Pharmacokinetic evaluations suggest that the discordant responses between the two diseases are due to marked differences in underlying tumor biology. In contrast to CRC, melanoma cells are not epithelial cells and do not typically express epidermal growth factor. The lack of response of BRAFV600ECRC to single-agent BRAF inhibition has been hypothesized to be due at least in part to feedback activation of EGFR resulting in MAPK signaling pathway reactivation.41,42 In vitro and in vivo models have shown decreased MAPK signaling and increased response with combined inhibition of BRAF and EGFR.40–42

Based on this hypothesis, 15 patients with BRAFV600E metastatic CRC who had previously progressed on prior chemotherapy were enrolled to a pilot study of vemurafenib plus panitumumab, both at previously approved doses.43 Initial responses were encouraging, with some tumor regression seen in 10 of the 12 evaluable patients and less cutaneous toxicity than would be expected with either single agent.

In another attempt to inhibit signaling through the MAPK pathway, a single-arm study treated 43 patients with BRAFV600E metastatic CRC with the combination of dabrafenib and the MEK inhibitor trametinib.44 Responses were improved compared to single-agent BRAF inhibition, with 12% of patients achieving a partial response or better and 56% with stable disease. Of note, there was one complete response, with the duration of response of >36 months, and 10 patients remained on study beyond 6 months. Adverse events were overall similar to what has been seen with combined BRAF and MEK inhibition in melanoma, notably with pyrexia occurring in 30% of patients and being the most common cause of treatment delay and dose reductions.

These approaches to MAPK signaling inhibition were combined in a Phase I/II clinical trial, which enrolled patients with BRAFV600E CRC to the combination of dabrafenib, trametinib, and panitumumab.45 In the Phase I component, there were no dose-limiting toxicities and the most common adverse event was acneiform dermatitis. The Phase II expansion of this study then compared the dabrafenib, trametinib, and panitumumab triplet with doublets of either dabrafenib plus panitumumab or trametinib plus panitumumab. In the trametinib plus panitumumab arm, there were no confirmed responses, suggesting that inhibition of BRAF is necessary but not sufficient for response. The response rate to dabrafenib plus panitumumab arm was 10% (95% CI 1.2–31.7) compared to 21% (95% CI 12.5–43.3) for the triplet arm.46 For the 91 patients treated with triplet therapy, disease control (response plus stable disease rates) was achieved in 86% of patients, with a median duration of response of 7.6 months and a median PFS of 4.2 months.47 These results suggest a role for combined targeted therapies as a therapeutic option for BRAF mutant disease (Figure 1).

Activation of the PI3K/AKT pathway has also been shown in BRAFV600E CRC cell lines to mediate both innate and acquired resistances to BRAF inhibition.48 Preclinical work confirmed that combining blockade of PI3Kα, BRAF, and EGFR resulted in synergistic antiproliferative effects in vitro and tumor regression in xenograft models.49 This spurred a Phase Ib/II study evaluating the RAF inhibitor encorafenib in combination with cetuximab with or without the PI3Kα inhibitor alpelisib in BRAFV600Emetastatic CRC.50 Although high rates of grade III/IV adverse effects of 69% in the two-drug arm and 79% in the three-drug arm were reported, the maximum tolerated dose was not reached in either arm.50 The overall response rates in the 54 patients treated were 19 and 18% in the two- and three-drug arms, with a median PFS of 3.7 and 4.2 months, respectively.50 Of note, this was a heavily pretreated population with the majority of patients having received two or more prior lines of therapy and >25% having received prior anti-EGFR therapy.

Concurrently with the development of targeted therapies, a Phase 1b study evaluated the combination of targeted therapies in combination with chemotherapy with a three-drug regimen of vemurafenib, cetuximab, and irinotecan.51 The combination was well tolerated with the most common adverse effects being fatigue, diarrhea, nausea, and rash, and responses were seen in 35% of evaluable patients. This led to the Phase II Southwest Oncology Group (SWOG) 1406 trial, the first-ever randomized controlled trial in BRAFV600E-mutant metastatic CRC, evaluating irinotecan plus cetuximab with or without vemurafenib.52 The primary endpoint was PFS, and cross-over to the experimental arm with vemurafenib was allowed. With heightened awareness and increased testing for BRAFV600E among local oncology providers, accrual to SWOG 1406 was swifter than projected; 106 patients enrolled in <1.5 years. Reflecting the insight of the investigators’ awareness of the unique challenges of measuring peritoneal metastasis and ascites in this patient population, SWOG 1406 included patients with nonmeasurable disease according to the RECIST 1.1 criteria. SWOG 1406 reported a median PFS of 4.4 vs 2.0 months for the three-drug regimen (irinotecan, cetuximab, and vemurafenib) vs the two-drug regimen (irinotecan and cetuximab) (HR 0.42; 95% CI 0.26–0.55; P<0.001). Response rates in this study were 16 vs 4%, respectively (P=0.09). Of note, neutropenia, anemia, and nausea occurred more frequently in the arm that included vemurafenib; however, the proportions of patients who developed rash and fatigue were comparable.

