Solving the BRAF Mystery in CRC: Genomic Clues Lead to Triplets and Immunotherapy

Diverse Molecular Drivers in CRC

Mutations in the BRAF kinase are found in a relatively small number of patients with metastatic colorectal cancer (mCRC), but they nonetheless have important implications for prognosis and response to standard therapy. Thus far, investigators have struggled to directly target BRAF activity in colorectal cancer (CRC), despite the progress that has been made in melanoma against a seemingly identical oncogenic driver. While searching for explanations for this discrepancy between the 2 tumor types, researchers have fostered a greater understanding of the molecular underpinnings of CRC. Novel treatment strategies including triplet regimens are yielding improved outcomes. Meanwhile, the role of BRAF mutations in CRC continues to be elucidated.In the United States, CRC is the third most common type of cancer and the third leading cause of cancer-related death.¹ Surgical resection remains the only curative option for patients with localized tumors. Approximately a quarter of patients present with metastases and another 25% develop them during the course of the disease.² The standard of care for these patients is influenced by several factors, including their performance status, age, comorbidities, and preferences. It involves the use of combination chemotherapy regimens, typically 5-fluorouracil and leucovorin with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI).³

Next-generation sequencing study results have revealed significant heterogeneity in the molecular nature of this cancer type. Several drugs targeting key molecular drivers of CRC have yielded significant survival gains in the past decade when added to chemotherapy in the frontline setting and beyond in patients with metastatic disease. The epidermal growth factor receptor (EGFR) antibodies, cetuximab (Erbitux) and panitumumab (Vectibix), and the antiangiogenic drug, bevacizumab (Avastin), which targets the vascular endothelial growth factor (VEGF), are all approved for first-line treatment in metastatic settings.³

To date, the only established predictive molecular biomarker to aid treatment selection for patients with mCRC is the presence of activating mutations in the KRAS or NRAS oncogenes, which predict lack of response to EGFR-targeted therapy.⁴

Another molecular driver of CRC is the BRAF gene, which encodes a serine/threonine protein kinase that is an integral part of the mitogenactivated protein kinase (MAPK) pathway, which governs cellular proliferation, differentiation, and survival. The MAPK pathway is activated downstream of extracellular growth signals transmitted via membrane-bound receptor tyrosine kinases (RTKs), such as the EGFR.

Inside the cell, these RTKs activate RAS proteins, which in turn stimulate the RAF family members, including BRAF, ultimately triggering the MEK and ERK proteins, which transduce the signal into the nucleus to effect changes to a range of cellular processes (Figure5 ).^3-6

Figure. Key Elements of BRAF Signaling Pathway⁵

Clinical Differences in BRAF-Driven CRC

According to a recent pooled analysis of randomized controlled trials, BRAF mutations are present in approximately 8% of mCRC cases.⁷ Since their discovery, more than 100 types of BRAF mutations have been described in CRC.⁸ The most common is a substitution mutation that results in the switching of a valine for a glutamine within the catalytic domain (V600E).⁹BRAF-mutant CRC constitutes a distinct subset of the disease, with unique clinical characteristics and behavior. It tends to be associated with colon ,rather than rectal tumors, and with tumors that are right-sided, high-grade, or with mucinous histology. Additionally, the mutations are correlated with older age and being female. They also have a different pattern of metastasis; in addition to being associated with multiple metastatic sites, they have higher rates of peritoneal and distant lymph node metastases and fewer lung metastases.^10,11

Best characterized, however, is the association of BRAF mutations with poor prognosis in CRC. Numerous reports have demonstrated a lack of response to standard therapies and reduced survival in advanced-stage patients with BRAF mutations. The impact of BRAF mutations in earlier-stage disease is not as clear-cut; some studies suggest a similar poorer prognosis, while others show no effect.¹¹

There has also been a great deal of interest in determining whether BRAF mutation status has any role in predicting response to molecularly targeted therapies. Because BRAF mutations activate the MAPK pathway downstream of EGFR, they are thought to drive resistance to EGFR inhibition. Indeed, a growing body of evidence supports this assumption.¹⁰

On the other hand, the currently available evidence also suggests that BRAF mutations have no impact on the benefits of bevacizumab; however, few studies have evaluated their effects in patients treated with other antiangiogenic therapies, such as ziv-aflibercept (Zaltrap) and ramucirumab (Cyramza), both approved for the second-line treatment of mCRC.³

Not All Mutations Are Created Equal

There is ample reason for pursuing the BRAF pathway as a therapeutic target in CRC, not just as a driver of this cancer type, but for its potential capacity as a mediator of resistance to molecularly targeted therapies, such as EGFR inhibitors.Although the majority of BRAF mutations are V600E substitutions, non-V600E mutations also have been described in patients with mCRC. Non-V600 mutations are extremely heterogeneous and found across 19 codons of the BRAF gene. Much less is known about them and their biological and clinical impact on disease, but that is beginning to change.¹²

In the largest cohort of non-V600 BRAF-mutant mCRC malignancies studied to date, tumors with these mutations were compared with those with V600 mutations and those with wild-type BRAF. The non-V600 mutations were found to represent another distinct subset of mCRC, with a better prognosis including for patients with wild-type disease.

