Management of BRAF Gene Alterations in Metastatic Colorectal Cancer: From Current Therapeutic Strategies to Future Perspectives

BRAF mutations constitute an important poor prognostic factor in metastatic colorectal cancer (mCRC) and the development of treatments in this context is of great necessity to prolong patient survival. Although the association between BRAF mutations and microsatellite instability (MSI) has been known for several years, previous clinical trials have revealed that the former has a limited prognostic impact and that immune checkpoint inhibitors offer a significant survival benefit to mCRC patients with both characteristics. Furthermore, the genomic classification of BRAF mutations according to their molecular functions enables greater understanding of the characteristics of mCRC patients with BRAF mutations, with therapeutic strategies based on this classification made more ideal to improve poor prognosis through the delivery of targeted therapies. Recently, a phase III trial was conducted in previously treated mCRC patients with BRAF V600E–mutated tumors and revealed that the combination therapy approach of BRAF inhibition and anti–epidermal growth factor receptor antibody therapy with or without MEK inhibition was more efficacious than standard chemotherapy alone. This review discusses current treatment strategies and future perspectives in BRAF-mutated mCRC.


INTRODUCTION
Colorectal cancer (CRC) is one of the most common cancers, with an associated mortality rate of 9.2% that makes it the second leading cause of cancer-associated deaths (1). Early-stage CRC can be curable under surgery, whereas metastatic or recurrent CRC is usually unresectable and carries a poor prognosis. In recent years, treatments targeting vascular endothelial growth factor (VEGF) receptors and epidermal growth factor receptors (EGFRs) have been reported to significantly extend the survival of metastatic CRC (mCRC). Furthermore, various biomarker studies, including concerning the RAS gene, have been also conducted and some findings have been deemed clinically useful in practice. In particular, BRAF mutations have been shown collectively to be a remarkably poor prognostic factor (2,3). BRAF is part of the RAS-RAF-MEK intracellular signaling pathway and its mutation is considered a genetic aberration that activates a signal that promotes tumor growth. BRAF is a kinase protein located downstream of EGFR, suggesting that patients with BRAF-mutated mCRC are less likely to benefit from anti-EGFR agents. However, a few clinical trials to date have suggested additional benefits of anti-EGFR therapy, so controversy therefore remains (4,5). The use of fluorouracil/folinic acid, oxaliplatin, and irinotecan (FOLFOXIRI) plus bevacizumab is considered to be a promising treatment regimen to prolong survival for BRAF V600E-mutated mCRC. However, recent reports indicate that further investigation is required (6). This article summarizes the data of previous clinical trials in BRAFmutated mCRC, discusses current treatment strategies, and offers future perspectives.

THE ROLES OF BRAF MUTATION IN COLORECTAL CANCER
BRAF is a serine/threonine-specific protein kinase involved in the signaling cascade of the mitogen-activated protein kinase (MAPK) pathway, which promotes cell growth and differentiation ( Figure 1). Activated RAF proteins trigger the activation of MEK1/2 and further activate ERK. Subsequently, ERK phosphorylates transcription factors and regulates significant cellular activity (7,8). Approximately 15% to 30% of all CRCs are thought to contribute to cancer in the serrated pathway, a multistage carcinogenic mechanism that is an alternative to the traditional adenomatous carcinoma model. Morphologically, serrated lesions can be classified as hyperplastic polyps, serrated adenomas/polyps, and classic serrated adenomas, respectively. These lesions exhibit a high incidence of BRAF mutations and also present the CpG island methylator phenotype (CIMP), which causes methylation of mismatch repair genes and can progress to microsatellite instability (MSI) (9).
BRAF mutations are found in 10% to 15% of CRCs, including early-stage cancers (10). In addition, the frequency of BRAF mutations in mCRC has been reported to be 8% to 10% according to prior retrospective analyses of several clinical trials (3,11,12). BRAF mutations are a key poor prognostic factor in this population, affecting the progression-free survival (PFS) and overall survival (OS) alike (13)(14)(15). The prognostic impact of BRAF mutations is generally similar in between CRCs and melanomas, whereas in lung cancers, there are conflicting results among studies, possibly due to their low frequency (16,17). Patients with BRAF-mutated CRC are more likely to be female, have tumors on the right side, exhibit more peritoneal metastases, and show more mucinous histology in comparison with patients with wild-type CRC (18)(19)(20). Most BRAF variants present with mutations in BRAF V600E. Most recently, it has become recognized that clinical features differ depending upon the site of the mutation.

