CCR FOCUS - Clinical Cancer Research...immune cells (i.e., natural killers cells or dendritic cells)...

The Intersection of Immune-Directed and MolecularlyTargeted Therapy in Advanced Melanoma: Where We HaveBeen, Are, and Will Be

Ryan J. Sullivan1, Patricia M. LoRusso2, and Keith T. Flaherty1

AbstractIn three years, four drugs have gained regulatory approval for the treatment of metastatic and

unresectable melanoma, with at least seven other drugs having recently completed, currently in, or

soon to be in phase III clinical testing. This amazing achievement has been made following a remarkable

increase of knowledge in molecular biology and immunology that led to the identification of high-

valued therapeutic targets and the clinical development of agents that effectively engage and inhibit

these targets. The discovery of either effective molecularly targeted therapies or immunotherapies would

have led to dramatic improvements to the standard-of-care treatment of melanoma. However, through

parallel efforts that have showcased the efficacy of small-molecule BRAF and MAP–ERK kinase (MEK)

inhibitors, as well as the immune checkpoint inhibitors, namely ipilimumab and the anti-PD1/PDL1

antibodies (lambrolizumab, nivolumab, MPDL3280), an opportunity exists to transform the treatment

of melanoma specifically and cancer generally by exploring rational combinations of molecularly

targeted therapies, immunotherapies, and molecular targeted therapies with immunotherapies. This

overview presents the historical context to this therapeutic revolution, reviews the benefits and

limitations of current therapies, and provides a look ahead at where the field is headed. Clin Cancer

Res; 19(19); 5283–91. �2013 AACR.

IntroductionMelanoma is a deadly disease that is rising in incidence.

In 2013, an estimated 76,000 new cases and 9,400 deathsare expected in the United States (1). Still, despite thesestark numbers, there is great optimism in the melanomaresearch and treatment communities due to a number ofbreakthroughs that are transforming the way this diseaseis being treated. To highlight the dramatic progress beingmade to treat patients with melanoma, it is very possiblethat for the second time in 3 years, the number of ther-apies approved by the U.S. Food and Drug Administration(FDA) for the treatment of advanced melanoma willdouble in a 12-month period (Fig. 1). These advanceshave been the result of extraordinary scientific discoverycombined with robust clinical and translational efforts.What follows is an overview of the past, current, and nearfuture states of melanoma therapeutics and an introduc-tion to the topics covered in this Clinical Cancer ResearchFocus section.

Immunotherapy and MelanomaMelanoma has long been considered a malignancy that

has a complex and unique interaction with the immunesystem. The first description of immune infiltrates in pri-mary tumorswasmade decades ago, as was the definition ofthe prognostic significance of these infiltrates (2, 3). Furtherinteractions between the immune system and melanomahave been posited as the explanation of two fascinatingphenomena: (i) the long latency from primary melanomaresection of early-stage disease to the development of wide-spread metastases and (ii) the spontaneous regression ofmetastatic melanoma in a small number of patients (4, 5).Because of these findings and beliefs, immunotherapy has along history in the treatment of melanoma starting withinjections of immune stimulants (i.e., Bacillus Calmette-Gu�erin), moving to treatment with mediators of immuneresponses (i.e., cytokines) with or without "educated"immune effectors such as primed T lymphocytes (adoptivecell transfer), and more recently, monoclonal antibodiesthat target critical immune checkpoints and thereby lead toT-lymphocyte (T-cell) activation (6–11).

Cytokine therapyIn the early days of tumor immunology, it was evident

that T-cell activation, in particular cytotoxic T-lymphocyte(CTL) activation, was required (12). Although the under-standing of how T cells become active has evolved over thepast four decades, one of the first major discoveries was that

Authors' Affiliations: 1Massachusetts General Hospital Cancer Center,Harvard Medical School, Boston, Massachusetts; and 2Karmanos CancerInstitute, Wayne State University, Detroit, Michigan

Corresponding Author: Keith T. Flaherty, Massachusetts General Hos-pital Cancer Center, 55 Fruit Street, Boston, MA 02114. Phone: 617-724-4800; Fax: 617-724-6898; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2151

�2013 American Association for Cancer Research.

