**2. Role of B-Raf in the cell**

Wild type B-Raf is a serine/threonine-specific protein kinase that acts as an important component of the MAPK pathway regulating cellular proliferation,

survival, and differentiation. The B-Raf protein is composed of three main conserved regions that act by maintaining a closed conformation to autoinhibit protein function and to activate downstream pathway targets. Conserved region 1 (CR1) binds to conserved region 3 (CR3) to autoinhibit B-Raf function until activated by Ras. It also contains a zinc finger motif that aids in B-Raf docking at the cell membrane after activation. During activation, Ras binds to the CR1 domain allowing release of the bound CR3 domain. Conserved region 2 (CR2) acts to connect CR1 and CR3 and contains serine and tyrosine residues that are constitutively phosphorylated after Ras binding to help keep the protein in an open, active conformation and allow ATP binding. CR3 contains the enzymatic kinase domain of B-Raf, binding ATP and substrate proteins to catalyze the transfer of a phosphate group from ATP to the substrate, activating downstream signaling proteins. Importantly, this region also contains the valine amino acid at position 600, an amino acid that is often mutated resulting in the constitutive activation of B-Raf [11, 12].

B-Raf acts as a signaling protein in the Ras-Raf-Mek-Erk cascade, one of the most important oncogenic pathways in cancer. In wild type cells, extracellular growth factors and cytokines bind to transmembrane receptors on the cell's surface such as epidermal growth factor receptor (EGFR) and insulin like growth factor-1 receptor (IGF-1R). Intracellular phosphorylated sites on these receptors attract guanine nucleotide exchange factors (GEFs) such as SOS that bind to Ras and activate it by exchanging GDP for GTP. Once activated, Ras promotes the homo- and heterodimerization and activation of Raf kinases such as A-Raf, B-Raf and C-Raf. In turn, Raf kinases activate the MAP kinase pathway by phosphorylating MEK1 and MEK2. MEK proteins activate ERK 1 and 2 and the MAPK signaling pathway phosphorylates hundreds of downstream proteins [10, 13–16]. Importantly, activation of this pathway also sends inhibitory feedback towards upstream signaling components, which turn off signaling. This ultimately results in downregulation of Ras by ERK-dependent feedback [6]. Although the MAPK pathway is the most important downstream target of B-Raf, the JNK cascade, p38-MAPK pathway, and ERK-5 pathway have also been shown to be activated by B-Raf signaling [10].

### **3. Mutations in B-Raf that drive melanoma and their clinical significance**

About 40–60% of melanomas will contain mutations in B-Raf at the V600 site, driving melanogenesis through upregulation of the Ras-Raf-Mek-ERK MAPK pathway [17]. Abnormal activation of the Ras-Raf-Mek Erk MAPK pathway is detected in approximately 90% of melanomas including the other genetic subsets such as Ras mutant, NF1 loss, and TWT [17]. Interestingly, other common mutations in melanoma, such as N-Ras, c-Kit, and NF1, also act through the MAPK pathway. B-Raf mutation alone is not considered sufficient to induce melanoma formation, as it has also been identified in benign and dysplastic nevi and can induce senescence [10, 18]. The vast majority (74–86%) of B-Raf mutations are substitutions of glutamic acid for valine at the 600th amino acid (V600E). However, substitutions of lysine for valine at amino acid 600 (V600K) in B-Raf is seen in 10–20% of melanomas and another 8% have other substations at the same site (V600M, V600D, and V600R) [6]. Case reports comparing the clinical significance of these different mutations show similar disease presentation and response to treatment [6, 9]. V600K mutations are more common in older patients and those with chronic sun exposure [9]. These B-Raf mutations occur in CR3 of the B-Raf protein and result in constitutive activation of the MAPK signaling pathway by destabilization of the inhibitory interaction between CR1 and CR3 through the introduction of a negatively charged or bulky amino acid at this site [10]. Mutations have also been identified in exon 15 (the region of DNA

**5**

*B-Raf-Mutated Melanoma*

activation [9].

cal trials [27].