ALTERATIONS IN THE BRAF GENE IN PATIENTS WITH mCRC
Most BRAF-mutated mCRC have mutations in V600E, but 2% to 3% show other mutations (19,21). Non-V600 BRAF-mutant mCRC was reported to involve different clinical characteristics relative to V600 BRAF-mutant mCRC; typically, patients with non-V600 BRAF-mutant mCRC are slightly younger and are less female, with low-grade tumors and with primary tumors located on the left side more often (19). In terms of prognosis, the median OS was longer in patients with non-V600 BRAF-mutant mCRC as compared with among both V600 BRAF-mutant mCRC and BRAF wild-type mCRC patients. In patients with NSCLC, the prognostic impact of non-V600 mutations is not consistent across reports (22,23), while in patients with melanoma, there was no significant survival difference between non-V600 mutations, V600 BRAF-mutation, and BRAF wildtype (24).
Based on recent analysis, BRAF mutations can be classified into three groups according to their function (25,26). Class 1, encompassing V600 mutations, is marked by high kinase activity and exhibit MEK/ERK signaling activation as RAS-independent monomers. Class 2 mutations involve intermediate kinase activity and exhibit RAS-independent activation of MEK/ERK signaling in a dimer with BRAF. Meanwhile, class 3 mutations exhibit reduced kinase activity and are dependent on RAS to activate signaling (27). Notably, class 3 mutations are more likely to be associated with long-term survival relative to both class 1 and class 2 mutations (28). Moreover, it has been reported that patients with BRAF mutations may respond differently to treatments depending on the mutation class. BRAF mutations of classes 2 and 3 are not responsive to RAF inhibitors, whereas class 3 mutants are RAS-dependent and potentially effective for EGFR inhibition (27). Confirming this, a large series analysis of non-V600 BRAF-mutant CRC patients receiving anti-EGFR therapy revealed that patients with class 3 BRAF mutations responded to anti-EGFR therapy, while those with class 2 BRAF mutations did not responded (29). On the contrary, two studies have reported that non-V600 BRAF mutations are unable to be mitigated with anti-EGFR therapy (30,31). Owing to the rarity of non-V600 BRAF mutations in this population, the efficacy of anti-EGFR therapy and the optimal regimen for non-V600 BRAF-mutant CRC remains unclear. A multicenter phase II trial of the combination of the MEK inhibitor binimetinib, the BRAF inhibitor encorafenib, and the anti-EGFR antibody drug cetuximab is currently ongoing, assessing patients with non-V600E BRAF-mutant mCRC who have not received any previous anti-EGFR antibody drugs. This study will also ultimately test the same regimen in class 3 patients with prior anti-EGFR antibody exposure (32).
Acquired BRAF alterations also may occur. BRAF amplification has been reported in patients with BRAF-mutated mCRC who have developed resistance to combination RAF/MEK inhibition. This alteration results in the onset of resistance to the RAF/EGFR or RAF/MEK combination through sustained MAPK pathway activity (33). Thus, therapeutic strategies to address the BRAF mutation class and resistance through BRAF amplification are also attracting attention regarding non-BRAF V600E.

THE EFFECT OF CONVENTIONAL CHEMOTHERAPY ON BRAF-MUTATED mCRC
Clinical trials assessing the impact of conventional chemotherapy, including both as first-line and second-line treatment, are shown in Table 1. The efficacy of anti-EGFR therapy for BRAF-mutated mCRC is controversial. In the pooled analysis of CRYSTAL and OPUS, two randomized clinical trials examining the effects of adding cetuximab to doublet chemotherapy, anti-EGFR treatment displayed numerical benefits in the objective response rate (ORR), PFS, and OS for BRAF-mutated mCRC, although no significant difference was recorded. Anti-EGFR plus chemotherapy presented an ORR of 21.9%, median PFS of 7.1 months, and median OS of 14.1 months; in contrast, chemotherapy alone demonstrated an ORR of 13.2%, median PFS of 3.7 months, and median OS of 9.9 months (3).