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a number of substances were produced and secreted byimmune cells and could interact with receptors on otherimmune cells as well as tumor cells (13–15). The substancesknown as cytokines were initially grouped as one of twotypes, type 1 associated with CTL activation (so-calledcellular immunity) and type 2, associated with antibodyformation (so-called humoral immunity; ref. 16). Interest-ingly, these two types of cytokines were typically antago-nistic, such that type 1 cytokines would inhibit humoralimmunity and type 2 cytokines would inhibit cellularimmunity. Not surprisingly, a number of type 1 cytokineswere tested as antineoplastic therapies for melanoma,among other malignancies; only IFN-a-2B (IFN2B) andinterleukin-2 (IL-2) showed sufficient benefit to supportregulatory approval for melanoma (17).

High-dose IFN2B is approved for the adjuvant treatmentofpatients with intermediate-to high-risk melanoma (definedas American Joint Committee on Cancer stage IIB, IIC, IIIA,IIIB, and IIIC) basedondata that showed an improvement inrelapse/disease-free survival (RFS) and overall survival (OS;ref. 18). Since this initial report, a number of studies havebeen conducted with high-dose IFN2B showing a consistentimprovement in RFS, yet not necessarily in OS (19). Similardata have been seen with pegylated IFN2B, an agent thatreceived FDA approval in 2011 (20). Although the data withIFN2B led to its FDA approval as an adjuvant therapy forpatients with intermediate-and high-risk melanoma, givenits toxicity profile and underwhelming efficacy, its use in thissetting is more by default due to a lack of more promisingoptions than an endorsement of its effectiveness.

High-dose IL-2 is a highly toxic therapy that leads to acapillary leak syndrome associated with hypotension/shock, massive fluid retention, and renal failure necessitat-ing that it be given in an inpatient, intensive-care levelsetting (8, 21). Its use is associated with a 16% to 23%response rate,with5%to10%ofpatients treated achieving a

durable response that can last for decades (8, 22). Given thehigh toxicity and low response rate, IL-2 is only given in asmall numberof centers, although thepotential for decades-long response is compelling and the reasonwhy this therapyis still considered for highly selected andmotivatedpatients.

Adoptive immunotherapyAnother therapy associated with long-term remissions is

adoptive T-cell therapy (23). This involves the harvesting oftumor-infiltrating lymphocytes (TIL) from metastatictumors, ex vivo expansion, and administration with IL-2following nonablating lymphodepleting chemotherapy(24). In a small series of patients, this has resulted in com-plete remissions in up to 40% of cases, even when otherimmunotherapies have failed (24, 25). This approachremains investigational but is being explored at an increas-ing number of centers. In addition, further manipulationsor genetic modifications of TILs (new culture conditions,altered cytokine secretion) and coadministrationwith otherimmune cells (i.e., natural killers cells or dendritic cells)and/or vaccines are being explored (26–30). Alternatively,tumor reactive T cells for clinical administration are alsobeing engineered from peripheral blood by the introduc-tion of receptors specific for tumor-associated antigens.And, most recently, the potential for molecularly targetedtherapy to augment tumor infiltrates and enhance effectorT-cell function following administration is being explored(reviewed by Kwong and colleagues in this issue; ref. 31).

Immune checkpoint inhibitionOver the past three decades, the complexities of immune

activation, and T-lymphocyte activation specifically, havebeen elucidated. Although cytokines play an important roleindirecting immune effectors, the process of T-cell activationrequires two major signals: (i) T-cell receptor (TCR) recog-nition of antigen in the context of MHC expressed on aprofessional antigen-presenting cell (APC) and (ii) costimu-lation in the form of TCR interactions between the T cell andtheAPC (32–34). This second step of costimulation involvesa number of so-called "checkpoints" that regulate whetherthis process occurs or not (35). The two checkpoints thathave garnered themost attention to date are the CTL antigen4 (CTLA4) and the program death 1 (PD1) molecule.