*DOI: http://dx.doi.org/10.5772/intechopen.86615*

**4. Diagnosis and diagnostic testing**

adjacent to V600), exon 11 and translocations involving the B-Raf gene in melanomas and melanoma cell lines. Despite the alternative locations of these mutations, some can also act to drive melanogenesis through activation of the MAPK pathway, but do not signal as a monomer like V600 mutant proteins and in some cases require Ras

Clinically, B-Raf mutations are associated with patients that are younger at initial diagnosis (<50 years old), locations with largely intermittent sun exposure, earlier diagnosis of distant metastasis (56 versus 63 years old), increased incidence of brain metastasis, a higher number of nevi and lesions with a truncal location. B-Raf mutations are not induced by UV (sun) DNA damage. Most concerning, some studies suggest that these mutations have been associated with shortened median survival (5.7 versus 8.5 months). However, these studies are often not powered to examine survival [10, 19–21]. This may relate to their association with increased ulceration in the tumor, a prognostic factor that independently is associated with decreased survival [22]. Additionally, B-Raf mutations are more common

Clinical detection of B-Raf mutations is a powerful tool in the management of advanced melanoma, allowing clinicians to make decisions regarding treatment plans with targeted therapy versus alternatives such as immunotherapy. Molecular testing for B-Raf mutations is recommended by both the National Comprehensive Cancer Network (NCCN) and European Society for Medical Oncology (ESMO) guidelines in patients requiring systemic therapy [23–25]. This now includes most patients with stage III melanoma based on recent adjuvant trials. The sensitivity and specificity of the screening tests chosen is critical, as B-Raf detection and targeting is the only biomarker that can predict a therapeutic response to B-Raf inhibitor treatment in melanoma [23, 26]. Additionally, inappropriate treatment of B-Raf negative tumors with B-Raf inhibitors may be associated with tumor progression through paradoxical activation of the MAPK pathway based on numerous preclini-

Due to advances in DNA sequencing, this method is being used more and more frequently as the initial method of mutation analysis. However, if there is not sufficient tissue or if rapid identification is needed diagnostic testing can be performed using immunohistochemistry with a VE1 monoclonal antibody to detect the V600E mutations. This method provides high sensitivity and is inexpensive. Unfortunately, it only detects this specific V600E mutation and misses other possible targets for B-Raf inhibitor therapy. Interpretation of immunohistochemistry by pathologists can also be subjective, making this method difficult to standardize. An alternative initial screening test is Sanger sequencing of the tumor DNA, often considered to be the gold standard. The tumor DNA is copied with amino acids attached to stop codons creating many copies of varying length that can be compared to determine the ultimate genetic sequence. This method is used less frequently, as a high ratio of mutant to wild type DNA is necessary to detect the B-Raf mutation and it has low sensitivity. If the immunohistochemistry or Sanger sequencing testing is negative it is often confirmed with pyrosequencing or RT-qPCR. Pyrosequencing is a method where DNA is sequenced using light tagged amino acids, allowing sequencing while the complementary DNA strand is being synthesized. This method is associated with a very high sensitivity for mutation detection; it also allows for quantification of mutated alleles in the tumor cell. However, pyrosequencing has a lower specificity than Sanger sequencing. Alternative confirmatory testing includes RT-qPCR,

in superficial spreading or nodular subtypes of melanoma [10].

#### *B-Raf-Mutated Melanoma DOI: http://dx.doi.org/10.5772/intechopen.86615*

*Cutaneous Melanoma*

survival, and differentiation. The B-Raf protein is composed of three main conserved regions that act by maintaining a closed conformation to autoinhibit protein function and to activate downstream pathway targets. Conserved region 1 (CR1) binds to conserved region 3 (CR3) to autoinhibit B-Raf function until activated by Ras. It also contains a zinc finger motif that aids in B-Raf docking at the cell membrane after activation. During activation, Ras binds to the CR1 domain allowing release of the bound CR3 domain. Conserved region 2 (CR2) acts to connect CR1 and CR3 and contains serine and tyrosine residues that are constitutively phosphorylated after Ras binding to help keep the protein in an open, active conformation and allow ATP binding. CR3 contains the enzymatic kinase domain of B-Raf, binding ATP and substrate proteins to catalyze the transfer of a phosphate group from ATP to the substrate, activating downstream signaling proteins. Importantly, this region also contains the valine amino acid at position 600, an amino acid that is

often mutated resulting in the constitutive activation of B-Raf [11, 12].