Two meta-analyses to date have evaluated the influence of anti-EGFR therapy in BRAF-mutated mCRC. Although these reports suggested a trend existed toward better hazard ratios (HRs) concerning PFS and OS in the context of EGFR therapy, it did not show a statistically significant difference (4,5). Recently, the VOLFI trial (AIO KRK0109), a randomized phase II trial investigating the addition of panitumumab to triplet chemotherapy with FOLFOXIRI, reported results for the subgroup of patients with BRAF-mutated mCRC. The ORR of the FOLFOXIRI plus panitumumab arm was 86% and was higher as compared with that of 22% in the FOLFOXIRI-alone arm. In contrast with the effect of ORR, the prolongation of PFS was modest, as PFS was 6.5 and 6.1 months for the FOLFOXIRI plus panitumumab and FOLFOXIRI-alone arms, respectively (34). These results support that anti-EGFR therapy may show some efficacy for the treatment of BRAF-mutated mCRC, although the magnitude of efficacy is less than that of RAS/ BRAF wild-type mCRC. Further accumulation of evidence and molecular analyses may be necessary to determine whether anti-EGFR therapy truly offers a degree of efficacy that is beneficial enough.
The efficacy of anti-VEGF therapy for BRAF-mutated mCRC has been also discussed. A biomarker analysis of a phase III study-the first report of the efficacy of bevacizumab in previously untreated patients with mCRC-included 10 patients with BRAF mutations. The median survival was 16 months among seven patients in the bevacizumab group and eight months for three patients in the placebo group, with an HR of 0.11 [95% confidence interval (CI): 0.01-1.06] (35). However, as the number of patients included was very small, it is not clear whether bevacizumab had the same effect in BRAF-mutated mCRC that it does in other types of mCRC. As second-line treatment, the efficacy of anti-VEGF targets against BRAFmutated tumors has been reported in a relatively large number of patients. Among 41 patients with BRAF mutation, the effect of ramucirumab, a monoclonal antibody drug targeting VEGFR2, relative to that of placebo was demonstrated in a subgroup analysis of the RAISE trial, with HRs of 0.54 (95% CI: 025-1.13) and 0.55 (95% CI: 0.28-1.08) for PFS and OS, respectively (36). A subgroup analysis of BRAF mutants in the VELOUR trial, a phase III study that proved the efficacy of aflibercept, also suggested that the efficacy of anti-VEGF therapy was preserved, with an HR of 0.59 (95% CI: 0.22-1.58) for PFS and that of 0.42 (95% CI: 0.16-1.09) for OS (37).
Meanwhile, in the CALGB/SWOG80405 and FIRE-3 trials, where bevacizumab was compared with cetuximab as first-line treatment, retrospective analyses were conducted for BRAFmutated mCRC (14,38). For BRAF-mutated mCRC, the median PFS in the bevacizumab group and the cetuximab group were similar in these studies; the median PFS outcomes in the bevacizumab group were 7.6 months and 6.6 months, and those in the cetuximab group were 6.2 months and 6.6 months, respectively. Further, the median OS tended to be slightly longer for bevacizumab, garnering median OS outcomes of 15.0 and 13.7 months in the bevacizumab group and 11.7 and 12.3 months in the cetuximab group. Ultimately, however, it remains uncertain which therapeutic option (bevacizumab or anti-EGFR antibody medication) as first-line treatment is more favorable for addressing BRAF-mutated mCRC.
