and differentiation in normal and transformed cells.
BRAF is a potent oncogene that is activated in approximately 8% of all cancers. BRAF point mutations have been identified in a wide range of solid tumours and a subset of haematological malignancies, and are particularly prevalent in melanomas [1,2]. The majority of BRAF mutations occur within the activation domain of the kinase, which results in constitutive activation of BRAF and phosphorylation of MEK, independent of upstream activation by receptor tyrosine kinases or RAS. This generates unopposed, constitutive activation of ERK, which leads to the promotion of cellular growth and evasion of apoptosis and, ultimately, neoplastic transformation. The most common BRAF mutation, found in more than 90% of BRAF-mutated tumours, is a substitution of a glutamic acid for valine at amino acid 600 (V600E) in the kinase activation domain. This substitution mimics phosphorylation of the activation loop, thereby inducing constitutive BRAF protein kinase activity.
BRAF Mutations in Melanoma
Activating BRAF mutations are present in approximately 50% of all melanomas. Approximately 90% of these mutations occur at amino acid 600, the majority of which are BRAF V600E mutations . Other mutations have been recorded at codon 600, including BRAF V600K, V600D and V600M.
The high frequency of BRAF mutations in melanoma implies that this oncogene may be an attractive therapeutic target and has led to a new era of targeted therapy for advanced melanoma.
BRAF as a Prognostic Biomarker in Melanoma
Prior to the approval of BRAF inhibitors, patients with BRAF-mutated melanoma faced a worse prognosis than that of patients whose disease expressed wild-type BRAF. A number of studies have demonstrated in patients with metastatic melanoma, an association between the presence of a BRAF mutation and poorer prognosis from diagnosis of first metastasis or resection of metastasis. However, the presence of a BRAF mutation appears to have no impact on the disease-free interval from diagnosis of the first melanoma to first distant metastasis . Patients with BRAF positive melanoma tend to be younger and have poorer survival than patients with wild-type melanoma at diagnosis. Whilst no specific clinical features of metastatic disease have yet been correlated with BRAF mutation status, the primary melanoma of BRAF mutants has been associated with specific clinicopathologic features including site (trunk), earlier age of onset, and lack of chronic sun damage in surrounding skin .
BRAF as a Predictive Biomarker in Melanoma
BRAF-targeted therapies show remarkable efficacy in BRAF-mutated melanoma with the presence of a BRAF V600 mutation serving as predictive biomarker of response. Two BRAF inhibitors, vemurafenib and dabrafenib, and one MEK inhibitor, trametinib have been approved in Europe for the treatment of adult patients with unresectable or metastatic melanoma with a BRAF V600 mutation [5-7]. Treatment of patients with BRAF-mutated melanoma with these targeted therapies has reversed the poor prognosis associated with this molecular alteration. These patients now have a longer median survival than those patients with wild-type BRAF melanoma .
The BRAF inhibitors vemurafenib and dabrafenib specifically bind to and suppress BRAF activity and downstream signaling in cells containing a BRAF V600 mutation. In clinical trials, both of these compounds have demonstrated profound clinical responses in patients with melanoma with BRAF V600 mutations, with a significant increase in progression-free and overall survival compared with standard-of-care chemotherapy [5,6,8,9].
The MEK inhihitor, trametinib, binds to unphosphorylated MEK, preventing RAF-dependent MEK phosphorylation and activation. In clinical trials, although the overall response rate achieved was not as high as that for vemurafenib or dabrafenib, trametinib demonstrated improved progression-free and overall survival compared with standard-of-care chemotherapy [7,10]. Evidence suggests that trametinib does not have clinical activity in patients who have progressed on a prior BRAF inhibitor therapy, but recent clinical trial data show that combined treatment with dabrafenib and trametinib improves efficacy without worsening side effects, compared with either agent alone . This combination has been approved by the FDA for the treatment of melanoma patients bearing BRAF V600 mutations.
The success of these targeted therapies in patients with BRAF-mutated melanoma leads to the recommendation that patients with metastatic or unresectable melanoma should be screened for BRAF V600 mutations in either a metastatic lesion (preferably) or the primary tumour to help guide therapeutic decision making .