B-Raf acts as a signaling protein in the Ras-Raf-Mek-Erk cascade, one of the most important oncogenic pathways in cancer. In wild type cells, extracellular growth factors and cytokines bind to transmembrane receptors on the cell's surface such as epidermal growth factor receptor (EGFR) and insulin like growth factor-1 receptor (IGF-1R). Intracellular phosphorylated sites on these receptors attract guanine nucleotide exchange factors (GEFs) such as SOS that bind to Ras and activate it by exchanging GDP for GTP. Once activated, Ras promotes the homo- and heterodimerization and activation of Raf kinases such as A-Raf, B-Raf and C-Raf. In turn, Raf kinases activate the MAP kinase pathway by phosphorylating MEK1 and MEK2. MEK proteins activate ERK 1 and 2 and the MAPK signaling pathway phosphorylates hundreds of downstream proteins [10, 13–16]. Importantly, activation of this pathway also sends inhibitory feedback towards upstream signaling components, which turn off signaling. This ultimately results in downregulation of Ras by ERK-dependent feedback [6]. Although the MAPK pathway is the most important downstream target of B-Raf, the JNK cascade, p38-MAPK pathway, and ERK-5 pathway have also been shown to be activated by B-Raf signaling [10].

**3. Mutations in B-Raf that drive melanoma and their clinical significance**

About 40–60% of melanomas will contain mutations in B-Raf at the V600 site,

driving melanogenesis through upregulation of the Ras-Raf-Mek-ERK MAPK pathway [17]. Abnormal activation of the Ras-Raf-Mek Erk MAPK pathway is detected in approximately 90% of melanomas including the other genetic subsets such as Ras mutant, NF1 loss, and TWT [17]. Interestingly, other common mutations in melanoma, such as N-Ras, c-Kit, and NF1, also act through the MAPK pathway. B-Raf mutation alone is not considered sufficient to induce melanoma formation, as it has also been identified in benign and dysplastic nevi and can induce senescence [10, 18]. The vast majority (74–86%) of B-Raf mutations are substitutions of glutamic acid for valine at the 600th amino acid (V600E). However, substitutions of lysine for valine at amino acid 600 (V600K) in B-Raf is seen in 10–20% of melanomas and another 8% have other substations at the same site (V600M, V600D, and V600R) [6]. Case reports comparing the clinical significance of these different mutations show similar disease presentation and response to treatment [6, 9]. V600K mutations are more common in older patients and those with chronic sun exposure [9]. These B-Raf mutations occur in CR3 of the B-Raf protein and result in constitutive activation of the MAPK signaling pathway by destabilization of the inhibitory interaction between CR1 and CR3 through the introduction of a negatively charged or bulky amino acid at this site [10]. Mutations have also been identified in exon 15 (the region of DNA

**4**

adjacent to V600), exon 11 and translocations involving the B-Raf gene in melanomas and melanoma cell lines. Despite the alternative locations of these mutations, some can also act to drive melanogenesis through activation of the MAPK pathway, but do not signal as a monomer like V600 mutant proteins and in some cases require Ras activation [9].

Clinically, B-Raf mutations are associated with patients that are younger at initial diagnosis (<50 years old), locations with largely intermittent sun exposure, earlier diagnosis of distant metastasis (56 versus 63 years old), increased incidence of brain metastasis, a higher number of nevi and lesions with a truncal location. B-Raf mutations are not induced by UV (sun) DNA damage. Most concerning, some studies suggest that these mutations have been associated with shortened median survival (5.7 versus 8.5 months). However, these studies are often not powered to examine survival [10, 19–21]. This may relate to their association with increased ulceration in the tumor, a prognostic factor that independently is associated with decreased survival [22]. Additionally, B-Raf mutations are more common in superficial spreading or nodular subtypes of melanoma [10].

## **4. Diagnosis and diagnostic testing**

Clinical detection of B-Raf mutations is a powerful tool in the management of advanced melanoma, allowing clinicians to make decisions regarding treatment plans with targeted therapy versus alternatives such as immunotherapy. Molecular testing for B-Raf mutations is recommended by both the National Comprehensive Cancer Network (NCCN) and European Society for Medical Oncology (ESMO) guidelines in patients requiring systemic therapy [23–25]. This now includes most patients with stage III melanoma based on recent adjuvant trials. The sensitivity and specificity of the screening tests chosen is critical, as B-Raf detection and targeting is the only biomarker that can predict a therapeutic response to B-Raf inhibitor treatment in melanoma [23, 26]. Additionally, inappropriate treatment of B-Raf negative tumors with B-Raf inhibitors may be associated with tumor progression through paradoxical activation of the MAPK pathway based on numerous preclinical trials [27].

