The BRAF mutant colorectal cancer subgroup is a small population with unique clinicopathological and molecular features. This subgroup has been associated with particularly poor prognosis and advanced disease. The poor response of these patients to available treatments has driven much of the effort in trialling combination targeted treatments involving BRAF and MEK inhibitors. Most recently, an observed survival benefit with intensive triplet chemotherapy agents would encourage its use as first-line treatment in suitable candidates given that few of these patients proceed to second- or third-line treatments.
- colorectal cancer
2. BRAF and the RAS/RAF/MEK/ERK signalling pathway
V-raf murine sarcoma viral oncogene homologue (RAF) is one of the most intensively researched mammalian effectors of RAS in the RAS/RAF/MEK/ERK signalling pathway (Figure 1) [5, 6]. The RAF protein itself is made up of three conserved regions: CR1, CR2, and CR3. CR1 and CR2 are situated in the N terminus. CR1 acts as the main binding domain for RAS. CR2 is the regulatory domain. CR3 is situated at the C terminus and functions as the catalytic kinase domain .
When GTP bound, RAS recruits RAF protein to the cell membrane and binds to it. This binding process activates RAF kinase by the phosphorylation of two amino acids (T599 and S602 of BRAF) situated in the activation segment of the kinase domain. RAF then phosphorylates its downstream effectors MEK1, MEK2, ERK1, and ERK2, leading to the activation of cellular proliferation, differentiation, and transcriptional regulation (Figure 1) .
In solid tumours, the highest incidence of
BRAFmutation detection methods
Sanger sequencing is the earliest form of first-generation direct sequencing. Sanger sequencing was developed in 1975 and relies on the chain-termination sequencing of amplified DNA by polymerase chain reaction (PCR) and detection through electrophoresis. It requires approximately 18 to 19 h to process and is also 10 times less sensitive than pyrosequencing. Sanger sequencing method also cannot detect the changes in chromosomal copy number and translocations .
Next-generation sequencing (NGS) differs in technology using a specific reagent wash of nucleotide triphosphates with synchronised optical detection and includes pyrosequencing, allele-specific (AS) PCR, mass spectrometry, and real-time qPCR with melt-curve analysis . NGS is the new gold standard test in
Pyrosequencing is referred to as sequencing by synthesis and relies on the release of pyrophosphate (PPi) by DNA polymerase. The test detects light emitted when nucleotides are added to the target DNA template by DNA polymerase releasing PPi via a chemiluminescence reaction. It is a more rapid and sensitive test in detecting
AS-PCR enriches known mutations in samples to increase the sensitivity of detection and is particularly useful in tissue with low tumour content. Mass spectrometry-based sequencing relies on the analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). This process is facilitated by the addition of mass-modified bases A, C, T, and G to the primed and amplified mutational hotspots. It is this flight time difference of the generated mass-modified complex that is measured by the mass spectrometer. Mass spectrometry-based sequencing is an even more sensitive test compared to pyrosequencing, with a detection ratio of 1:10 and 1:8, respectively .
Melt-curve analysis involves detecting the melting temperature for WT
|Low||99.9%||20 min–3 h||2400||Requires the time-consuming|
step of PCR of plasmid cloning;
impractical for larger sequencing
|Medium||99.9%||24 h||10||Homopolymer errors|
|High||99.9%||1–11 days||0.05–0.15||Expensive equipment;|
requires high DNA
|High||99.9%||1–2 weeks||0.13||Slower; issues sequencing|
|High||98%||2 h||1||Homopolymer errors|
|High||87%||30 min–4 h||0.13–0.60||Expensive equipment;|
The IHC detection of
Recently, examination of CTC in peripheral blood has been explored as a new non-invasive means for detecting
BRAFmutation and its frequency in CRC
A meta-analysis of 10 studies reported
|PRIME [29, 30]||8%||Bidirectional Sanger sequencing|
|CRYSTAL ||6%||PCR clamping/melt-curve analysis|
|MAX ||10.6%||High-resolution melting point/PCR|
|NORDIC-VII ||12%||Wobble enhanced ARMS*/real-time PCR|
|AGITG/NCIC CO.17 ||3.2% (overall) and 4.8% (KRAS WT)||PCR/sequencing|
|COIN ||8%||MALDI-TOF (Sequenom)/Sanger sequencing|
BRAFmutation and its clinical significance in CRC
3.1. CRC tumourigenesis pathways
The two main separate pathways observed in CRC development and progression are the chromosomal instability pathway (CIN), which accounts for 75% of the cases, and the microsatellite instability (MSI) pathway in 25% of the cases. Two processes are observed to contribute towards the MSI pathway: (1) germ-line mutations from Lynch syndrome and (2) sporadic MLH1 methylation from the serrated methylated pathway (Figure 3)  .
