T his review examines progress in the treatment of metastatic

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1 Progress in the treatment of metastatic cutaneous melanoma John G. Lenehan, MD, FRCPC, Department of Oncology, The Schulich School of Medicine and Dentistry, University of Western Ontario, and D. Scott Ernst, MD, FRCPC, Division of Medical Oncology, London Regional Cancer Program Abstract T his review examines progress in the treatment of metastatic melanoma seen in the past 5 years. It focuses on the development of targeted therapies and immune checkpoint inhibitors that have dramatically changed the prognosis for this group of patients. Results from clinical trials of agents used alone and in combination are described. Toxicities specific to new classes of drugs are discussed. Current knowledge about the duration and sequencing of treatments is presented, along with a look at emerging therapeutic agents now entering into early trials. The incidence of cutaneous melanoma in Canada has been rising annually for more than a decade, with an estimated 6,500 new diagnoses and over 1000 deaths in Results from the Surveillance, Epidemiology and End Results (SEER) database between 2008 and 2012 show that 4% of melanomas are metastatic and incurable at diagnosis, with a 5-year survival of 16.6%. 2 Up to 9% of melanoma diagnoses are locally advanced when discovered, but many recur and prove incurable despite adjuvant treatment. 3-6 Systemic therapy has historically offered little hope to patients, who succumbed relatively quickly to the disease despite the best efforts of oncologists. Dacarbazine (DTIC) has been the gold standard treatment for metastatic melanoma since Health Canada s approval in the 1970s, but offers only modest benefit for patients who respond. In general, the response rate is 10 20% and the median survival is approximately 6 months. 7,8 High-dose interleukin-2 (IL-2) was approved in the late 1990s and is commonly used as immune therapy to treat metastatic melanoma, but is associated with severe toxicity and benefits only a few select patients. 9 Several other cytotoxic agents have been combined with DTIC or used in novel combinations in an effort to improve survival. These combinations increase the response rate, but also the toxicity, and have minimal impact on survival In 2012, the immune checkpoint inhibitor ipilimumab became the first new single-agent drug approved by Health Canada as initial treatment for metastatic melanoma in more than a decade, 15 providing significantly increased clinical John G. Lenehan, MD, FRCPC, is a Clinical Fellow in Personalized Medicine in the Division of Medical Oncology at the University of Western Ontario. D. Scott Ernst, MD, FRCPC, is Head of the Division of Medical Oncology at the London Regional Cancer Program. FIGURE 1: Timeline of Health Canada approval for drugs indicated for the treatment of metastatic cutaneous melanoma DTIC IL-2 Vemurafenib Ipilimumab Dabrafenib Trametinib Pembrolizumab Nivolumab benefit with a tolerable side effect profile Additional immune therapies have since been approved, along with targeted agents designed to inhibit the mutated signaling proteins that drive some melanomas. In less than 5 years, the landscape of metastatic melanoma treatment has been revolutionized. Conventional chemotherapies like DTIC have been relegated to a tertiary role in treatment algorithms. Figure 1 highlights the pace of new drug approvals for melanoma in Canada. 15 Targeted therapies Cellular growth and proliferation are tightly controlled in normal cells through a signaling cascade that begins with a mitogen binding to a growth factor receptor on the cell surface. The receptor transmits the growth signal to the nucleus via a series of activating protein kinases, such as RAS, RAF, MEK and ERK of the mitogen-activated protein kinase (MAPK) pathway (Figure 2). Melanoma cells frequently harbour mutations in these signaling kinases. Up to 50% have a BRAF mutation and 30% an NRAS mutation; these 2 mutations are generally mutually exclusive. 19,20 Although BRAF mutations constitutively activate the MAPK pathway, they also represent a vulnerability that can be exploited with specifically targeted inhibitors. Vemurafenib and dabrafenib are two BRAF inhibitors 20 oe VOL. 14, No. 4, november 2015