Due to advances in DNA sequencing, this method is being used more and more frequently as the initial method of mutation analysis. However, if there is not sufficient tissue or if rapid identification is needed diagnostic testing can be performed using immunohistochemistry with a VE1 monoclonal antibody to detect the V600E mutations. This method provides high sensitivity and is inexpensive. Unfortunately, it only detects this specific V600E mutation and misses other possible targets for B-Raf inhibitor therapy. Interpretation of immunohistochemistry by pathologists can also be subjective, making this method difficult to standardize. An alternative initial screening test is Sanger sequencing of the tumor DNA, often considered to be the gold standard. The tumor DNA is copied with amino acids attached to stop codons creating many copies of varying length that can be compared to determine the ultimate genetic sequence. This method is used less frequently, as a high ratio of mutant to wild type DNA is necessary to detect the B-Raf mutation and it has low sensitivity. If the immunohistochemistry or Sanger sequencing testing is negative it is often confirmed with pyrosequencing or RT-qPCR. Pyrosequencing is a method where DNA is sequenced using light tagged amino acids, allowing sequencing while the complementary DNA strand is being synthesized. This method is associated with a very high sensitivity for mutation detection; it also allows for quantification of mutated alleles in the tumor cell. However, pyrosequencing has a lower specificity than Sanger sequencing. Alternative confirmatory testing includes RT-qPCR,

another highly sensitive method that is relatively rapid and inexpensive but relies on primer design and selection for mutation detection and may miss uncommon mutations. As Next Generation Sequencing (NGS) becomes less expensive and more readily available in the clinic, it is becoming a more common method of mutation detection, allowing high sensitivity and specificity as well as the detection of rare mutations [6, 28]. Finally, studies evaluating levels of circulating tumor DNA show promise in evaluating disease response and relapse [28].

The above testing modalities are all laboratory based and are used in diagnostic centers that have been certified by the Clinical Laboratory Improvement Amendments and have been reviewed by the US Centers for Medicare and Medicaid Services. However, there are two testing modalities that were developed in concert with the testing and approval of targeted therapies that are considered companion diagnostic tests. These have been reviewed by the FDA and approved for diagnostic testing prior to initiation of these specific drug therapies. B-Raf mutations are detected using two primary companion diagnostic tests, the cobas 4800 BRAF V600 Mutation Test (Roche Molecular Systems, Inc) and the THxID-BRAF kit (BioMerieux, Inc). Both RT-qPCR based, these tests were developed with vemurafenib plus cobimetinib and dabrafenib plus trametinib, respectively. Despite their high sensitivity, laboratory-based tests such as Sanger sequencing can be used to confirm negative results from companion diagnostic tests [28].

### **5. B-Raf inhibitor monotherapy**

Given the frequency and importance of B-Raf in the development and progression of melanoma, interest in the development of B-Raf inhibitors was a high priority to all in the melanoma world. Three kinase inhibitors, vemurafenib, dabrafenib and encorafenib, are currently approved in the treatment of B-Raf V600-mutated melanoma. While they have been FDA approved in the treatment of B-Raf V600E and V600K-mutated melanomas, case studies and small trials suggest that these agents are also active in V600R mutants [6, 9]. However, based on published case reports, V600E mutations have improved response rates and longer progressionfree survival after dabrafenib or vemurafenib treatment than other mutations [27].

Vemurafenib (PLX4032) is a B-Raf inhibitor that acts by binding to the ATP binding site in B-Raf, inhibiting the active form of the serine-threonine kinase [29, 30]. The BRIM3 trial was a phase III trial by Chapman et al. comparing vemurafenib targeted therapy (960 mg twice daily) with dacarbazine chemotherapy in 675 patients with untreated metastatic melanoma containing the B-Raf V600E or V600K mutation. The 6-month overall survival (OS) was 84% in the vemurafenib-treated group (95% confidence interval (CI) 78–89) versus 64% in the dacarbazine-treated group (95% CI, 56–73). Interim analysis showed a 63% reduction in the risk of death (p < 0.001) and a 74% reduction in the risk of either death or disease progression (p < 0.001) compared to dacarbazine. Overall response rates, a secondary endpoint, were 48% for vemurafenib and 5% for dacarbazine [31]. In follow up of this same population, McArthur et al. showed a median OS of 13.6 months in the vemurafenib-treated group (95% CI 12–15.2) versus 9.7 months in the dacarbazine-treated group (95% CI 7.9–12.8, p < 0.001). Progression-free survival (PFS) also improved with a median PFS of 6.9 months in the vemurafenib-treated group (95% CI 6.1–7) versus 1.6 months in the dacarbazinetreated group (95% CI 1.6–2.1, p < 0.001). Overall response rate increased with time to 57% in the vemurafenib group versus 9% in the dacarbazine group. Complete responses were seen in 6% of the vemurafenib-treated group versus 1% of the dacarbazine-treated group [32]. Based on these results, vemurafenib was the first approved drug for the treatment of B-Raf V600E and V600K-mutated advanced melanoma.