FOLFOXIRI plus bevacizumab is considered to be one of the leading regimens against BRAF-mutated mCRC based on evidence from both retrospective and prospective trials involving small numbers of patients as well as the results of a subgroup analysis of the TRIBE trial. A retrospective analysis of a phase II study of FOLFOXIRI plus bevacizumab indicated that the response rate was 90%, the PFS was 12.8 months, and the OS was 23.8 months among 10 patients with BRAF-mutated mCRC (39). Elsewhere, a prospective study was conducted involving 15 patients with BRAF-mutated mCRC and FOLFOXIRI plus bevacizumab ensured a response rate of 60%, a PFS of 9.2 months, and an OS of 24.1 months, with these results being comparable to those of the previous report (40). In the TRIBE trial, FOLFOXIRI plus bevacizumab (triplet arm) versus FOLIRI plus bevacizumab (doublet arm) in the context of mCRC were compared. A subgroup analysis of BRAF-mutated tumors was also completed to confirm the previously described efficacy of the triplet regimen for BRAF-mutated mCRC. The results showed that the response rate was 56% in the triplet group (n = 16 patients) and 42% in the doublet group (n = 12 patients), PFS outcomes of 7.5 months versus 5.5 months and OS outcomes of 19.0 months versus 10.7 months, indicating that the triplet group was more favorable for BRAF-mutated tumors (6). In the subgroup analysis of the STEAM and CHART trials, the HRs for the median PFS were 0.8 (95% CI: 0.2-3.0) and 0.72 (95% CI: 0.25-2.07), respectively, which were not significantly different but suggested that the triplet regimen was a slightly better option (41,42). A pooled analysis including these studies reported an HR of 0.65 (95% CI: 0.33-1.26) for the triplet group, indicating no significant difference, but suggesting that the triplet regimen was again preferable (41). However, recently, a subgroup analysis of the TRIBE2 trial presented a different trend as compared with in previous studies (43). A total of 33 patients with BRAF-mutated mCRC were present in both the experimental and control groups, with an HR for the PFS of 1.02 (95% CI: 0.61-1.71), while the HR for PFS2, defined as the time from randomization to disease progression on any treatment given after the first instance of disease progression, was 1.23 (95% CI: 0.72-2.09). Furthermore, the OS presented an HR of 1.35 (95% CI: 0.79-2.30), which indicates considerable uncertainty regarding whether the triplet therapy approach is beneficial for BRAF-mutated mCRC. A metaanalysis of these five studies using individual patient data reported an HR of 1.11 (95% CI: 0.75-1.73) for the OS of the triplet regimen versus the doublet regimen, with an uncertain level of benefit noted in patients with BRAF-mutated mCRC (44). In addition, using real-world data from the United States, a retrospective evaluation of triplet and doublet regimens for patients with BRAF-mutated mCRC was conducted and showed that 16 patients receiving the triplet regimen and 423 patients receiving the doublet regimen showed median OS outcomes of 13.8 months and 15.5 months (p = 0.38), respectively (45). In summary, the benefit of the triplet plus bevacizumab regimen in BRAF-mutated tumors is controversial and adequate prospective validation in BRAF-mutated tumors is becoming more crucial. Also, there is insufficient evidence of the additive effect of anti-VEGF-targeted drugs in BRAF-mutated mCRC, but no negative evidence is currently available; thus, anti-VEGF drugs are recommended for patients for whom anti-VEGF therapy is accessible. There is no specific regimen recommended for first-line therapy of BRAF-mutated tumors, as the National Comprehensive Cancer Network guidelines recommend using the same regimen as that for the RAS wild type tumors.

CURRENT TREATMENT STRATEGIES FOR BRAF-MUTATED mCRC
Therapeutic strategies encompassing BRAF and MEK inhibitors for melanoma and thyroid cancer with BRAF mutation have been successful and several BRAF and MEK inhibitors have already been approved to date by the United States Food and Drug Administration for these diseases (46). In this section, the current development of BRAF and MEK inhibitors for the management of BRAF-mutated mCRC will be described ( Table  2). In contrast with in melanoma, monotherapy with BRAF inhibitors has not been quite as successful in mCRC. For example, a phase I dose-escalation study of encorafenib was conducted in 18 patients with BRAF V600E-mutated mCRC and none of the patients showed a confirmed response. In 12 patients, however, the best response was achieved with stable disease, with a median PFS of 4.0 months (47). Elsewhere, the effects of vemurafenib were assessed in 21 patients with BRAF-mutated mCRC, with only one partial response. The best response with stable disease was seen in seven patients and the median PFS was 2.1 months (48). In a basket study of vemurafenib in BRAF V600E-mutated cancers, 10 patients with mCRC who received vemurafenib monotherapy experienced no response. Both in vitro and in vivo studies have reported that the cause of the failure to respond to BRAF inhibition alone was the rapid reactivation of ERK via the feedback activation of EGFR. Given this, the combination of BRAF inhibitor and anti-EGFR antibody medications was expected to increase efficacy in mCRC and the combination of vemurafenib and cetuximab was therefore attempted in another cohort. Ultimately, however, one of the 27 patients experienced a partial response (49). Moreover, in a phase I/II trial of another anti-EGFR antibody combination, 13 patients received the combination of vemurafenib and panitumumab, and two patients responded to treatment (50).