Patients with non-V600 mutations tended to be younger, were less likely to be female, and were less likely to have peritoneal metastases and have right-sided or high-grade tumors. The median overall survival for these patients was 60.7 months compared with 43 months for BRAF wild-type tumors and 11.4 months for patients with BRAF V600 mutations.¹³

Three classes of BRAF mutations have been described: class 1 and 2 lead to RAS-independent activation of BRAF as a monomer (class 1) and as dimers (class 2) and have well-established oncogenic activity. Class 3 mutations do not result in activation of BRAF kinase activity and their oncogenic role is less clear.

Melanoma Success Does Not Translate

In 2 recently published Nature papers, class 3 mutations were shown to have increased capacity for binding RAS and CRAF (another member of the RAF protein family) and activated MEK and ERK in this manner, thus suggesting that they do play a role in tumorigenesis. According to the study authors, the distinct mechanisms by which they upregulate the MAPK pathway have important implications for treatment selection in patients with these types of mutations who may respond differently to certain drug classes compared with patients with class 1 and 2 BRAF-mutant tumors.^14,15Inappropriate activation of the BRAF pathway is observed in other types of cancer, most notably in melanoma, in which BRAF-mutant disease constitutes nearly 50% of all cases. Small-molecule inhibitors of the BRAF protein have demonstrated astounding efficacy as monotherapy and in combination regimens for patients with BRAF-mutant melanoma.

The selective small-molecule BRAF inhibitors, dabrafenib (Tafinlar) and vemurafenib (Zelboraf), have demonstrated response rates in the 50% range in this setting and have shown even more impressive activity in combination with inhibitors of the MEK protein, among which trametinib (Mekinist) and cobimetinib (Cotellic) have attained regulatory approval. In striking contrast to melanoma, vemurafenib monotherapy yields response rates of only 5% in patients with BRAF-mutant CRC and the other drugs fare no better (Table 1).

Table 1. Key Findings in Targeted Therapy Trials in BRAF-Mutant CRC

16-25 Although BRAF and MEK inhibitor combinations are slightly more effective, CRC response rates are not comparable even to monotherapy in BRAF-mutant melanoma.²⁶ Significant efforts have been made to understand the reasons for this disparity in the preclinical setting and by using paired pre- and posttreatment biopsies from clinical trials of these drugs. These studies’ results have highlighted the difficulty in achieving sustained blockade of the BRAF pathway in CRCs, which are able to rapidly deploy numerous mechanisms of resistance. Indeed, 1 study’s results demonstrated recovery of MAPK signaling as early as 3 to 6 hours after vemurafenib treatment.^3,27

Common resistance mechanisms include BRAF gene amplification or alternative splicing; activating mutations in other components of the BRAF pathway, such as MEK1 and NRAS; and activation of alternative pathways, such as those governed by insulin-like growth factor 1 receptor (IGF1R), hepatocyte growth factor (HGF) and phosphoinositide 3-kinase (PI3K), which enable the cell to bypass the need for BRAF signaling.^3,28

Capitalizing on Synergy of Combinations

In particular, high levels of activated EGFR appear to be associated with development of resistance, a phenomenon that is not observed in melanoma cells, most likely because they intrinsically express much lower levels of EGFR.²⁹In addition to further delineating these mechanisms of resistance, much of the focus in the field has shifted onto identifying ways to more stably inhibit the MAPK pathway, predominantly through rational combinations of drugs (Table 1).

Using BRAF and EGFR inhibitors together has shown particular promise in producing more sustained suppression of the MAPK pathway in the preclinical setting, prompting clinical trials of this combination. In a pilot trial of vemurafenib and panitumumab, 10 of 12 patients with BRAF-mutated mCRC had tumor regression.²⁰

Vemurafenib has also been combined with cetuximab and the small-molecule tyrosine kinase inhibitor erlotinib (Tarceva), and dabrafenib with panitumumab, but for the most part any observed enhancements to efficacy have been modest.³

Triplet Therapy Emerges

The combination of EGFR and BRAF inhibition with chemotherapy has proved to be much more effective than BRAF monotherapy in mCRC and presents a possible new standard of care in BRAF-mutant CRC. In a phase Ib study, the combination of vemurafenib, cetuximab, and irinotecan produced a radiographic response rate of 35% and had manageable toxicity.³⁰