**7**

*B-Raf-Mutated Melanoma*

*DOI: http://dx.doi.org/10.5772/intechopen.86615*

melanomas through similar mechanisms of action [6, 10].

B-Raf inhibition induces an overall response in approximately 50–60% of melanomas with B-Raf mutations. Predictors of response include B-Raf V600E mutations, higher PTEN levels at baseline (patients with deleted or mutant PTEN showed shorter PFS with dabrafenib therapy), initially increased levels of phosphorylated ERK followed by downregulation of phosphorylated ERK after treatment initiation, absence of MEK1p124 mutation, absence of CDKN2a gene deletion or chromosomal gains of the CCND1 gene [10]. Unfortunately, median progressionfree survival with B-Raf targeted therapy is only 7 months [27, 31, 33]. Clinical factors that may be associated with a shorter PFS include an ECOG performance status of greater than 2, an elevated LDH at treatment initiation and M1C disease [10].

Dabrafenib (GSK2118436), another approved targeted therapy for the treatment of B-Raf V600-mutated melanoma, acts as a competitive inhibitor for ATP binding on the B-Raf protein and decreases its activity. Break-3 was a phase III trial by Hauschild et al. in which dabrafenib treatment (150 mg twice daily) was compared to dacarbazine administration. A total of 250 patients with previously untreated B-Raf V600E-mutated melanoma were enrolled. Dabrafenib therapy resulted in a median PFS of 5.1 months while the dacarbazine treatment group had a median PFS of 2.7 months. The hazard ratio for progression was 0.3 (95% CI 0.18–0.51, p < 0.0001). The OS hazard ratio was 0.61 (95% CI 0.25–1.48), suggesting significantly improved survival with dabrafenib treatment. About 50% of patients treated with dabrafenib had an objective response (95% CI 42.4–57.1) versus 7% with dacarbazine therapy (95% CI 1.8–15.5). Complete response was seen in 3% of patients treated with dabrafenib versus 2% of those treated with dacarbazine. Median time to response was 6.3 weeks (95% CI 6.1–6.3) with a median duration of response of 5.5 months. Patients that progressed on dacarbazine were allowed to cross over to treatment with dabrafenib, at the end of the study 44% of patients had crossed to dabrafenib treatment [33]. Based on these results, dabrafenib was approved by the FDA for treatment of B-Raf V600E-mutated advanced melanoma. There has not been a direct head-to-head trial comparing dabrafenib and vemurafenib monotherapy in advanced melanoma with a B-Raf V600 mutation. However, extrapolating from the above trials suggests that they have very comparable clinical activity. Despite this, there is evidence suggesting that patients experience different drug-related toxicities. Vemurafenib was associated with toxicity requiring dose reduction due to grade 2 side effects in 38% of patients, while 28% of dabrafenib-treated patients required a dose reduction for grade 2 or greater side effects [32, 33]. Common toxicities of both drugs include rash, secondary skin malignancies (squamous cell carcinoma and keratoacanthomas), fatigue, arthralgia, and nausea. Vemurafenib was associated with higher rates of hepatic transaminitis, photosensitivity, and cutaneous hyperproliferative lesions; while, dabrafenib was associated with higher rates of pyrexia and chills. Despite the higher association with vemurafenib treatment, secondary skin hyperproliferative disorders and malignancies are seen with all B-Raf inhibitors. Median time to development of a squamous cell carcinoma after B-Raf inhibitor initiation is approximately 8 weeks and is seen in 20% of patients [10, 32]. These cutaneous side effects are primarily mediated by loss of feedback inhibition on the MAPK pathway after B-Raf suppression. In wild type cells, these B-Raf inhibitors accelerate B-Raf and C-Raf dimerization to activate the MAPK pathway. However, in B-Raf-mutated cells, signaling through negative feedback inhibition results in downregulation of MAPK signaling. After the addition of B-Raf inhibitors this negative feedback is lost, resulting in upregulation of MAPK signaling through C-Raf and Ras. Uncontrolled Ras activity has been associated with skin tumor formation, particularly squamous cell carcinomas. These patients are also at increased risk of new primary B-Raf wild type