2 Table 1: Efficacy of targeted therapy for BRAF-mutant metastatic cutaneous melanoma in phase 3 trials Reference trial treatment arms N ORR (%) Chapman et al. NEJM (2011) 12 * Flaherty et al. NEJM (2012) 26 Hauschild et al. Lancet (2012) 23 * Larkin et al. NEJM (2014) 30 Long et al. NEJM (2014) 27 * NEJM (2015) 29 BRIM-3 METRIC BREAK-3 cobrim COMBI-d COMBI-v vemurafenib DTIC mttr mpfs mos survival (%) trametinib chemotherapy (months) 6 month 9 month 1 year 2 year dabrafenib DTIC vemurafenib + cobimetinib vemurafenib dabrafenib + trametinib dabrafenib dabrafenib + trametinib NR 72 vemurafenib N, number of patients; ORR, objective response rate; mttr, median time to response; mpfs, median progression-free survival; mos, median overall survival; NR, not reached. * updated results included; DTIC or carboplatin approved by Health Canada in 2012 and 2013, respectively (Figure 1). Vemurafenib was compared with DTIC in a randomized trial that included 675 BRAF mutation-positive patients who had not received prior therapy for advanced disease. All patients had good performance status (Eastern Cooperative Oncology Group [ECOG] 0 1). Patients treated with vemurafenib had a significantly improved response rate and estimated median progression-free survival (mpfs) of FIGURE 2: Components of the mitogen-activated protein kinase (MAPK) pathway From Petrella, Ernst S, Spatz A et al. Canadian perspective on the clinical management of metastatic melanoma. New Evid in Oncol 2012: months, compared to 1.6 months with DTIC (Table 1). However, while long-term responses were seen in a minority of patients, the median time to response (mttr) was only 1.45 months. 21,22 Similar results were seen with dabrafenib. 23,24 Despite a relatively quick and sometimes dramatic response, tumours ultimately develop resistance to BRAF inhibitors, and the disease rebounds with rapid progression. 25 In order to overcome BRAF-inhibitor resistance, MEK inhibitors were developed to target the MEK kinase downstream of BRAF. A randomized trial compared the MEK inhibitor trametinib with DTIC or paclitaxel in 322 BRAF mutation-positive patients who had received no more than 1 previous chemotherapy regimen. Trametinib led to a statistically significant increase in response rate, a mpfs of 4.8 months, and a hazard ratio (HR) for death of 0.54 (p=0.01). 26 Trametinib was approved by Health Canada in 2013 (Figure 1). Combining both a BRAF inhibitor and a MEK inhibitor in BRAF-mutated patients delayed the time to resistance and improved outcomes further. For example, dabrafenib and trametinib were combined and compared to dabrafenib alone in 423 BRAF mutation-positive patients who had not received prior treatment for advanced melanoma. The combination treatment was superior for all outcomes and achieved a median overall survival (mos) of 25.1 months, compared with 18.7 months in the dabrafenibalone arm (p=0.0107). 27,28 The combination was also more effective than vemurafenib alone. 29 Comparable results were observed when vemurafenib and cobimetinib were combined in a similar population of patients, although survival data are not expected until Study results are summarized in Table 1. Despite the addition of a MEK inhibitor, patients with inhibited mutant BRAF eventually progress, and several oe VOL. 14, No. 4, november

3 possible mechanisms have been explored in order to develop newer targeted treatments. Resistance to BRAF inhibitors may be the result of mutations in MEK that can be temporarily overcome, as seen with in vitro studies of melanoma cell lines. 31 Alternatively, tumours may develop a de novo NRAS-activating mutation that phosphorylates CRAF instead of BRAF, resulting in subsequent activation of downstream kinases Resistance occurs even in the presence of combined BRAF and MEK inhibitors, and additional in vitro data suggest that a separate pathway such as PI3K becomes activated by mutated NRAS. A study by Greger et al showed that, in models with NRAS mutations and BRAF inhibition, use of the drug GSK , a PI3K inhibitor, can downregulate the phosphorylation of the terminal kinase ERK. 31 Drug-related toxicities due to BRAF inhibition are different from those seen with standard cytotoxic chemotherapies. Many of the unique toxicities are cutaneous, including the development of squamous cell carcinomas of the skin, papular rash and photosensitivity Many of the skin-related toxicities seen with BRAF inhibitors were