CTLA4 is a surface protein onT cells that interactswith theAPC membrane-bound costimulatory molecules, B7-1(CD80) and B7-2 (CD86), and functionally competes withthe T-cell costimulatory molecule CD28 (35, 36). After itsidentification as a potent, negative regulator of T-cell acti-vation, it became an attractive target for monoclonal anti-body therapy (37). Ipilimumab is a fully humanizedmono-clonal antibody that binds and inhibits CTLA4 function,thereby releasing a critical brake to T-cell costimulation(10). The preclinical discoveries of CTLA4 and its role inT-cell costimulatory regulation and subsequent clinicaldevelopment of ipilimumab offer an amazing example oftranslational research, as ipilimumab was the first agent tobe proven to prolong OS in patients with metastatic mel-anomaand thefirst agent since IL-2 to achieve FDAapproval

Nivolumab, lambrolizumab

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Dacarbazine

1980 1990 2000 2010

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© 2013 American Association for Cancer Research

Figure 1. FDA-approval timeline for metastatic melanoma. Dacarbazine(1976) and high-dose IL-2 (1998) were the only approved agents between1976 and 2011. In 2011, both vemurafenib and ipilimumab wereapproved, thereby doubling the number of approved agents. In 2013,dabrafenib and trametinib were approved, and based on the emergingdata with nivolumab and lambrolizumab, regulatory approval isexpected in the near future, thereby setting up the possibility that thenumber of approved agents will double again within a 12- to 18-monthtime period.

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for this treatment indication (10, 38). Clinical activity ofipilimumab has also been associated with the induction ofserious immune-mediated adverse events,most prominent-ly colitis, implying a broad role for CTLA4 in suppressingautoimmunity. Notably, an agonist CD28 antibody provedto induce a life-threatening cytokine release syndrome andhighlights the delicate balance of immune cell activation/inactivation that must be respected in designing safe andeffective immune checkpoint–targeted therapies (39).A second example of the increased knowledge of immune

checkpoint biology leading to clinical improvement is thedevelopment of antagonists of PD1 and one of its ligands,PDL1. Following chronic T-cell activation, the inhibitoryreceptor PD1 is induced on T cells, and expression of one ofits ligands, PDL1, on tissue-based macrophages and tumorcells can offer protection from immune destruction (40). Asa result, targeting either PD1 or PDL1 offers an opportunityto disable a major mechanism of tumor-mediated immuneevasion. The clinical development of monoclonal antibo-dies that inhibit either PD1 or PDL1 is under way and theresults of early-stage clinical trials of the PD1 antibodies,nivolumab and lambrolizumab, aswell as the PDL1antago-nists, MDX-1107 and MPDL1-3280, are impressive. Tumorresponses (at least 50% appearing to be durable) are seen ina sizable minority of patients, whereas toxicity seems to beless prominent as compared with ipilimumab (11, 41–44).It is not an understatement to say that these agents are themost promising treatments for melanoma that have everbeen developed given the high clinical benefit rate, reason-able tolerability, and potential for being added to otherstandard and experimental agents. This is reviewed ingreater detail by Ott and colleagues in their article onimmunotherapy in this CCR Focus section (45).

Molecular Signaling and MelanomaIn parallel to the amazing developments in the field of

immunotherapy, there has been a remarkable advancementin the understanding of the molecular biology of tumorcells. Perhaps the most profound discovery, as it relates tothe field of targeted therapy development, is the identifica-tion that tumors generally, and melanoma specifically, co-opt and then become dependent upon a small number ofsignal transduction pathways to stimulate cell-cycle pro-gression and angiogenesis, prevent apoptosis, and abrogatehost defense responses. In melanoma, the mitogen-activat-ed protein kinase (MAPK) and phosphoinositide 3-kinase(PI3K) pathways are the two major pathways that mediategrowth and survival signals (Fig. 2; refs. 46, 47). The role ofthe PI3Kpathway is reviewed in detail byKwong andDaviesin this CCR Focus section (48).