The CIN pathway involves a defect in replication, mitosis, or DNA repair leading to genetic abnormalities, both structural and numeric, which are acquired sequentially. As a result, oncogenes are activated or tumour suppressor gene function is lost, which contributes towards malignant growth. This pathway is also often associated with aneuploidy by karyotyping. The genetic changes found in CRC arising via the CIN pathway include APC mutations (90%),
The MSI pathway is a result of defective mismatch repair (MMR) and occurs in a subset of CRC that arise from either adenomas or serrated polyps. It contributes towards tumour progression via the accumulation of tiny insertions and deletions in the repetitive sequences of microsatellites in coding genes, thereby retaining a near-diploid karyotype. This mechanism of tumourigenesis is readily recognized through a test for MSI, which categorises each tumour as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS), based on the proportion of microsatellites mutated. MSI-H cases usually imply an acquired or inherited defect in DNA repair.
In inherited cases of MSI-H CRC, germ-line mutation in one of the four genes that encode proteins responsible for MMR (MLH1, MSH2, PMS2, and MSH6) is responsible for a familial predisposition to cancer. This familial predisposition to CRC is known as Lynch syndrome , and the CRC that arise in this condition develop in adenomas.
In sporadic cases of MSI-H CRC, the serrated methylated pathway is increasingly implicated. Serrated polyps, not driven by CIN but by
BRAFtesting to distinguish between sporadic versus germ-line MSI-H cases (Lynch syndrome)
Approximately 12% of MSI-H cases are sporadic in nature and
MLH1 methylation testing is an alternative assay to distinguish sporadic from familial cases of CRC. However, given that methylation testing is more technically challenging than
3.3. Clinicopathological and molecular features of
The relationship between
Another study  reported a significantly increased rate of peritoneal (46% vs 24%; p<0.001) and distant lymph node metastases (53% vs 38%; p=0.001) and a lower rate of lung metastases (35% vs 49%; p=0.049) in
|1. Age >70 years||1. More prevalent in MSI-H>MSS CRC|
|2. Female patients||2. More CIMP|
|3. Proximal right-sided tumours||3. More |
|4. High-grade and poorly differentiated||4. Mutually exclusive to KRAS mutation|
|6. More peritoneal and lymph node metastases|
|7. Less lung metastases|
Table 3 summarises the clinicopathological and molecular characteristics of
BRAFmutation and its prognostic and predictive significance
4.1. Prognostic role and nature of progression
Multiple studies have reported poorer median overall survival (OS) in the
|CRYSTAL (2011) ||1198||First line: FOLFIRI|
|6%||5.6 vs 8.0 (HR=0.93; p=0.87)||10.3 vs 14.1 (HR=0.91; p=0.74)||8.8 vs 10.9 (HR=0.67; p=0.001)||21.6 vs 25.1 (HR=0.83; p=0.055)|
|PRIME (2013) [29, 30]||1183||First line: FOLFOX|
|8%||5.4 vs 6.1 (HR=0.58; p=0.12)||9.2 vs 10.5 (HR=0.90; p=0.76)||RAS/BRAF|