#### *B-Raf-Mutated Melanoma DOI: http://dx.doi.org/10.5772/intechopen.86615*

*Cutaneous Melanoma*

another highly sensitive method that is relatively rapid and inexpensive but relies on primer design and selection for mutation detection and may miss uncommon mutations. As Next Generation Sequencing (NGS) becomes less expensive and more readily available in the clinic, it is becoming a more common method of mutation detection, allowing high sensitivity and specificity as well as the detection of rare mutations [6, 28]. Finally, studies evaluating levels of circulating tumor DNA

The above testing modalities are all laboratory based and are used in diagnostic centers that have been certified by the Clinical Laboratory Improvement Amendments and have been reviewed by the US Centers for Medicare and Medicaid Services. However, there are two testing modalities that were developed in concert with the testing and approval of targeted therapies that are considered companion diagnostic tests. These have been reviewed by the FDA and approved for diagnostic testing prior to initiation of these specific drug therapies. B-Raf mutations are detected using two primary companion diagnostic tests, the cobas 4800 BRAF V600 Mutation Test (Roche Molecular Systems, Inc) and the THxID-BRAF kit (BioMerieux, Inc). Both RT-qPCR based, these tests were developed with vemurafenib plus cobimetinib and dabrafenib plus trametinib, respectively. Despite their high sensitivity, laboratory-based tests such as Sanger sequencing can be used to

Given the frequency and importance of B-Raf in the development and progression of melanoma, interest in the development of B-Raf inhibitors was a high priority to all in the melanoma world. Three kinase inhibitors, vemurafenib, dabrafenib and encorafenib, are currently approved in the treatment of B-Raf V600-mutated melanoma. While they have been FDA approved in the treatment of B-Raf V600E and V600K-mutated melanomas, case studies and small trials suggest that these agents are also active in V600R mutants [6, 9]. However, based on published case reports, V600E mutations have improved response rates and longer progressionfree survival after dabrafenib or vemurafenib treatment than other mutations [27]. Vemurafenib (PLX4032) is a B-Raf inhibitor that acts by binding to the ATP binding

site in B-Raf, inhibiting the active form of the serine-threonine kinase [29, 30]. The BRIM3 trial was a phase III trial by Chapman et al. comparing vemurafenib targeted therapy (960 mg twice daily) with dacarbazine chemotherapy in 675 patients with untreated metastatic melanoma containing the B-Raf V600E or V600K mutation. The 6-month overall survival (OS) was 84% in the vemurafenib-treated group (95% confidence interval (CI) 78–89) versus 64% in the dacarbazine-treated group (95% CI, 56–73). Interim analysis showed a 63% reduction in the risk of death (p < 0.001) and a 74% reduction in the risk of either death or disease progression (p < 0.001) compared to dacarbazine. Overall response rates, a secondary endpoint, were 48% for vemurafenib and 5% for dacarbazine [31]. In follow up of this same population, McArthur et al. showed a median OS of 13.6 months in the vemurafenib-treated group (95% CI 12–15.2) versus 9.7 months in the dacarbazine-treated group (95% CI 7.9–12.8, p < 0.001). Progression-free survival (PFS) also improved with a median PFS of 6.9 months in the vemurafenib-treated group (95% CI 6.1–7) versus 1.6 months in the dacarbazinetreated group (95% CI 1.6–2.1, p < 0.001). Overall response rate increased with time to 57% in the vemurafenib group versus 9% in the dacarbazine group. Complete responses were seen in 6% of the vemurafenib-treated group versus 1% of the dacarbazine-treated group [32]. Based on these results, vemurafenib was the first approved drug for the

treatment of B-Raf V600E and V600K-mutated advanced melanoma.

show promise in evaluating disease response and relapse [28].

confirm negative results from companion diagnostic tests [28].