Other BRAF inhibitors were also tested in combination with anti-EGFR antibody drugs. In a phase Ib study of the combination of encorafenib and cetuximab in patients with BRAF-mutated mCRC, five of 26 (18%) patients showed a response. The trial also included the PI3K inhibitor alpelisib in combination with encorafenib and cetuximab, with responses observed in five of 28 (18%) patients; for these two study populations, the median PFS outcomes were 3.7 months and 4.2 months, respectively (51). Subsequently, a phase II part of the same study enrolled 50 patients in the encorafenib plus cetuximab arm and 52 patients in the encorafenib plus cetuximab plus alpelisib arm, respectively, and reported response rates of 22% and 27% and median PFS outcomes of 4.2 months and 5.4 months, respectively (52). Vemurafenib has also been assessed in combination with irinotecan, a cytotoxic anticancer agent: a phase I trial of vemurafenib plus cetuximab with or without irinotecan in BRAF-mutated mCRC showed promising outcomes, with a response rate of 35% and a median PFS of 7.7 months (53). In the subsequent phase II study, however, the results were not as expected, with a response rate of just 16% and a median PFS of 4.4 months (54).
As the inhibition of BRAF leads to the activation of MEKdependent signaling, simultaneously inhibiting MEK while administering a BRAF inhibitor is expected to result in a better therapeutic response. Henceforth, further studies that have attempted to inhibit both BRAF and MEK will be described. In a phase I/II study of the MEK inhibitor trametinib in combination with dabrafenib, five of 43 patients (12%) with BRAF-mutated mCRC achieved a partial response (55). MEK inhibitors were further studied in combination with anti-EGFR therapy: a phase I/II study of dabrafenib and trametinib plus panitumumab in comparison with dabrafenib plus panitumumab or trametinib plus panitumumab, achieved a response rate of 21% and a median PFS of 4.2 months with the triplet regimen (56). Recently, a large phase III (BEACON CRC) trial demonstrated that encorafenib plus cetuximab with or without binimetinib was superior in terms of the response rate, PFS, and OS compared to FOLFIRI/irinotecan plus cetuximab. As a result of this study, the Food and Drug Administration approved encorafenib plus cetuximab for the treatment of BRAF V600E-mutated mCRC (57). The updated National Comprehensive Cancer Network guidelines also recommend combining encorafenib and cetuximab for patients with BRAF V600E-mutated mCRC who previously progressed on firstline therapy.

TREATMENT FOR BRAF-MUTATED mCRC WITH SIGNIFICANT MSI (MSI-HIGH)
In previous research, BRAF-mutated mCRC showed a higher percentage of MSI than BRAF wild-type mCRC (12.6% vs. 3%) (12). A pooled analysis of four phase III studies in mCRC reported significantly worse PFS and OS outcomes for BRAFmutant mCRC in comparison with BRAF wild-type mCRC with microsatellite-stable tumors, but no significant difference was apparent between BRAF-mutant and BRAF wild-type mCRC with MSI-high tumors (13). Therefore, MSI may have a stronger impact on prognosis as compared with BRAF mutation. Several clinical trials testing the effects of anti-programmed cell death protein 1 (PD-1) antibody in MSI-high/mismatch repairdeficient mCRC included patients with BRAF mutations. These results showed that BRAF mutation was not a predictor for the efficacy of anti-PD-1 antibody in MSI-high mCRC. In the KEYNOTE-164 phase II trial of pembrolizumab in previously treated MSI-high mCRC, there were nine patients included with BRAF V600E mutations in cohort A (who received two or more lines of treatment) and five patients with BRAF V600E mutations in cohort B (who received one or more lines of treatment) and the response rates were 55% and 20%, respectively. The response rates for BRAF-mutant and BRAF wild-type mCRC were 42% and 38%, respectively, and seemed to be independent of the BRAF mutation status (58). In the recently reported phase III KEYNOTE-177 trial of pembrolizumab versus chemotherapy in MSI-high mCRC, a subgroup analysis of patients with BRAF V600E mutations showed an HR of 0.48 (95% CI: 0.27-0.86) for PFS, similar to that for BRAF wild-type mCRC (HR: 0.50, 95% CI 0.31-0.80) (59).
