**8. B-Raf targeted therapy in brain metastases**

As discussed previously, B-Raf inhibitor therapy is an effective treatment option for patients with inoperable or metastatic melanoma. Unfortunately, melanoma has one of the highest cerebral tropisms of any malignancy. Approximately 20% of stage IV patients have brain metastases at time of diagnosis and up to 40–50% of patients with stage IV melanoma will ultimately develop intracranial disease [56]. This development contributes significantly to mortality in 20–54% of metastatic melanoma patients; and once brain metastases are diagnosed, median survival decreases to 4–5 months [56–58]. Therefore, in evaluating the efficacy of targeted and immunotherapies in advanced melanoma, it is important to evaluate whether these agents are active in the central nervous system. The BREAK-MB trial showed that dabrafenib (150 mg twice a day) had an acceptable safety profile and induced a response in the metastatic brain lesions of 39% of B-Raf V600E mutant advanced melanoma if no prior local therapy had been used and in 31% of patients with prior local therapy. Median progression-free survival was 16 weeks and median overall survival was 31 weeks [59]. The COMBI-MB trial by Davies et al. was a phase II trial of dabrafenib (150 mg twice a day) and trametinib (2 mg daily) in 125 patients with V600 mutant melanoma. About 58% of patients with asymptomatic brain metastases and no prior therapy showed a response (95% CI 46–69) with a median progression-free survival of 5.6 months (95% CI 5.3–7.4) and a median overall survival of 10.8 months (95% CI 9.7–19.6). About 56% of patients who had received prior therapy showed a response with a median PFS of 7.2 month (95% CI 1.7–6.5), while 59% of patients with symptomatic brain metastases showed a response with a median PFS of 5.5 months (95% CI 2.8–7.3). About 44% of patients with V600D/K/R mutations responded to dabrafenib and trametinib with a median PFS of 4.2 months (95% CI 1.7–6.5) [60]. Vemurafenib has been studied in a phase 2 trial with similar results [57, 58]. Interestingly, these

trials show that there is a decreased response in the brain lesions when compared to extracranial lesions after B-Raf inhibition and overall the duration of response is approximately 50% that of extracranial sites, which may be due to higher concentrations of drug at the extracranial tumor site [61, 62].

Unfortunately, investigators have also found that the brain is a frequent site of disease recurrence or metastases after B-Raf inhibition [58]. This is thought to be related to signaling changes in the metastatic cell. MAPK downregulation is associated with upregulation of the PI3K/AKT pathway. Increased signaling through this pathway is often found in brain metastases [62, 63]. Therefore, it is also important to continue to investigate optimal treatment for intracranial disease after treatment with B-Raf inhibitors.

## **9. Mechanisms of resistance**

Initial response rates to B-Raf inhibitors in B-Raf-mutated melanoma ranged between 50 and 70%, suggesting that 30–50% of these tumors have a mechanism of primary resistance prior to therapy. Additionally, approximately 50% of patients treated with B-Raf targeted therapy develop resistance within 1 year and only 10% of patients will respond to combination B-Raf and MEK targeted therapy for at least 3 years [10]. On average, resistance to B-Raf inhibition occurs after 6–8 months of treatment, although this is prolonged with dual MEK inhibition [38]. Evaluation of tumor samples after the development of B-Raf inhibitor resistance showed 38% of the mechanisms of resistance were non-genomic in origin, while 56% were due to both genomic and non-genomic changes [64]. About 79% of these mechanisms are associated with MAPK signaling reactivation [38]. Adjusting treatment regimens to address B-Raf inhibitor resistance is made even more difficult by the finding that several resistance mechanisms often coexist within the same tumor or between different tumor sites in patients treated with B-Raf inhibitors [27, 38].

Although mechanisms of primary resistance have been defined, it is difficult to conclusively establish that there was no response to treatment. Almost all patients with B-Raf-mutated melanoma respond initially to B-Raf inhibition; however, the duration of response is so short that there is evidence of progression at the time of disease evaluation. Alterations in the MAPK pathway such as predominance of signaling through C-Raf or the PI3K pathway increases immunity to B-Raf inhibition. NF1 is a tumor suppression that acts to inhibit Ras, and loss of NF1 function leads to constitutive Ras activation and activation of the MAPK pathway irrelevant of B-Raf inhibition. Through similar signaling changes, alterations in the PI3k-AKTmTOR pathway (such as loss of function in PTEN) lead to constitutive activation of AKT and cell survival. Alterations in the RB1 pathway through mutations in cyclin D1, CDK4, or CDK6 can also lead to cell cycle progression irrelevant of B-Raf signaling [6].