**5. B-Raf inhibitor monotherapy**

**6**

Dabrafenib (GSK2118436), another approved targeted therapy for the treatment of B-Raf V600-mutated melanoma, acts as a competitive inhibitor for ATP binding on the B-Raf protein and decreases its activity. Break-3 was a phase III trial by Hauschild et al. in which dabrafenib treatment (150 mg twice daily) was compared to dacarbazine administration. A total of 250 patients with previously untreated B-Raf V600E-mutated melanoma were enrolled. Dabrafenib therapy resulted in a median PFS of 5.1 months while the dacarbazine treatment group had a median PFS of 2.7 months. The hazard ratio for progression was 0.3 (95% CI 0.18–0.51, p < 0.0001). The OS hazard ratio was 0.61 (95% CI 0.25–1.48), suggesting significantly improved survival with dabrafenib treatment. About 50% of patients treated with dabrafenib had an objective response (95% CI 42.4–57.1) versus 7% with dacarbazine therapy (95% CI 1.8–15.5). Complete response was seen in 3% of patients treated with dabrafenib versus 2% of those treated with dacarbazine. Median time to response was 6.3 weeks (95% CI 6.1–6.3) with a median duration of response of 5.5 months. Patients that progressed on dacarbazine were allowed to cross over to treatment with dabrafenib, at the end of the study 44% of patients had crossed to dabrafenib treatment [33]. Based on these results, dabrafenib was approved by the FDA for treatment of B-Raf V600E-mutated advanced melanoma.

There has not been a direct head-to-head trial comparing dabrafenib and vemurafenib monotherapy in advanced melanoma with a B-Raf V600 mutation. However, extrapolating from the above trials suggests that they have very comparable clinical activity. Despite this, there is evidence suggesting that patients experience different drug-related toxicities. Vemurafenib was associated with toxicity requiring dose reduction due to grade 2 side effects in 38% of patients, while 28% of dabrafenib-treated patients required a dose reduction for grade 2 or greater side effects [32, 33]. Common toxicities of both drugs include rash, secondary skin malignancies (squamous cell carcinoma and keratoacanthomas), fatigue, arthralgia, and nausea. Vemurafenib was associated with higher rates of hepatic transaminitis, photosensitivity, and cutaneous hyperproliferative lesions; while, dabrafenib was associated with higher rates of pyrexia and chills. Despite the higher association with vemurafenib treatment, secondary skin hyperproliferative disorders and malignancies are seen with all B-Raf inhibitors. Median time to development of a squamous cell carcinoma after B-Raf inhibitor initiation is approximately 8 weeks and is seen in 20% of patients [10, 32]. These cutaneous side effects are primarily mediated by loss of feedback inhibition on the MAPK pathway after B-Raf suppression. In wild type cells, these B-Raf inhibitors accelerate B-Raf and C-Raf dimerization to activate the MAPK pathway. However, in B-Raf-mutated cells, signaling through negative feedback inhibition results in downregulation of MAPK signaling. After the addition of B-Raf inhibitors this negative feedback is lost, resulting in upregulation of MAPK signaling through C-Raf and Ras. Uncontrolled Ras activity has been associated with skin tumor formation, particularly squamous cell carcinomas. These patients are also at increased risk of new primary B-Raf wild type melanomas through similar mechanisms of action [6, 10].

B-Raf inhibition induces an overall response in approximately 50–60% of melanomas with B-Raf mutations. Predictors of response include B-Raf V600E mutations, higher PTEN levels at baseline (patients with deleted or mutant PTEN showed shorter PFS with dabrafenib therapy), initially increased levels of phosphorylated ERK followed by downregulation of phosphorylated ERK after treatment initiation, absence of MEK1p124 mutation, absence of CDKN2a gene deletion or chromosomal gains of the CCND1 gene [10]. Unfortunately, median progressionfree survival with B-Raf targeted therapy is only 7 months [27, 31, 33]. Clinical factors that may be associated with a shorter PFS include an ECOG performance status of greater than 2, an elevated LDH at treatment initiation and M1C disease [10].

Even after clinical evidence of progression during treatment with vemurafenib or dabrafenib, studies suggest that continued treatment with B-Raf inhibitors may prolong survival through impedance of disease growth while preventing a disease flare that can be seen with cessation of treatment [10, 34, 35].