Resistance to BRAF Inhibition
A small subset of patients with BRAF-mutated melanoma do not respond to treatment with BRAF or MEK inhibitors because of intrinsic mechanisms of resistance, and most patients who initially respond to these therapies ultimately develop a mechanism of acquired resistance, leading to progressive disease. There are multiple possible mechanisms of resistance and most mechanisms described so far involve reactivation of the MAPK pathway [13,14]. NRAS mutations and splice variants of BRAF V600E mRNA are common mechanisms identified to date . Activation of the PI3K-PTEN-AKT pathway has been also shown to play a role in the acquired resistance to BRAF inhibitors.
There are a number of RAF inhibitors currently in preclinical and clinical development, each with different properties aimed at overcoming the resistance mechanisms that can limit the effectiveness of these drugs.
BRAF Testing Recommendations in Melanoma
Vemurafenib, dabrafenib, and trametinib are indicated for patients with unresectable or metastatic melanoma, who have a BRAF V600 mutation-positive tumour confirmed by a validated test carried out in an accredited (certified) institute that includes appropriate quality controls [5-7].
Several methods are available for detecting BRAF V600 mutations including, mutation-specific real-time polymerase chain reaction (RT-PCR), Sanger sequencing, pyrosequencing, conformation analysis and high-resolution melting analysis.
Two RT-PCR companion diagnostic assays were developed alongside vemurafenib and dabrafenib to assess patient eligibility for enrolment into the clinical trials; the cobas® 4800 BRAF V600 Mutation Test  and the bioMerieux (bMx) THxID®-BRAF assay , respectively. Both tests have FDA and CE-IVD approval for the detection of BRAF mutations and involve the extraction of genomic DNA from formalin-fixed, paraffin-embedded (FFPE) tumour sample and an RT-PCR assay that detects both wild-type and mutant BRAF. The cobas® 4800 Test is designed to detect the predominant BRAF V600E mutation with high sensitivity (down to 5% V600E sequence in a background of wild type sequence from FFPE-derived DNA) and also detects the less common BRAF V600D and V600K mutations with lower sensitivity . The THxID®-BRAF assay was designed to detect the BRAF V600E and V600K mutations with high sensitivity (down to 5 % V600E and V600K sequence in a background of wild-type sequence using DNA extracted from FFPE tissue) and also detects the less common BRAF V600D mutation and V600E/K601E mutation with lower sensitivity . However, several different CE-IVD methods for BRAF testing are available in Europe.
Ensuring Quality and Timely BRAF Mutation Testing Results
It is important that all melanoma patients eligible for BRAF-inhibitor therapies are assessed for BRAF mutation in an accurate, reliable and timely manner so that the results can be appropriately applied to the clinical management of the patient.
Availability of BRAF mutation results to clinicians can be affected by:
- the point during the patient's pathway at which the test is requested,
- the stage and clinical urgency of patients selected for BRAF mutation testing,
- the turnaround time of the BRAF mutation test itself,
- and how the results are transmitted to the treating clinicians .
Which Technique and Which Algorithm Should be Used for the Analysis of the BRAF Status in Melanoma?
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 .
In line with the European license for vemurafenib, dabrafenib and trametinib, BRAF mutation testing is recommended in patients diagnosed with unresectable or metastatic melanomas. European guidelines state that BRAF mutation testing of primary tumours in patients without metastases is not recommended .
- Holderfield M, Deuker MM, McCormick F, et al. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nature Reviews Cancer 2014;14:455-67.
- Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417: 949-54.
- 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.
- Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol 2011;29:1239-46.
- Vemurafenib. Summary of Product Characteristics. 2014.
- Dabrafenib. Summary of Product Characteristics. 2015.
- Trametinib. Summary of Product Characteristics. 2015.
- Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364:2507-16.
- Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012;380:358-65.
- Flaherty KT, Robert C, Hersey P, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma.N Engl J Med2012;367:107-14.
- Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med 2012;367:1694-703.
- Dummer R, Hauschild A, Guggenheim M, et al. ESMO Guidelines Working Group. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2012;23(Suppl 7):vii86-91.
- Chapman PB. Mechanisms of resistance to RAF inhibition in melanomas harboring a BRAF mutation Am Soc Clin Oncol Educ Book 2013; doi: 10.1200/EdBook_AM.2013.33.e80.
- Shi H, Hugo W, Kong X, et al. Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov 2014;4(1):80-93. doi: 10.1158/2159-8290.CD-13-0642. Epub 2013 Nov 21.