Results from the follow-up randomized phase II SWOG 1406 trial were presented at the 2017 Gastrointestinal Cancers Symposium in January. The addition of vemurafenib to cetuximab and irinotecan significantly prolonged progression-free survival (PFS): 2 months for doublet cetuximab and irinotecan versus 4.4 months with the addition of vemurafenib to the combination (HR, 0.42; P = .002).²⁴

Triplet targeted therapy approaches are also being explored, including simultaneous inhibition of 3 points in the BRAF pathway with BRAF, MEK, and EGFR inhibition and cross-pathway blockade with EGFR, BRAF and PI3K inhibition (Table 2).

Table 2. Selected Combination Trials in BRAF-Mutant CRC

In a phase I/II study, the addition of trametinib to a combination of dabrafenib and panitumumab increased the response rate from 10% with the doublet to 26% with triplet therapy.23 Array Biopharma is developing the novel BRAF and MEK inhibitors, encorafenib and binimetinib, and is seeking FDA approval for the combination for the treatment of BRAF-mutant advanced melanoma.

Meanwhile, in BRAF-mutant CRC, these drugs are being combined with cetuximab in the phase III BEACON-CRC trial (NCT02928224). The trial began in 2016 and is expected to be completed by 2019, but initial data from the safety lead-in of the study were recently presented. As of August 2017, 30 patients (29 of whom had a BRAF V600E mutation) had been treated with the triplet combination. The confirmed response rate was 41%, including 1 complete response (CR). The response rate was 59% among patients with BRAF V600E mutations who had received only 1 prior therapy (n = 17).

The most common grade 3/4 adverse events (AEs) were nausea, vomiting, increased blood creatinine kinase, and urinary tract infection.²⁵

Epigenetics Open Door to Immunotherapy

The combination of encorafenib, cetuximab, and the PI3K inhibitor alpelisib has also shown promising activity. The results of a phase II study showed that the doublet encorafenib and cetuximab and the triplet regimen had significant activity. In the doublet arm, the response rate was 22% and the median PFS was 4.2 months; in the triplet cohort, the results were 27% and 5.4 months, respectively. The improved PFS in the triplet arm came at the expense of increased toxicity; grade 3/4 AEs were reported in 79% of patients compared with 58% with the doublet.²²BRAF V600E mutations are strongly associated with a CpG island methylator phenotype (CIMP).³¹ CpG islands are short rare sequences of DNA that contain an unusually high frequency of a cytosine followed by a guanine and have a tendency to be methylated. Methylated CpG islands located in the promoter region of genes silence the expression of that gene; thus, CIMP in the promoter region of tumor suppressor genes can potentiate tumorigenesis^.32

One of the genes that may be epigenetically inactivated as a result of the CIMP phenotype is MLH1, which plays an important role in the DNA pathway that repairs the genetic mismatches that can occur when the DNA is replicated. Thus, CIMP can lead to mismatch repair deficiency (dMMR); in turn, dMMR is often associated with microsatellite instability (MSI), which are fluctuations in the length of repetitive sequences of DNA within the genome whose function is unknown. Approximately one-fifth of BRAF-mutant CRCs display dMMR.^10,28

Tumors that have high levels of MSI (MSI-H) or dMMR are usually hypermutable, displaying hundreds or thousands of DNA mutations. These can serve as antigens that stimulate the immune system. Accordingly, MSI-high or dMMR tumors often contain many infiltrating T cells. In order to combat this increased exposure to the immune response, these tumors have also evolved mechanisms for evading the antitumor immune response, such as displaying high levels of PD-L1 protein on their surface.^33,34

It stands to reason, therefore, that BRAF-mutant CRC tumors may be sensitive to immunotherapy, particularly the immune checkpoint inhibitors that target PD-L1 and its receptor PD-1.

Clinical trials of immune checkpoint inhibitors have demonstrated impressive activity in patients with mCRC with high levels of MSI and dMMR, although responses are either not dependent upon BRAF mutation status or the effect of BRAF mutations has not been evaluated.

In June, pembrolizumab (Keytruda), a PD-1—targeting antibody, became the first immunotherapy approved for the treatment of mCRC. The approval was based on durable responses achieved in 5 uncontrolled single-arm trials involving 149 patients with MSI-high or dMMR tumors, including 90 participants with mCRC. Specifically, pembrolizumab is indicated for use in patients with MSI-high or dMMR mCRC that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. This decision was followed in August by the approval of nivolumab (Opdivo) for the same indication, based on the results of the phase II CheckMate-142 trial, in which the objective response rate was 28%, including 1 CR and 14 partial responses.³⁵

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