WT 9.2 vs 10.8 (HR=0.68; p<0.01)
WT 20.9 vs 28.3 (HR=0.74; p=0.02)
|FIRE-3 (2013) ||400||First line: Avastin+FOLFIRI vs|
|10.5%||6 vs 4.9 (HR=0.87; p=0.65)||13.7 vs 12.3 (HR=0.87; p=0.65)||RAS WT|
10.2 vs 10.4 (HR=0.93; p=0.54)
25.6 vs 33.1 (HR=0.70; p=0.011)
|1630||First line: FOLFOX/XELOX vs cetuximab+FOLFOX/XELOX||8%||5.6 vs 9.0|
(RAS/BRAF WT) p<0.0001
|8.8 vs 14.4|
(KRAS MT) p<0.001
|8.6 vs 8.6 (HR=0.96; p=0.60)||17.9 vs 17.0 (HR=1.04; p=0.67)|
VII (2012) 
|566||First line: NORDIC FLOX+cetuximab vs FLOX alone vs intermittent FLOX+cetuximab||12%||5.1 vs 8.3|
(BRAF WT) p<0.001
|9.5 vs 22|
(BRAF WT) p<0.001
|8.7 vs 7.9 vs|
|22.0 vs 20.1|
vs 21.4 (HR=1.08–1.14;
|572||Chemorefractory: cetuximab vs BSC||3.2%||Median PFS not reported (HR=0.76; p=0.69)||1.77 vs 2.97 (HR=0.84; p=0.81)||Favours cetuximab (HR=0.4; p<0.001)||9.7 vs 5.0 (HR=0.52; p<0.0001)|
|471||First line: capecitabine (C) vs capecitabine/bevacizumab (CB) or capecitabine/bevacizumab/|
|10.6%||2.5 vs 5.5 (HR=0.86; p=0.71)||6.3 vs 9.2 (HR=0.67; p=0.34)||5.9 vs 8.8 (HR=0.66; p=0.006)||20 vs 19.8 (HR=0.86; p=0.38)|
|460||Second line: irinotecan vs irinotecan/panitumumab (IrPan)||14.8%||Favours|
IrPan (~6M) (HR=0.78;
|10.5 vs 10.4|
Peeters M, Oliner KS, Price TJ, Cervantes A, Sobrero AF, Ducreux M, et al. Updated analysis of KRAS/NRAS and BRAF mutations in study 20050181 of panitumumab (pmab) + FOLFIRI for 2nd-line treatment (tx) of metastatic colorectal cancer (mCRC). J Clin Oncol 2014;32(Suppl.). Abstract 3568.
|1015||Second line: FOLFIRI|
1.8 vs 2.5
5.7 vs 4.7
5.5 vs 6.9
15.4 vs 18.7
|508||First line: Avastin/FOLFIRI vs Avastin/FOLFOXIRI||7.5%||5.5 vs 7.5 (HR=0.56)||10.8 vs 19.1 (HR=0.55)||RAS WT|
11.3 vs 13.3
34.4 vs 41.7
To further analyse the impact of MSI status in the
In accordance with their aggressive nature,
Recently, other rare (<1%) subtypes of
4.2. Predictive role
To date, the predictive role of
5. Treatment strategies
5.1. Triplet chemotherapy effect
The updated analyses of the same study reported a BRAF mutation rate of 7.5%. In the
5.2. Maintenance treatment
A recent meta-analysis on five RCTs had failed to demonstrate a statistically significant OS benefit (HR=0.93; 95% CI=0.85–1.02; p=0.12;
In terms of the choice for maintenance treatment, there is no current recommended standard. However, practice trends could perhaps be extrapolated from the AIO KRK 0207 trial, which confirmed the prognostic impact of mutation status . In all patients (irrespective of BRAF or RAS status), at a median follow-up of 27 months, the authors reported a time to failure of strategy of 3.6, 6.2, and 4.6 months among all patients receiving no treatment, fluoropyrimidine plus bevacizumab, or bevacizumab alone, respectively (p<0.001). However, in RAS/BRAF WT patients, bevacizumab monotherapy was as effective as combination treatment (fluoropyramidine/bevacizumab) for maintenance. In contrast, in the RAS or BRAF MT subgroup, the combination treatment was favoured, as single-agent bevacizumab was equivalent to no maintenance at all.
