|Year : 2019 | Volume
| Issue : 2 | Page : 37-42
The mala fides of BRAF in oncogenesis
Anurag Mehta1, Garima Gupta2, Shrinidhi Nathany3
1 Departments of Laboratory; Transfusion Services; Research; Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
2 Department of Research, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
3 Department of Laboratory, Rajiv Gandhi Cancer Institute and Research Centre, Delhi, India
|Date of Web Publication||30-Dec-2019|
Dr. Anurag Mehta
Director Laboratory, Molecular Diagnostic Services & Research Rajiv Gandhi Cancer Institute & Research Centre, Sector-V, Rohini, Delhi- 110085.
Source of Support: None, Conflict of Interest: None
BRAF is a proto-oncogene that encodes a serine threonine kinase belonging to intracellular RAS-RAF-MEK& ERK signaling cascade downstream of surface Receptor Tyrosine Kinase. The gain of function mutations in BRAF gene have been shown to have a powerful oncogenic effect especially a transversion at nucleotide 1,799 from thymidine to adenine (p.V600E), accounting for greater than 80% of the observed mutations in BRAF. This one mutation has been identified as an oncogenic driver in a diverse set of solid and hematologic cancers. Fortunately, introduction of BRAF & MEK inhibitors has modestly transformed the treatment outcomes in patients with BRAF mutations, especially those with non-small cell lung carcinoma, melanoma and thyroid cancers. Besides, the predictive nature of BRAF mutation, the mutational analysis also helps predict prognosis and secure diagnosis of a varied group of malignancies. This review comprehensively addresses to the various mala fides of BRAF in oncogenesis, as well as elucidates the nucleotide variants which have been reported in literature, the diagnostic and prognostic utility, and the testing methods which are available to test the same.
Keywords: BRAF, BRAF V600E, RAS–RAF–MEK–ERK, significantly mutated genes
|How to cite this article:|
Mehta A, Gupta G, Nathany S. The mala fides of BRAF in oncogenesis. J Curr Oncol 2019;2:37-42
| Introduction|| |
Gain-of-function mutations or rearrangements in proto-oncogenes confer survival advantage and endow transformative attributes on the host cell pushing it on the path of carcinogenesis. Of the 25,000 genes in human genome, approximately 400 genes have been identified as “Significantly Mutated Genes.”,, Among these, RAS, PIK3CA, and BRAF have exceptional oncogenic potential, accounting for 15%–30%, 13%, and 7% of driver mutations in all cancer types, respectively.,,
BRAF mutation and fusion rearrangements have been recognized as primary driver mutation in diverse solid and hematologic malignancies. Their mechanistic intricacies, potential in diagnosis, therapy selection, and prognosis are being elucidated regularly and integrated into clinical practice. This review discusses these aspects of BRAF genetic alterations.
| BRAF Gene and Protein|| |
The BRAF gene is located on the long arm of chromosome 7 (7q34) and has 18 exons. It encodes a serine–threonine kinase named BRAF that belongs to the RAF family, which also includes ARAF and CRAF (RAF1).
BRAF protein is an intermediate signal transducer of RAS–RAF–MEK–ERK signaling pathway, which regulates:
Binding of “growth factor, hormone, or cytokine” to their receptor tyrosine kinase initiates conversion of membrane-bound inactive GDP-RAS to its active form GTP-RAS. The GTP RAS binds the BRAF protein. The high density KRAS molecules tethered to the inside of cell membrane bring BRAF protein molecules close enough to trigger BRAF protein dimerization or heterodimerization with CRAF. This activates BRAF especially the heterodimer and initiates a cascade that relays signal downstream activating ERK as the terminal effector [Figure 1].
|Figure 1: The MEK–ERK pathway. Receptor tyrosine kinase (RTK) binds with its ligand growth factor (GF) that leads to binding of docker protein GRB2 to its intracytoplasmic portion, which in turn binds to Son of Sevenless (SOS). SOS transfers GTP to RAS and activates it. The GTP RAS activates BRAF by dimerization. Resultant downstream signaling activates MEK and ERK proteins. The ERK protein internalizes to nucleus and initiates transcription of several proteins involved in cell division, differentiation, inhibition of apoptosis, and secretion. ER, FOS, GLUT1, CCND, cMYC, B3 integrin, and VEGF are positive regulators. DUSP and SPRY are autoregulatory proteins of the MEK–ERK pathway. BAD and BIM are inactivated. (Copyright Dr A. Mehta)|
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Activated ERK internalizes to nucleus and regulates cellular functions as listed below:
Promotes cellular proliferation through expression of cyclin D1, cyclin D2, cyclin D3, ER, FOS, and GLUT1 genes
Negatively regulates p53 by expression of MDM2
Inhibits apoptosis by inactivation of proapoptotic “bcl2-associated agonist of cell death”’ (BAD) and BIM
Induces insensitivity to growth inhibition signals through expression of MYC protein
Promotes angiogenesis by promoting expression of VEGF
Controls cellular migration through β3 integrin expression.