Mechanisms of secondary resistance that develop after treatment with B-Raf inhibitors predominantly occur through changes allowing MAPK signaling despite B-Raf inhibition. Signaling through the MAPK pathway can be restored through N-Ras or MEK1/2 activating mutations. Upregulation and activation of the receptor tyrosine kinases and the PI3K-AKT-mTOR pathway (through IGF1-R, PDGFRβ, MET, mTORC1/2, EGFR, and ERBB3) can also activate MAPK signaling regardless of B-Raf inhibition. These changes have been identified in cell lines and in biopsies from the tumors of B-Raf inhibitor-treated patients after progression. Feedback activation of EGFR following B-Raf inhibition causes resistance through deactivation of MIG6 and increased expression of SOX10, restoring downstream signaling. But the most important pathways effect the B-Raf V600 molecule themselves,

**13**

*B-Raf-Mutated Melanoma*

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

monotherapy (82 versus 50%) [66, 67].

with B-Raf inhibition [10].

**10. Future directions**

including alternative splicing of the B-Raf V600E protein resulting in loss of the RAS binding domain and decreased sensitivity to the inhibitor as well as amplification of the B-RAF V600 gene inducing an overabundance of ligand. Copy number amplification of the B-Raf mutation can result in drug saturation and lead to dimerization despite inhibitor exposure, allowing downstream activation. Upregulation of C-Raf can increase signaling through a similar mechanism. MAP3K8 encodes COT, a protein that phosphorylates MEK independently of Raf signaling. Mutations in MAP3K8 have been identified in resistant tumors. Shifts in cellular metabolism to favor oxidative metabolism through increased expression of PGC1alpha have also been associated with B-Raf inhibition [6, 10]. Increased signaling through the YAP pathway and escape from cell death through upregulation of Bcl-XL have been identified in resistant cells after treatment with B-Raf inhibitors [9]. Mutations in the PI3K-AKT pathway (either through positive regulation of the pathway or negative regulation of its inhibitors PIK3R2 or PHLPP1) can upregulate signaling through this pathway, allowing cell survival despite B-Raf inhibition [6, 10]. The tumor microenvironment can also upregulate MAPK signaling through increased MAPK signaling in melanoma-associated fibroblasts after B-Raf inhibitor exposure. These fibroblasts act to promote matrix formation and remodeling, creating a protective environment for the tumor cell [38, 65]. In a study of 132 melanoma samples collected after the development of B-Raf inhibitor resistance, 20% had a N-Ras/K-Ras mutation, 16% had developed a B-Raf splice variant, 13% showed B-Raf amplification, 7% had a MEK1/2 mutation, and 11% developed an alteration in a non-MAPK signaling pathway [66]. Combined treatment with B-Raf and MEK inhibitors has shown development of resistance through similar mechanisms [67]. In fact, resistance after treatment with combination therapy is more often mediated through MAPK signaling reactivation than after treatment with B-Raf inhibitor

Due to the relative high rate of primary and secondary resistance to B-Raf inhibitors, alternative dosing schedules are being studied to see if these slow the rate of treatment escape. Intermittent dosing schedules show some promise in increasing the average time to progression for B-Raf-mutated melanomas treated

In vivo data and studies involving patient tumor samples have found that soon

after B-Raf inhibitor initiation, immune activation is enhanced in the tumor microenvironment through multiple mechanisms [6]. The microphthalmiaassociated transcription factor (MITF) is activated by MAPK signaling to suppress the expression of melanocyte-lineage antigens. Blockade of this pathway with B-Raf inhibitors upregulates expression of these melanoma-specific antigens, increasing the immune system's ability to recognize and target tumor cells. By the time tumor progression is noted on B-Raf inhibitors, these markers are usually downregulated and suppressed. B-Raf inhibition is also associated with an increase in tumor infiltrating lymphocytes early after treatment initiation. Finally, B-Raf inhibition often results in decreases in the immunosuppressive cytokines interleukin (IL)-6 and IL-8. Associated with a better tumor response to B-Raf inhibition, these findings suggest that adding immunotherapy or employing immunotherapy somewhere in the treatment course may be beneficial [38, 68]. Mouse studies have also demonstrated that treatment with dabrafenib, trametinib, and an anti-PD1 immunotherapy resulted in improved outcomes compared to either therapy alone [38, 69]. Attempts at combining vemurafenib and ipilimumab have been terminated

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

*Cutaneous Melanoma*

with B-Raf inhibitors.

**9. Mechanisms of resistance**

trials show that there is a decreased response in the brain lesions when compared to extracranial lesions after B-Raf inhibition and overall the duration of response is approximately 50% that of extracranial sites, which may be due to higher concen-

Unfortunately, investigators have also found that the brain is a frequent site of disease recurrence or metastases after B-Raf inhibition [58]. This is thought to be related to signaling changes in the metastatic cell. MAPK downregulation is associated with upregulation of the PI3K/AKT pathway. Increased signaling through this pathway is often found in brain metastases [62, 63]. Therefore, it is also important to continue to investigate optimal treatment for intracranial disease after treatment