Correlative laboratory studies performed in conjunction with these studies provide a sobering reminder of how little we know. A study of molecular alterations in paired exposure biopsies obtained before and after exposure to BRAF inhibition showed acquisition of KRAS amplification, BRAF amplification, and MEK1 mutation following exposure to BRAF inhibitors.53 Similarly, analysis of circulating tumor DNA revealed the emergence of RAS mutations at disease progression in nearly half of patients following treatment with dabrafenib, trametinib, and panitumumab.47 Each of these alterations results in reactivation of the MAPK pathway, and these findings point to the critical importance of signaling through this pathway for the growth of BRAFV600E-mutant CRC cells. Additionally, analysis of pERK suppression from tumor samples after treatment with the combination of dabrafenib, trametinib, and panitumumab showed less suppression than what is seen with treatment with dabrafenib alone in melanoma, suggesting that we are currently still unable to inactivate MAPK pathway signaling in BRAFV600E-mutant CRC. This pathway dependence intimates that ongoing efforts to inhibit MAPK signaling may be necessary to achieve improvements in clinical response.

THE INTERSECTION OF BRAF AND MSI

As noted earlier, there is a significant overlap between BRAFV600E and sporadic high-level MSI in CRC. In previously completed randomized trials, 16–33% of MSI-high tumors also harbored BRAFV600Emutations.44,46,50 In a population-based cohort of 1,253 patients, 52% of the 193 MSI-high cases were also BRAF mutant, while 55% of the BRAF mutant cases were also MSI-high.26 In this population, MSI-high status was associated with significantly lower CRC-specific mortality (multivariable HR =0.28, 95% CI 0.17–0.46; P<0.001) and the presence of a BRAFV600E mutation was associated with significantly higher CRC-specific mortality (multivariable HR =1.64, 95% CI 1.18–2.27; P=0.003), as seen in other studies. Within each subgroup, MSI and BRAFV600E status remained prognostic, such that BRAFV600E was associated with higher mortality in both MSS and MSI-high patients, and MSI-high tumors were associated with decreased mortality in both BRAF-wild-type and -mutated patients.26These results suggest that the presence of a BRAFV600E mutation may diminish the more favorable prognosis afforded by MSI-high status in metastatic CRC.

However, it is not clear that this relationship holds true for stage III CRC, where studies have had conflicting results. A study performed on tumor specimens from the Intergroup 0135 trial for stage III CRC found that MSI-high and BRAF-wild-type patients had the best long-term survival but found no differences between MSI-high/BRAFV600E-mutant, MSS/BRAFV600E-mutant, and MSS/BRAF-wild-type patients.54 A similar analysis performed on specimens from the adjuvant CALGB 89803 trial also showed that MSI-high and BRAF-wild-type tumors are associated with the best prognosis but identified MSI/BRAFV600E-mutant and MSS/BRAF-wild-type patients to have an intermediate prognoses, while MSS/BRAFV600E-mutant patients have the worst prognosis.55 This finding parallels findings from patients with metastatic CRC.

The recent US Food and Drug Administration (FDA) approvals of two checkpoint inhibitors, pembrolizumab and nivolumab, for the treatment of MSI-high metastatic CRC transformed the therapeutic landscape for the small subset of metastatic CRC patients with MSI-high tumors (~4%). Given the substantial overlap between MSI-high status and BRAF mutation, there is a considerable interest in the application of checkpoint inhibitors in the subset of patients with MSI-high BRAFV600ECRC.

Pembrolizumab is a monoclonal antibody against the programed death receptor 1 (PD-1), which has been shown to increase immune recognition of tumor across a variety of tumor types. In a Phase II study of pembrolizumab given to 10 MSI-high and 18 MSS metastatic CRC patients, the response rates were 40 and 0%, respectively.56 This led to an expansion cohort for a total of 28 MSI-high patients, in whom the response rate was 50% (95% CI 31–69%).57 Median PFS had not been reached at the time of presentation at the 2016 ASCO Annual Meeting. However, there were no identified patients with BRAF mutations in this cohort; thus, it is not yet clear from this study if presence of a BRAFV600E was associated with the likelihood of response.

Nivolumab, the second checkpoint inhibitor to be FDA approved for MSI-high metastatic CRC, is also a monoclonal antibody directed against PD-1. The Phase II Checkmate-142 study reported a 31% (95% CI 20.8–42.9) response rate and a 69% (95% CI 57–79) disease control rate in 74 MSI-high metastatic CRC patients treated with nivolumab.58 Similar to what has been seen in other studies of checkpoint blockade, responses were often durable and median PFS has not yet been reported. Of the 16% of patients with a BRAF mutation (n=12), an objective response was seen in 25%. Although this response rate is similar to what has thus far been observed with the aforementioned targeted BRAFinhibition combinations, the possibility of durable disease control may be much higher with checkpoint inhibition; this remains an area of active investigation.