Melanoma accounts for 4% of incident cancers and its mortality rate is increasing.  Surgical excision remains the treatment of choice for early disease, and adjuvant therapy with interferon alfa has shown benefit in some stage II and III cases.  However, advanced melanoma is an aggressive disease for which there are few therapies, and which portends a poor prognosis.
The US Food and Drug Administration (FDA) previously approved interleukin-2 and dacarbazine in this setting; in clinical trials, each had response rates of 10-20% and neither showed an overall survival benefit. [3, 4] More recently, ipilimumab (Yervoy), a fully humanized antibody that binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and sustains an immune attack on neoplastic cells, was approved by the FDA for use in the metastatic setting. Approval was based on a randomized study showing that ipilimumab improved survival from 6 months to 10 months compared with an experimental vaccine.  This advance promised new effective treatment for what was once a treatment-refractory disease and more research is underway to investigate other molecular pathways that may be targeted in an effort to produce further significant results.
The use of immune checkpoint inhibitors for the treatment of advanced melanoma has evolved beyond monotherapies to combination strategies. This combination approach results in response rates around 60% and superior progression-free survival compared with ipilimumab monotherapy (median 11.5 versus 2.9 months). 
One potential advance that showed a lot of promise in the preclinical, and now in the clinical setting, is the targeting of the BRAF kinase in melanoma. Vemurafenib and dabrafenib are BRAF kinase inhibitors available in the United States. Trametinib, a mitogen-activated extracellular signal regulated kinase (MEK) inhibitor, is also FDA-approved.
The mitogen-activated protein (MAP) kinase pathway is an important driver in melanoma and is made up of several potential targets providing therapeutic options.  In this pathway, the activation of RAS proteins stimulates the RAF kinases ARAF, BRAF, and RAF1. This process causes the phosphorylation of the MEK kinases, which phosphorylate the ERK kinases. Activated ERK regulates cyclin D1, which, in turn, regulates multiple cellular processes involved in cell division (see Figure).
Approximately 40-60% of melanomas contain a mutation in the gene that encodes BRAF that leads to constitutive activation of downstream signaling in the MAP kinase pathway. In 80-90% of these cases, the activating mutation consists of the substitution of glutamic acid for valine at amino acid 600 (V600E). [7, 8] In a series of 197 patients, Long and colleagues showed that BRAF mutations were associated with a features of high-risk melanoma, including truncal primary, earlier age ofonset, lack of chronic skin damage, and shortened survival,  suggesting the value of inhibiting mutated BRAF.
Clinical Implications and Genetic Testing
First-line treatment of patients with BRAF V600 wild-type or mutation-positive, unresectable or metastatic melanoma is with nivolumab as a monotherapy or in combination with the immunotherapy ipilimumab.
The November 2015 approval for nivolumab monotherapy is based on data from the randomized phase 3 CheckMate-066 trial, which compared nivolumab monotherapy with dacarbazine in the first-line treatment of 418 patients with advanced BRAF wild-type melanoma. In an interim analysis, nivolumab demonstrated superior overall survival, which was the primary outcome. The overall survival rate at 1 year was 72.9% (95% CI, 65.5 to 78.9) in the nivolumab group and 42.1% (95% CI, 33.0 to 50.9) in the dacarbazine group. A significant benefit with respect to overall survival was observed in the nivolumab group, as compared with the dacarbazine group (hazard ratio for death, 0.42; 99.79% CI, 0.25 to 0.73; P<0.001). Median progression-free survival was also improved in the nivolumab-treated patients compared with dacarbazine (5.1 vs 2.2 months; HR, 0.43; P <0.001). 
The FDA approved the combination regimen of nivolumab plus ipilimumab on September 30, 2015 in previously untreated patients with BRAF V600 wild-type unresectable or metastatic melanoma. Approval was based on results from the phase 2 CheckMate-069 study Of the 142 patients enrolled, 109 had both BRAF wild-type and BRAF mutation-positive melanoma. The primary endpoint was objective response rate (ORR) in patients. In patients with BRAF wild-type melanoma treated with the combination regimen, the overall response rate was 61% (95% CI: 48-71) compared to 11% (95% CI: 3-25) in patients given ipilimumab monotherapy (P <0.001).
Additional analysis showed that complete responses were seen in 22% of patients. Partial responses were seen in 43% of the combination group and 11% of the ipilimumab monotherapy group. The combination group had a 60% reduction in the risk of progression compared with ipilimumab alone (HR=0.40; 95% CI: 0.22-0.71; P <0.002). Median PFS was 8.9 months with the combination (95% CI: 7.0, NA) and 4.7 months with ipilimumab alone (95% CI: 2.8-5.3). 
In January 2016, this indication was expanded to include mutation-positive melanoma, making nivolumab effective across BRAF status. 
Selective inhibitors of BRAF have shown early clinical activity and have rapidly progressed through clinical trials.  A multicenter phase 1/2 trial of vemurafenib (Zelboraf), an oral inhibitor of BRAF, demonstrated that vemurafenib inhibits the kinase activity of BRAF containing the V600E mutation at low nanomolar concentrations, blocking the proliferation of cells. [9, 10] In the dose escalation phase of the trial, 55 patients were enrolled; an additional 32 patients were enrolled during the extension phase. Of the 32 patients in the extension phase, all of whom were confirmed to have the V600E mutation, 24 had partial response and 2 had complete responses, for an overall response rate of 81%. Thirty-one percent of patients enrolled in the extension phase developed squamous cell carcinoma of the keratoacanthoma type. Other side effects included arthralgias, rash, photosensitivity, nausea, palmar-plantar dysesthesia, and pruritus.  A phase 2 trial enrolling 132previouslytreatedpatientsshowed an overall response rate of 52% and a median progression-free survival of 6.2 months. 