reduced when they were combined with a MEK inhibitor The common toxicities associated with BRAF inhibitors, alone and in combination with MEK inhibitors, are shown in Table 2. Immune checkpoint inhibitors The adaptive immune system functions in part to keep transformed malignant cells in check by eradicating them when identified. Consequently, a tumour is more likely to develop when the immune system is overwhelmed. A great deal has been discovered over the past 2 decades about the regulatory components of the immune system, which has led to new opportunities for treatment. CTL inhibition Antigen-presenting cells (APCs) identify melanoma cells and present tumour antigens to naive T cells within the nearby lymphatic tissue. 35 The T cells become activated to mount an attack specifically on the antigenic source wherever it is located. The process is complex and requires a well-orchestrated system of checks and balances to attain maximum effectiveness in eradicating aberrant cells, and to minimize damage to the host s normal tissues T cell activation leads to the proliferation of effector T cells, also known as cytotoxic T lymphocytes (CTLs), and to increased expression of signaling proteins that guide the activation and duration of immune response. One such protein is the cytotoxic T lymphocyte antigen 4 (CTLA-4), which appears on the surface of activated T cells between 24 to 48 hours after priming. The B7 receptor on the surface of the APC binds to CTLA-4, triggering deactivation of the T cell to ensure a contained immune response. 36,39 Ipilimumab is a human anti-ctla-4 monoclonal antibody that binds directly to the CTLA-4 receptor and blocks its interaction with B7, thereby amplifying the cytotoxic immune response by permitting continued proliferation of primed T cells (Figure 3). 39 Between 2004 and 2008, 676 patients who failed previous chemotherapy or IL-2 participated in a randomized trial that compared ipilimumab to a melanoma-derived glycoprotein vaccine (gp100). All patients were required to have good performance status and were excluded if untreated brain metastases were present. Patients in the ipilimumab arm experienced a significantly improved mos of 10.1 months and, at the time of publication, 60% of those treated with ipilimumab who experienced an objective response maintained that response for at least 2 years. 16 Table 2: Common adverse events associated with BRAF inhibitors and MEK inhibitors, alone or in combination Toxicity Vemurafenib Dabrafenib Trametinib Vemurafenib and cobimetinib Dabrafenib and trametinib BRIM-3 trial 12,22 BREAK-3 trial 24 METRIC trial 26 cobrim trial 30 COMBI-v trial 29 grade 2 grade 3 4 grade 2 grade 3 4 grade 2 grade 3 4 grade 2 grade 3 4 grade 2 grade 3 Arthralgia < <1 Rash Fatigue Cutaneous SCC * 4* < percent Keratoacanthoma Nausea Pruritus Hyperkeratosis < Diarrhea 5 < <1 Headache 4 < Vomiting <1 * combined cutaneous SCC and keratoacanthoma 22 oe VOL. 14, No. 4, november 2015

4 Further analysis showed that, in a small subset of those who responded, a rechallenge with ipilimumab at progression showed additional benefit for several months. 18 Subsequently, 502 patients who had not yet received treatment for metastatic disease were included in a randomized study that compared DTIC alone to the combination of DTIC and ipilimumab. All patients had good performance status but were excluded if they had brain metastases. The addition of ipilimumab significantly improved outcomes compared to DTIC alone, with a mos of 11.2 months vs 9.1 months (p<0.001). 17 The study results for ipilimumab are shown in Table 3. Figure 3: Activation of T cells through B7/CD28 costimulation and inhibition of deactivation signal by ipilimumab by blocking the interaction between B7 and CTLA-4 Toxicities Immune checkpoint therapy produces specific immune-related side effects that can occur in addition to side effects associated with conventional chemotherapy. Most immune-related side effects are tolerable, although some can be life threatening and require vigilance to detect and treat early. Diarrhea is a common toxicity and can progress to a severe and life-threatening colitis requiring the early From Petrella, Ernst S, Spatz A et al. Canadian perspective on the clinical management of metastatic melanoma. New Evid in Oncol 2012: use of corticosteroids and, on occasion, the anti-tnf-alpha antibody infliximab. 