Initial response rates to B-Raf inhibitors in B-Raf-mutated melanoma ranged between 50 and 70%, suggesting that 30–50% of these tumors have a mechanism of primary resistance prior to therapy. Additionally, approximately 50% of patients treated with B-Raf targeted therapy develop resistance within 1 year and only 10% of patients will respond to combination B-Raf and MEK targeted therapy for at least 3 years [10]. On average, resistance to B-Raf inhibition occurs after 6–8 months of treatment, although this is prolonged with dual MEK inhibition [38]. Evaluation of tumor samples after the development of B-Raf inhibitor resistance showed 38% of the mechanisms of resistance were non-genomic in origin, while 56% were due to both genomic and non-genomic changes [64]. About 79% of these mechanisms are associated with MAPK signaling reactivation [38]. Adjusting treatment regimens to address B-Raf inhibitor resistance is made even more difficult by the finding that several resistance mechanisms often coexist within the same tumor or between different tumor sites in patients treated with B-Raf inhibitors [27, 38].

Although mechanisms of primary resistance have been defined, it is difficult to conclusively establish that there was no response to treatment. Almost all patients with B-Raf-mutated melanoma respond initially to B-Raf inhibition; however, the duration of response is so short that there is evidence of progression at the time of disease evaluation. Alterations in the MAPK pathway such as predominance of signaling through C-Raf or the PI3K pathway increases immunity to B-Raf inhibition. NF1 is a tumor suppression that acts to inhibit Ras, and loss of NF1 function leads to constitutive Ras activation and activation of the MAPK pathway irrelevant of B-Raf inhibition. Through similar signaling changes, alterations in the PI3k-AKTmTOR pathway (such as loss of function in PTEN) lead to constitutive activation of AKT and cell survival. Alterations in the RB1 pathway through mutations in cyclin D1, CDK4, or CDK6 can also lead to cell cycle progression irrelevant of

Mechanisms of secondary resistance that develop after treatment with B-Raf inhibitors predominantly occur through changes allowing MAPK signaling despite B-Raf inhibition. Signaling through the MAPK pathway can be restored through N-Ras or MEK1/2 activating mutations. Upregulation and activation of the receptor tyrosine kinases and the PI3K-AKT-mTOR pathway (through IGF1-R, PDGFRβ, MET, mTORC1/2, EGFR, and ERBB3) can also activate MAPK signaling regardless of B-Raf inhibition. These changes have been identified in cell lines and in biopsies from the tumors of B-Raf inhibitor-treated patients after progression. Feedback activation of EGFR following B-Raf inhibition causes resistance through deactivation of MIG6 and increased expression of SOX10, restoring downstream signaling. But the most important pathways effect the B-Raf V600 molecule themselves,

trations of drug at the extracranial tumor site [61, 62].

**12**

B-Raf signaling [6].

including alternative splicing of the B-Raf V600E protein resulting in loss of the RAS binding domain and decreased sensitivity to the inhibitor as well as amplification of the B-RAF V600 gene inducing an overabundance of ligand. Copy number amplification of the B-Raf mutation can result in drug saturation and lead to dimerization despite inhibitor exposure, allowing downstream activation. Upregulation of C-Raf can increase signaling through a similar mechanism. MAP3K8 encodes COT, a protein that phosphorylates MEK independently of Raf signaling. Mutations in MAP3K8 have been identified in resistant tumors. Shifts in cellular metabolism to favor oxidative metabolism through increased expression of PGC1alpha have also been associated with B-Raf inhibition [6, 10]. Increased signaling through the YAP pathway and escape from cell death through upregulation of Bcl-XL have been identified in resistant cells after treatment with B-Raf inhibitors [9]. Mutations in the PI3K-AKT pathway (either through positive regulation of the pathway or negative regulation of its inhibitors PIK3R2 or PHLPP1) can upregulate signaling through this pathway, allowing cell survival despite B-Raf inhibition [6, 10]. The tumor microenvironment can also upregulate MAPK signaling through increased MAPK signaling in melanoma-associated fibroblasts after B-Raf inhibitor exposure. These fibroblasts act to promote matrix formation and remodeling, creating a protective environment for the tumor cell [38, 65]. In a study of 132 melanoma samples collected after the development of B-Raf inhibitor resistance, 20% had a N-Ras/K-Ras mutation, 16% had developed a B-Raf splice variant, 13% showed B-Raf amplification, 7% had a MEK1/2 mutation, and 11% developed an alteration in a non-MAPK signaling pathway [66]. Combined treatment with B-Raf and MEK inhibitors has shown development of resistance through similar mechanisms [67]. In fact, resistance after treatment with combination therapy is more often mediated through MAPK signaling reactivation than after treatment with B-Raf inhibitor monotherapy (82 versus 50%) [66, 67].

Due to the relative high rate of primary and secondary resistance to B-Raf inhibitors, alternative dosing schedules are being studied to see if these slow the rate of treatment escape. Intermittent dosing schedules show some promise in increasing the average time to progression for B-Raf-mutated melanomas treated with B-Raf inhibition [10].