In normal physiological state, the process is switched off by negative feedback from activated ERK that undoes the BRAF heterodimerization by phosphorylation at S365 [Figure 2] and by conversion of GTP-RAS to GDP-RAS by RAS protein intrinsic GTPase activity further potentiated by cytosolic GTPase-activating protein (GAP). DUSP and SPRY are two autocrinal proteins blocking the relay of signaling from MEK to ERK. Once the signaling is switched off, the ERK protein returns to cytoplasm abrogating the transcription of aforementioned genes.
|Figure 2: Structure of the BRAF protein. The conserved regions superimposed on their exonic locations are shown as CR1, CR2, and CR3. S365 in CR2 is the negative regulatory phosphorylation site. The N terminal of kinase domain (KD) comprising SSDD amino acid sequence provides negative charge and phosphorylation site at S446. The S446 phosphorylation is constitutive and necessary for function of kinase domain. T599 and S602 in the activation segment TVKS are the phosphorylation site activated by binding of RAS GTP and are necessary for activation of downstream MEK. RBD = GTP-RAS binding domain, CRD = cysteine-rich domain. (Copyright Dr A. Mehta)|
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The BRAF protein is 766-amino acid (AA) long with 84kDa molecular weight. It has three conserved regions labeled as CR1, CR2, and CR3 [Figure 2]. The functionally significant domains in these conserved regions are as follows:
CR1 region has GTP-RAS binding domain (RBD) and autoinhibits BRAF kinase domain.
CR2 contains a negative regulatory phosphorylation site at S365.
CR3 contains the kinase domain with N terminal of SSDD AAs that provides negative charge to this site for its function. T599 and S602 are the activation-induced phosphorylation sites and are crucial for downstream relay of signal.
| Cancer and BRAF Gene Alterations|| |
BRAF-activating somatic mutations account for nearly 7% of all cancers. Such mutations bestowed with manifold higher kinase activity amplify the downstream signaling causing unregulated cellular proliferation, genomic instability due to loss of p53 function, angiogenesis and migration along with suppression of apoptosis, and the classical makings of a transformed cell. Approximately 45 disease-producing somatic missense mutations have been identified in different types of cancer [Table 1].
|Table 1: Clinically significant variants of BRAF gene and their frequency as described in My Cancer Genome database|
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A great majority (~90%) of these mutations reside at V600 and K601 of the TVKS activation segment. T1799A that causes the AA substitution BRAFV600E accounts for 80% of driver mutations ascribed to BRAF gene. The oncogenic potential of this mutation stems from its 500- to 700-fold increase of kinase activity. This mutated protein is active in monomeric form and does not require activation by GTP–RAS. Another roughly 10% deleterious mutations reside in exon 15 either at AA K601 or in vicinity of V600 at AA sequence D594, G596, or L597. The other 10% exist in the glycine of the G-loop in the exon 11 at G464, G466, and G469.
In addition to missense deleterious mutations, approximately 80 fusion rearrangements have also been identified in several cancers, predominantly in melanoma and non-small-cell lung carcinoma (NSCLC). Pilocytic astrocytoma, colorectal adenocarcinoma, and papillary thyroid carcinoma are other cancers with BRAF fusion rearrangement. The fusion rearrangements derive their oncogenic potential through loss of autoregulatory N terminal portion of the BRAF protein caused by fusion rearrangement., A large fraction of these are caused by inversion 7 causing rearrangement with several genes as shown in [Table 2].
|Table 2: Some of the examples of fusion rearrangements involving BRAF gene|
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| Diagnostic Methods|| |
A variety of molecular assays are used for detection of BRAF mutations. Sanger sequencing, Next-Generation Sequencing (NGS), and pyrosequencing are best suited to identify all the single nucleotide variants. Because of the dominant nature of BRAFV600E and its druggability, the current emphasis is on identifying BRAFV600E along with a few other mutations at codon 600 and 601. For this limited objective, strategies based on real-time polymerase chain reaction (PCR) using allele-specific PCR or amplification refractory mutation–specific system with approximate limit of detection of 5% are commonly used. A BRAFV600E mutation–specific antibody (VE1) for immunohistochemistry is also available and has the US Food and Drug Administration (FDA) approval for use in colorectal cancer for determination of MLH1-deficient tumors that should undergo germ line testing for Lynch syndrome. [Table 3] lists the various FDA-approved tests and the variant called by each of these assays.