16,17,40 The toxicity profile of ipilimumab was tolerable and is shown in Table 4. A comprehensive approach to managing ipilimumab-related immune toxicities is detailed by Fecher et al. 41 Table 3: Efficacy of immune checkpoint inhibitors for metastatic cutaneous melanoma in phase 3 trials Reference trial treatment arms n ORR (%) Hodi et al. NEJM (2010) 16 NEJM (2011) 17 MDX NEJM (2015) 48 CheckMate 066 Weber et al. Lancet Oncol (2015) 49 CheckMate 037 NEJM (2015) 50 KEYNOTE-006 Larkin et al. NEJM (2015) 51 CheckMate 067 mttr mpfs mos survival (%) (months) 1 year 2 year 3 year ipilimumab + gp ipilimumab gp ipilimumab + DTIC DTIC nivolumab NR 72.9 DTIC nivolumab chemotherapy pembrolizumab every 2 weeks pembrolizumab every 3 weeks ipilimumab nivolumab nivolumab + ipilimumab ipilimumab N, number of patients; ORR, objective response rate; mttr, median time to response; mpfs, median progression-free survival; mos, median overall survival; NR, not reached. DTIC or paclitaxel oe VOL. 14, No. 4, november

5 Response Delayed response and even early progression may be observed with ipilimumab treatment during the time it takes for amplification of the CTLs and their subsequent migration to the tumour site. The fourth and final dose of ipilimumab is given at day 64 of treatment, and the median time to response (mttr) seen in clinical trials is approximately 95 days. 16,17 A pseudoprogression may be observed as the number and size of tumours increase due to CTL infiltration into the tumour and corresponding inflammatory response. 17,42 It is important for oncologists not to stop treatment before a true antitumour response can be determined. Tumour cells have developed elaborate mechanisms to evade attack by CTLs, one of which exploits a deactivation signal separate from CTLA-4. Under normal inflammatory conditions, epithelial and other tissue cells in the area of inflammation express a ligand known as programmed cell death ligand 1 (PD-L1). This ligand binds to a receptor expressed on CTLs known as the programmed death receptor 1 (PD-1), which transmits a death signal to the CTLs, resulting in downregulation and apoptosis. 36,43,44 The evolutionary role of this interplay is most likely to ensure that CTLs do not attack normal healthy tissue in the area of inflammation (Figure 4). 36,44 PD-1 inhibition Recently developed monoclonal antibodies specific to PD-1 are able to block the death signal, resulting in a sustained and tumour-specific cytotoxic response directly against the melanoma cells in the tumour microenvironment Two of these antibodies, pembrolizumab and nivolumab, are Table 4: Common adverse events associated with immune checkpoint inhibitors Toxicity ipilimumab nivolumab pembrolizumab* MDX trial CheckMate 067 trial KEYNOTE-006 trial any grade grade 3 any grade grade 3 any grade grade 3 percent (%) Fatigue Nausea Diarrhea Pruritis Vomiting Rash Arthralgia Dyspnea Headache Colitis Hypopituitarism Hypophysitis Hypothyroidism * Administered at 10 mg/kg every 2 weeks furthest along in clinical development and were approved by Health Canada in 2015 (Figure 1). Other PD-1 and PD-L1 antibodies are also under active clinical development. Nivolumab was directly compared to DTIC in a randomized trial of 418 previously untreated patients with metastatic melanoma who had good performance status, no untreated brain metastases, and who were BRAF wild-type. Compared to treatment with DTIC, nivolumab dramatically and significantly increased 1-year survival to 72.9%, from 42.1%. 48 Nivolumab was also compared to chemotherapy of the investigator s choice in 405 BRAF wild-type patients with good performance status who had progressed on ipilimumab, or BRAF mutation-positive patients who had progressed on a BRAF inhibitor and ipilimumab. The results Table 5: Efficacy of immune checkpoint inhibitors based on programmed death ligand 1 (PD-L1) expression Reference trial treatment PD-L1 positive* PD-L1 negative N ORR (%) mpfs (months) N ORR (%) mpfs (months) NEJM (2014) 48 CheckMate 066 nivolumab Weber et al. Lancet Oncol (2015) 49 CheckMate 037 nivolumab Larkin et al. NEJM (2015) 51 CheckMate 067 nivolumab nivolumab + ipilimumab ipilimumab ORR, objective response rate; mpfs, median progression-free survival. * Considered positive if >5% of cells stain for PD-L1 by immunohistochemistry; Includes indeterminate PD-L1 expression 24 oe VOL. 14, No. 4, november 2015