Following on these results, the BRAF Inhibitor in Melanoma (BRIM)-3 phase 3 trial randomly assigned 675 patients to vemurafenib or dacarbazine 1000 mg/m2 every 3 weeks. In January 2011, investigators reported that the trial had closed early due to meeting its overall survival and progression-free survival endpoints, and that patients randomized to dacarbazine would be able to cross over upon progression. Final data from BRIM-3 were presented at the American Society of Clinical Oncology meeting in June 2011 and simultaneously published in The New England Journal of Medicine.  Vemurafenib was associated with a 63% relative risk reduction for death and a 74% risk reduction for disease progression or death; the response rate with vemurafenib was 48% vs only 5% with dacarbazine. At 6 months, 84% of patients who had been given vemurafenib were still alive vs only 64% of those who received dacarbazine. Of note, the median time to response to vemurafenibwas1.45months,andthemedianprogression-free survival 5.3 months.
Vemurafenib was FDA-approved in August 2011 for patients with unresectable or metastatic melanoma who have the BRAF V600E mutation as detected by the companion diagnostic FDA-approved cobas® 4800 BRAF V600 Mutation Test (Roche Molecular Systems). Vemurafenib is not approved for use in patients with wild-type BRAF melanoma. [12, 13, 14] Dabrafenib (Tafinlar, GSK2118436) was approved by the FDA in May 2013 for patients with unresectable or metastatic melanoma with BRAF V600E mutation confirmed by the THxID BRAF mutation test. [15, 16]
Two other agents that block BRAF have been studied in patients with metastatic melanoma: dabrafenib and sorafenib (Nexavar).  Preliminary results of a phase 1/2 study of dabrafenib demonstrated a 63% objective response rate in the 57 patients with V600 BRAF mutations.  The BREAK 3 study was conducted in 250 patients with previously untreated BRAF V600 mutation–positive metastatic melanoma. Results showed that dabrafenib significantly improved the median progression-free survival compared with chemotherapy with dacarbazine (5.1 vs 2.7 months; P < 0.0001). 
Sorafenib is a tyrosine kinase inhibitor that blocks wild-type BRAF but not the V600E mutated oncogenic BRAF. A phase 3 trial of sorafenib combined with carboplatin and paclitaxel failed to show an impressive response rate.  It is thought that this is due to the fact that sorafenib alone does not inhibit the V600E mutated BRAF and that BRAF is only the major driver in melanoma when it is mutated. It remains to be seen whether sorafenib in combination with other targeted agents in melanoma could overcome treatment resistance.
Trametinib (Mekinist) is a MEK inhibitor that was approved by the FDA in May 2013. It is indicated for unresectable or metastatic melanoma with BRAF V600E or V600K mutations confirmed by the THxID BRAF mutation test. Approval was based on a phase 3 open-label trial that compared trametinib to either dacarbazine or paclitaxel. Median progression-free survival was 4.8 months in the trametinib group and 1.5 months in the chemotherapy group (P< 0.001). At 6 months, the rate of overall survival was 81% in the trametinib group and 67% in the chemotherapy group despite crossover (hazard ratio for death, 0.54; 95% CI, 0.32 to 0.92; P=0.01). 
Dabrafenib and trametinib have been approved for use as monotherapy, and not as a combination treatment. Preliminary results from a clinical trial with the combination suggest that use of the 2 drugs together significantly improved progression-free survival, although the incidence of pyrexia was increased. [21, 22]
In January 2014, the FDA approved trametinib for use in combination with dabrafenib for treating patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations. Approval was based on the demonstration of response rate and median duration of response in a Phase I/II study. Median progression-free survival in the combination full dose 150 mg/2 mg group was 9.4 months compared with 5.8 months in the dabrafenib monotherapy group. The rate of complete or partial response with combination therapy was 76% compared with 54% with monotherapy. Improvement in disease-related symptoms or overall survival has not been demonstrated for this combination. 
It is as yet unknown how vemurafenib or other BRAF inhibitors might be most effectively incorporated into the clinic. Data from the phase 3 trial of vemurafenib indicate that the drug has a rapid onset of response but a short duration of response; thus, the drug might be best suited for patients with rapidly progressing and/or symptomatic disease. By contrast, patients with asymptomatic and/or slowly progressing disease could be considered first for ipilimumab in an attempt to elicit an immune response and then be switched to vemurafenib upon signs of progression.  Note that there are no data yet on sequencing ipilimumab and vemurafenib so it is unknown whether the use of ipilimumab might affect response to vemurafenib.
If one were to follow this approach, patients with localized disease who are considered at “high risk” for metastasis should be tested for BRAF mutations so that the information would available if there is a need to initiate vemurafenib immediately. The definition of “high risk” in this setting is variable, and could include patients with stage IIB disease or higher, signs of ulceration, positive lymph nodes, larger tumors, etc.
For patients with newly diagnosed, rapidly progressing metastatic melanoma, a BRAF mutation analysis should be done immediately. The cobas® 4800 BRAF V600 Mutation Test detects the mutation in formalin-fixed, paraffin-embedded tissue. Although the manufacturer states that the test can be performed in fewer than 8 hours from receipt of the specimen, physicians in private practice who do not routinely process specimens for genetic testing might find that waiting for a positive or negative test result might constitute an unacceptable delay in treatment initiation. It would be considered off-label use to start treatment immediately while waiting for test results and then discontinuing if the test results show that a mutation is not present. Whether this practice will nevertheless gain traction in the community remains to be seen.
Ultimately, as more studies are conducted with this class of agents, the community will hopefully come to a consensus on how best to incorporate BRAF mutation testing into clinical practice.