6. Investigated treatments targeting EGFR/RAF/MEK
6.1. BRAF/MEK inhibitors
As mentioned above, RAS proteins normally activate BRAF along with A-RAF and C-RAF . BRAF mutations lead to the constitutive activation of BRAF kinase activity, resulting in phosphorylation and activation of the MEK kinases (MEK1 and MEK2). Once activated, MEK kinases phosphorylate and activate ERK kinases, which phosphorylate a multitude of cellular substrates involved in cell proliferation and survival (Figure 1).
RAF inhibitors, such as vemurafenib and dabrafenib, have produced response rates of 50 to 80% in melanomas that harbour the BRAF V600 mutations [79, 80]. This is disappointingly contrasting to the response rate of only 5%, and median PFS of 2.1 months achieved in
Many RAF inhibitor combinations were hence evaluated in clinical trials in recent years and have shown promising results. A phase I to II clinical trial of combined RAF/MEK inhibition with dabrafenib (150 mg BD) and trametenib (2 mg OD) in 43
6.2. Dual and triplet targeting EGFR/BRAF/MEK inhibitors
The observations above have also led to a number of studies assessing the combined blockade at other sites in the EGFR pathway in addition to RAF/MEK inhibition. It was observed that the dual inhibition of anti-EGFR therapy in combination with RAF inhibition in resistant cell lines might still produce a lower degree of mitogen-activated protein kinase (MAPK) pathway inhibition in BRAF MT CRC compared to single-agent RAF inhibitors in
Encouragingly, the triplet combination of EGFR/RAF/MEK inhibition in
6.3. Acquired resistance to EGFR/RAF/MEK targeted therapies
Although trials have demonstrated early efficacies of combination targeted therapies in these
Interestingly, the group also discovered an ERK inhibitor that retained the ability to suppress MAPK signalling and overcome each of these mechanisms identified . In conjunction with these findings, early-phase clinical trials are currently incorporating ERK inhibitors as potential future treatment strategies for
6.4. Other possible EGFR/RAF targeted combination treatments
6.4.1. Vemurafenib/irinotecan/cetuximab combination
The phase I vemurafenib/irinotecan/cetuximab triplet study reported a RR of 35% (partial response) in 18 mCRC patients with a median PFS of 7.7 months. The most common adverse effects were fatigue (94%), diarrhoea (89%), nausea (83%), and rash (78%). Following this, a U.S. cooperative group randomised phase II trial (NCT01787500) of irinotecan and cetuximab±vemurafenib in BRAF-mutated mCRC (SWOG 1406) is now ongoing .
7. Alternative target signalling pathways
Although our increasing understanding of the complexity of the EGFR/RAF pathway has led to some advances in our understanding of possible mechanisms of resistance to BRAF inhibition, additional complex interactions with related pathways are likely to be involved, including the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, mammalian target of rapamycin (mTOR), and Wnt signalling.
7.1. PI3K/AKT and mTOR pathway
The PI3K/AKT pathway is an alternative resistance mechanism to BRAF inhibition in
Based on the above observations, the combination triplet inhibition treatment was studied with encorafenib (BRAF inhibitor), cetuximab, and PI3K inhibitor (alpelisib) in 28 patients and reported an overall RR of 32.1% with a median PFS of 4.3 months. The most common grade 3/4 adverse events reported were hyperglycemia (11%) and increased lipase (7%) .
Sustained PI3K/mTOR activity was demonstrated also by Corcoran et al.  in BRAF MT CRC cell lines upon BRAF inhibition. Pleasingly, a potent growth-inhibitory effect was recently observed in xenografts of BRAF MT CRC with the combined BRAF/PI3K/mTOR inhibition .