6 were significantly in favour of nivolumab with respect to ORR (31.7% vs 10.6%). The median time to response was 2.1 months and, of those who responded to nivolumab, 87% were still on treatment and without progression at the time of analysis (Table 3). 49 In 834 patients with good performance status treated with no more than 1 line of systemic therapy, pembrolizumab was compared to ipilimumab, and demonstrated superior results for all primary endpoints, including an estimated 1-year survival rate of 68.4% compared to 58.2% for the 10 mg/kg every 3 weeks dose. The study was stopped early due to superior survival (HR for death 0.69, p=0.0036). At a median followup of 7.9 months, 96.7% and 87.9% of patients treated with pembrolizumab or ipilimumab, respectively, had ongoing responses. 50 PD-1 inhibitors are not associated with the pseudoprogression seen with CTLA-4 inhibition. Immune-related toxicities caused by PD-1 inhibition are tolerable and are listed in Table 4. Table 6: Adverse events associated with combination immune checkpoint inhibitors Toxicity nivolumab + ipilimumab CheckMate 067 trial any grade (%) grade 3 (%) Diarrhea Rash Fatigue Pruritus Nausea Pyrexia Anorexia Emesis Hypothyroidism Colitis Arthralgia Headache Combinations Due to their independent mechanisms of action, nivolumab was combined with ipilimumab and compared with each agent alone in a study that included 945 patients with good performance status not previously treated for advanced disease. Both nivolumab and the combination treatment were superior to ipilimumab alone for all outcome measures. When nivolumab and ipilimumab were compared to ipilimumab alone, the mpfs was 11.5 months vs 2.9 months (HR 0.42, p<0.001; Table 3). 51 Compared to nivolumab alone, the combination treatment reduced the risk of progression by 26%, although the study was not designed to compare these 2 arms. In a subgroup analysis, the results for each treatment arm were stratified according to PD-L1 status, with patients expressing PD-L1 showing a better response to combination therapy than to nivolumab or ipilimumab alone. Interestingly, for patients not expressing PD-L1, the mpfs and mos for the combination was significantly better than for nivolumab alone; both treatments were more effective than ipilimumab alone (Table 5). 51 This finding suggests that in patients with PD-L1-expressing tumours, nivolumab alone may be as effective as the combination, but without the combined toxicities. For patients who do not express PD-L1, both drugs seem to be required to maximize the antitumour effect. There is significant interest in using biomarkers to identify patients who will likely benefit from treatment and those who may be harmed by therapy. The expression of PD-L1 has been assessed in some clinical trials to explore its potential as a biomarker for response to PD-1 inhibitors. 45,48,49,51 At this time, there are several limitations to Table 7: Therapeutic agents undergoing clinical trials for metastatic melanoma Molecular target drug clinical trial phase comment identifier* BRAF (mutated) encorafenib (LGX818) 3 COLUMBUS trial: combined with binimetinib in advanced melanoma NCT CDK4/6 ribociclib (LEE011) 1/2 combined with binimetinib in NRAS-mutant advanced melanoma NCT CD137 (4-1BB) PF CTLA-4 tremelimumab (CP ) 1/2 MEK1/2 binimetinib (MEK162) 3 * From ClinicalTrials.gov JAVELIN Medley trial: combined with avelumab in advanced solid tumours including melanoma in combination with IMCgp100 and durvalumab in solid tumours including advanced melanoma NEMO trial: comparing binimetinib with DTIC in NRAS-mutant advanced melanoma NCT NCT NCT PD-1 AMP advanced solid tumours including advanced melanoma NCT PD-L1 avelumab (MSB C) 1 JAVELIN Solid Tumour trial including advanced melanoma NCT PI3K buparlisib (BKM120) 2 LOGIC-2 trial: combined with encorafenib and binimetinib in 1 of 4 treatment arms in advanced melanoma NCT oe VOL. 14, No. 4, november