The phase II trial of nivolumab or the combination of nivolumab plus ipilimumab in MSI-high mCRC, CheckMate 142, also included patients with BRAF-mutated mCRC and reported a relatively high response rate. In this trial, patients with BRAF-mutated mCRC were grouped into three arms; patients who received two or more lines of treatment were assigned to the nivolumab group (n = 25 patients) and patients who received one or more lines of treatment (n = 29 patients) or no prior treatment (n = 17 patients) were assigned to the nivolumab plus ipilimumab group. The response rates reported for these patients were 25%, 55%, and 76%, respectively, and were comparable to those for BRAF wild-type patients (41.4%, 55%, and 62%, respectively) (60)(61)(62). Therefore, BRAF-mutated mCRC with concomitant MSI-high should be preferred for treatment strategies according to the MSI-high status.

FUTURE PERSPECTIVES
Several clinical trials in BRAF V600E-mutated mCRC are currently ongoing (  (65). Although not specific to first-line therapy, a phase II trial, the IMPROVEMENT study (NCT03727763) is also ongoing to evaluate the efficacy and the safety of the combination of FOLFIRI with cetuximab and vemurafenib (66). Combination therapy with immune checkpoint inhibitors and targeted therapies is being investigated in two trials. NCT03668431 is a phase II trial investigating the combination of dabrafenib and trametinib with the PD-1 inhibitor, PDR001 (spartalizumab) in previously treated BRAF-mutated mCRC patients (67). A similar trial of anti-PD-1 antibody medication in combination with BRAF inhibition (NCT04017650) is ongoing in patients receiving second-and third-line treatments with encorafenib in combination with cetuximab and nivolumab (68).
Preclinical data indicate that Wee1 and ERK1/2 are located downstream of BRAF in the MAP kinase signaling cascade and are potentially important therapeutic targets, and based on this, AZD1775 (Wee1 inhibitor) and LY3214996 (ERK1/2 inhibitor) have been tested in phase I trials (69)(70)(71)(72)(73). In addition, activation of the Wnt pathway by RNF43 mutation may contribute to the resistance of BRAF-mutated mCRC to BRAF inhibitors. Therefore, a phase I/II trial of combination therapy of Wnt pathway inhibition with WNT974 (porcupine inhibitor) and BRAF inhibition has been conducted for BRAF-mutant CRC with RNF43 mutation (74)(75)(76).
Although clinical trials have not progressed since then, preclinical studies have reported that CDK1 and MCL-1 are involved in apoptosis resistance in BRAF-mutant CRC (77,78). Combination therapy with these inhibitors and BRAF inhibitors is expected to be developed.
The future development of treatments for BRAF-mutated mCRC is likely to be based on combination of BRAF inhibitors and anti-EGFR agents for untreated BRAF-mutated mCRC, while further investigation of combination therapy with novel agents to overcome resistance to BRAF inhibitors is expected to promoted.

CONCLUSION
Advances in genetic analysis techniques and the development of therapies via clinical trials have significantly improved the treatment of BRAF-mutated mCRC to date. In BRAF-mutant mCRC with MSI-high, the administration of immune checkpoint inhibitors as first-line treatment led to a significant survival benefit. In contrast, standard of care in first-line treatment for microsatellite-stable BRAF-mutated mCRC remains chemotherapy, as the optimal regimen is still uncertain and controversial. As second-line therapy, the combination of encorafenib plus cetuximab was demonstrated to provide a survival benefit, which is one of the most promising regimens for BRAF V600E-mutated mCRC. However, even with this new treatment, the OS with second-line therapy is only about 10 months, which is not sufficiently longer than that in RAS/BRAF wild-type mCRC. Therefore, additional therapeutic strategies must be elucidated to control the disease in the longer term; such may require the adoption of molecularly targeted therapies in first-line treatment or the further investigation of the molecular mechanisms of resistance to BRAF treatment.

AUTHOR CONTRIBUTIONS
HT drafted the manuscript and YS revised it precisely. All authors contributed to the article and approved the submitted version.