|Table 3: FDA-approved testing methods for BRAFV600E and codon 600 and 601 testing,,|
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| Value of Identifying BRAF Mutations|| |
As stated before, BRAFV600E is by far the most common gain-of-function mutation in BRAF gene, and its detection is used for diagnostic, prognostic, and predictive purposes. Some important indications are listed as follows under each category implication:
Diagnostic utility of BRAFV600E detection
The correct diagnosis of hairy cell leukemia (HCL) is essential because it can be effectively treated by purine analogues, whereas its mimics such as splenic marginal zone lymphoma or splenic lymphoma/leukemia unclassifiable including HCL variant (HCL-v) do not respond to these drugs. All cases of HCL carry BRAFV600E mutation and its presence is confirmatory of HCL in this context.,
Indeterminate fine-needle aspirate (FNA) or aspirates suspicious for papillary thyroid carcinoma are molecularly tested for BRAFV600E and its presence is used to confirm the diagnosis of papillary thyroid carcinoma. A study has shown that the supplementing diagnostic effort by molecular detection of BRAFV600E significantly improves the sensitivity of FNA procedure, from 67.5% with FNAC alone to 89.6% with FNAC combined with molecular testing for BRAFV600E.
MLH1-deficient colorectal carcinomas are negatively selected for the presence of BRAFV600E mutation to proceed further for germ line testing to diagnose a case of Lynch syndrome.
Identification of BRAFV600E can help conclude the diagnosis of Pleomorphic Xanthoastrocytoma, ganglioglioma and pilocytic astrocytoma in appropriate clinical and pathological settings.
Prognostic utility of BRAFV600E
BRAFV600E-mutated colorectal carcinomas (CRC) that are microsatellite stable (MSS) have inferior outcomes compared to those that are BRAFV600E wild type along with those that are BRAFV600E mutated but in addition show microsatellite instability (MSI-H) and carry CpG island methylator phenotype. It has been shown in a meta-analysis of 27 independent studies, comprising approximately 24,000 CRC patients, that compared MSS–BRAFwt with MSS–BRAFmut, that latter had a shorter overall survival (OS) with hazard ratio of 2.018. A trend toward inferior OS was also noted in MSI–BRAFmut with hazard ratio of 1.324; however, there was no association of MSI–BRAFwt with OS. Additionally, BRAFmut CRC have been reported to be associated with a high rate of peritoneal metastases.
The prognostic value of two most common genetic alterations KIAA1549–BRAF fusion and BRAFV600E mutation in pediatric glial tumors is controversial with some studies showing better outcomes and others showing neutral effect of such genetic alterations.
Predictive value of BRAF mutations
The predictive value of BRAF gain-of-function mutation has been established in melanoma and NSCLC and several BRAF inhibitors alone or in combinations with MEK inhibitors have been approved for treatment of advanced melanoma [Table 3]. Dual inhibition with dabrafenib + trametinib is approved for BRAFV600E-positive NSCLC. Newer strategies combining BRAF inhibitors with immunotherapy are focus of many ongoing trials and have shown superior survival nevertheless higher rates of adverse events have been observed.
BRAFV600E mutation in metastatic CRC makes response to anti-EGFR antibodies highly unlikely unless given with a BRAF inhibitor. BRAFV600E has been included as a predictive biomarker for treatment of metastatic colorectal cancer National Comprehensive Cancer Network (NCCN) version 3.2019—September 26, combinations such as (a) irinotecan + anti EGFR antibody + vemurafenib (b) Dabrafenib + Trametinib+anti EGFR antibody (c) encorafenib + binimetinib + anti EGFR antibody.
A phase 2, open-label VE-BASKET study with 26 enrolled subjects has shown gratifying results with vemurafenib in BRAFV600E-mutated Erdheim–Chester disease and Langerhans cell histiocytosis exhibiting objective response rate of 62% and the 2-year progression-free survival rate of 86%.
Vemurafenib has recently been approved by US FDA for BRAFV600E-mutated advanced radioactive iodine refractory thyroid cancer. Further, dabrafenib (Tafinlar) has also been granted approval for BRAFV600E-mutated metastatic papillary thyroid carcinoma (PTC). A combination therapy with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib has also been approved for the treatment of patients with unresectable or metastatic BRAFV600E-positive anaplastic thyroid cancer. The study showed an impressive overall response rate of 69%.
Early trials with BRAF–MEK and ERK inhibitors are exploring utility of genome-directed therapy in pediatric low-grade gliomas.
| Conclusion|| |
BRAF is an intermediate signal transducer of the RAS–RAF–MEK–ERK signaling pathway, which drives fundamental cell processes of proliferation, differentiation, and secretion. A gain-of-function genetic alteration in the BRAF gene has high oncogenic potentials. Of approximately 45 single-nucleotide variants and 80 fusion rearrangements, BRAFV600E is the archvillain. Fortunately, introduction of BRAF inhibitors used alone or in combination with MEK inhibitor has improved outcomes in BRAF-mutated advanced melanoma and NSCLC. Combination of immune checkpoint inhibitors and BRAF inhibitors are showing promise in further expanding the survival, though serious adverse events with such combinations is a challenge that needs to be addressed. Determining BRAF mutation status in a variety of cancer shall help explore therapeutic options beside helping in diagnosis and anticipating prognosis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]