7 using PD-L1 expression as a biomarker, such as the absence of assay standardization, the lack of an established threshold for positivity, and a lack of long-term outcome data supporting its clinical benefit. Nivolumab combined with ipilimumab was tolerable but considerably more toxic than either drug alone. Fifty-five percent of patients experienced grade 3 or 4 toxicity, compared to 27.3% with ipilimumab and 16.3% with nivolumab alone. There was no apparent amplification of immunerelated toxicity with combination treatment (Table 6). 51 Figure 4: Anti-PD-1 inhibitors block the interaction between PD-1 on the activated cytotoxic T cell and PD-L1 on the cancer cell Sequencing In general, the majority of patients diagnosed with metastatic melanoma will require treatment shortly after diagnosis, and all tumours should be assessed for BRAF mutations from primary or metastatic tissue. The choice of initial treatment is influenced by the estimated rate of growth (with imaging and/or LDH levels) and the presence of a BRAF mutation. For rapidly progressing tumours that are BRAF mutation-positive, firstline treatment should include a BRAF inhibitor, with or without a MEK inhibitor, in order to gain control quickly. A similar approach may be considered in BRAF mutationpositive patients who are moderately to severely symptomatic due to the location of disease and mass effect on nearby organs. There is retrospective data to suggest decreased survival in patients treated with a BRAF inhibitor followed by ipilimumab, when compared to initial treatment with ipilimumab followed by a BRAF inhibitor. 52,53 Trials assessing the impact of treatment sequencing are currently underway. 54 Regardless of BRAF mutation status, patients who are asymptomatic or minimally symptomatic may have slowgrowing disease. In this case, immune checkpoint inhibitors may be the most appropriate first-line treatment when a longer time to response is permissible. Patients with BRAFmutated tumours can be switched to targeted therapy, should they progress on immune therapy. At this time, the combination of ipilimumab and nivolumab is not approved for use in Canada. When available, patients should be started on a PD-1 inhibitor, given its superior performance and more tolerable side-effect profile. Nivolumab has recently been approved for first-line use only in patients who are BRAF wild-type; however, the benefit of ipilimumab following progression on a PD-1 inhibitor is unknown. In jurisdictions where nivolumab is not funded, ipilimumab is the most appropriate first-line choice. Pembrolizumab is approved for use in ipilimumab-refractory patients regardless of BRAF status and should be used in the second line. Within the tumour microenvironment, cytotoxic T cells (CTL) interact with tumour cells through the TCR. The binding of the PD-L1 on the tumour cell to the PD-1 on the CTL leads to transmission of a death signal triggering apoptosis in the CTL. When an anti-pd-1 antibody binds the PD-1 on the CTL, the death signal is blocked allowing the CTL to survive and continue to eradicate tumour cells. PD-L1 = programmed death ligand 1; PD-1 = programmed death receptor 1; MHC = major histocompatibility complex; TCR = T-cell receptor Currently, there is insufficient evidence for PD-L1 status to influence treatment choices. Finally, traditional cytotoxic chemotherapies such as DTIC remain options in the second and third line, but may not provide any meaningful benefit to patients who have progressed on targeted and/ or immune therapies. All patients should be encouraged to participate in clinical trials when available. Future directions The availability of new therapies for metastatic melanoma has dramatically increased within a few years, and there is no indication that this pace will slow in the near future. On the contrary, several clinical trials are currently investigating new targeted and immune therapies, analyzing the combination of targeted and immune therapies with conventional chemotherapy, and determining the optimal sequence for the use of available drugs. A full assessment of ongoing studies is beyond the scope of this review, however Table 7 summarizes a broad selection of therapeutics currently in clinical trials Conclusion Metastatic melanoma is relatively uncommon, but is one of the most lethal malignancies. The annual incidence rate in Canada continues to grow in the context of our aging population. Within the last 5 years, there has been a dramatic improvement in treatment with the emergence of new targeted and immune therapies that have displaced conventional chemotherapies. The development of these new agents presents a great opportunity to provide effective treatments 26 oe VOL. 14, No. 4, november 2015

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