7.2. Wnt/β-catenin pathway
A study by Lemieux et al. demonstrated the Wnt/β-catenin pathway (Figure 3) as a potential novel target in MEK/ERK signalling involved in CRC tumourigenesis . The Wnt/β-catenin pathway is activated via the binding of Wnt1 protein to the G-protein coupled receptor, Frizzled. After the activation by Wnt1, Dishevelled protein (Dsh) induces the dissociation of the destruction complex that usually degrades β-catenin. Without the destruction complex, β-catenin is accumulated in the cytoplasm and transported to the nucleus to act as a transcriptional coactivator of transcription factors as shown in Figure 4. The aforementioned destruction complex comprises Axin (A), adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK3β). In the absence of Wnt1 activation, the destruction complex phosphorylates the downstream ubiquinating process. Here, the β-transducin repeat containing protein (βTrCP) binds β-catenin, ubiquinating it and marks it for degradation by the proteasome. Although there is conflicting literature with regards to the role of MAPK signalling in activating Wnt/β-catenin pathway, this group found Wnt signalling induction in high-grade
8. Other possible therapeutic mechanisms
Recently, a number of other early studies have reported additional potential mechanisms of targeted treatment, which had shown promise in
8.1. Multi-targeted angiokinase inhibitor (dovitinib)
Dovitinib is a multi-target angiokinase inhibitor with activity against fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and VEGF receptors, which participate in tumour growth, survival, angiogenesis, and vascular development. Although not effective
8.2. Proteasome inhibitor (carfilzomib)
A novel use of proteasome inhibitors (carfilzomib, bortezomib), known more for utility in haematological malignancy, has shown promising preclinical results in
8.3. microRNA (miR-145)
miR-145, a short RNA molecule of microRNA gene, which was observed to have tumour suppressor function, was found to be down-regulated in vemurafenib-resistant
In situcancer vaccine (Allostim)
AlloStim is an innovative design based on immunotherapy principles. It is derived from the blood of normal blood donors and is intentionally mismatched to the recipient. CD4+ T cells are initially separated from the blood and differentiated and expanded for 9 days in culture to make an intermediary called T-Stim. AlloStim is made by incubating T-Stim cells for 4 h with antibody-coated microbeads. The cells with the beads still attached are suspended in infusion media and loaded into syringes. The syringes are shipped refrigerated to the point-of-care. A phase I study was completed in May 2011 and a phase II/III study is due to recruit in 2016. It involves an
8.5. Apoptosis regulator (BCL-2/BCL-XL) inhibitor (Navitoclax)
Apoptosis regulator (BCL-2/BCL-XL) inhibitor (Navitoclax) was explored as a novel approach in sensitising
9. Ongoing trials for BRAF MT CRC
Many phase I/II trials are currently ongoing for
|NCT01543698||I/II||RAF inhibitor (dabrafenib)+MEK inhibitor|
(trametenib)+CDK4/6 inhibitor (LEE011)
|NCT 01719380||IB/II||RAF inhibitor (LGX818)+cetuximab+PI3K inhibitor|
(BYL-719) vs LGX818+ BYL-719
|NCT01902173||I/II||Dabrafenib+trametenib: in stage IIIC+IV CRC||Recruiting|
|NCT02034110||II||Dabrafenib+trametenib: BRAF MT rare cancers||Recruiting|
|NCT00265824||III||Avastin±erlotinib: maintenance treatment|
in unresectable CRC
|II||Regorafenib: single-agent second-line|
|NCT02041481||I||MEK inhibitor+FOLFOX: CRC failing|
treatment in KRAS/BRAF MT CRC
|NCT02278133||IB/II||Wnt ligand inhibitor (WNT974), RAF inhibitor|
|NCT01351103||I||Wnt ligand inhibitor (LGK974)||Recruiting|
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