Molecular classification of melanomaThe MAPK pathway is almost always overexpressed in

melanoma and is constitutively activated through geneticaberrations, most commonly via specific point mutations,in the greatmajority of the cases (Fig. 3). Themost commonof these genetic aberrations is mutation at the 600 positionof BRAF (V600), present in 40% to 50% of melanomas

(49, 50). Mutations of the N-isoform of RAS are found inanother 15% to 25% of cases and tend to occur at eitherposition 12 or 61 (51). NRAS and BRAF mutations aremutually exclusive (the co-occurrence rate is <<1%), lead tohyperactivation of the MAPK pathway, and are associatedwith a worse prognosis than are melanomas with wild-typeNRASandBRAF (52, 53). A loss-of-functionmutation in thetumor suppressor gene, neurofibromatosis 1 (NF1), wasrecently identified in approximately 10% to 15% of casesand also is associated with abnormal MAPK signaling (54).Whenmutated, NF1 is no longer capable of keeping RAS inits inactive RAS-GDP form and thus leads to constitutiveactivation of RAS and the pathways downstream (55).Genetic mutations or amplifications are seen in other genesleading to upregulation of theMAPKpathway aswell. Theseinclude cKIT mutations, seen in less than 1% of all mela-nomas, although in upwards of 10% to 30% of acral ormucosal melanomas, and mutations in small G-proteinsubunits called GNAQ and GNA11 that are present inmorethan 80%of ocular melanomas, though rarely seen in othermelanoma subtypes (56–58).

In addition to the oncogenicmutations that lead to hyper-activation of the MAPK pathway (BRAF, NRAS, CKIT, NF1,andGNAQ/GAQ11), anumberofother genesmaybealteredin melanoma and serve to complement the primary onco-genic mutations described above. In particular, abnormali-ties of genes involved in cell-cycle regulation, includingcyclin-dependent kinases (CDK), CDK inhibitors [such asP16Ink4a (CDKN2A)], and cyclin D are seen in more than70% of the patients (reviewed in detail by Sheppard andMcArthur in this CCR Focus section; refs. 59, 60). Also, thefunctionof thenegative regulatorof thePI3Kpathway,PTEN,is either lostor impaired inup to30%ofcases (61, 62). This ismost commonly seen in a subset of patients with BRAFmutations, thereby allowing for unregulated signaling ofboth the MAPK pathway, via BRAF mutation, and the PI3Kpathway, through PTEN loss of function (52, 63).

MAPK inhibition and resistanceWith thefirst descriptionof oncogenic BRAFmutations in

melanoma, efforts were made to identify and developclinical inhibitors of both BRAF specifically and the MAPKpathway in general. The initial targeted therapy studies inmelanoma were with agents that are now considered non-specific inhibitors of BRAF, sorafenib and RAF265, andlower potency inhibitors of MAP–ERK kinase (MEK)-1/2,such as selumetinib, PD-325901, andCI-1040 (64–72). It isimportant to note that these studies were open to anypatient with melanoma independent of mutational status.Thus, these trials were doomed for failure as the agents werenot able to inhibit the MAPK pathway sufficiently at toler-able doses, and the patients treated were not preselected toinclude only those most likely to benefit. Mechanisms ofresistance to these agents were impossible to determinegiven the fact that the pathway was suboptimally inhibitedand thus few patients received benefit.

BRAF-directed therapy in BRAF mutants. The first so-called targeted therapy to show substantial efficacy in

Immunotherapy and Molecular Targeted Therapy in Melanoma

www.aacrjournals.org Clin Cancer Res; 19(19) October 1, 2013 5285



melanoma was vemurafenib (73). In the initial phase Istudy, it was determined early that only patients withoncogenic (i.e, V600) mutations experienced clinical ben-efit and that almost every one of these patients had someevidence of tumor regression with treatment. Furthermore,responses occurred early with improvement of symptomswithin days and near complete 2[18F]fluoro-2-deoxy-D-glu-cose positron emission tomography (FDG-PET) responseswithin 2 weeks from the onset of therapy. A subsequentphase II study confirmed the remarkable response dynamicsand frequency of vemurafenib, and a phase III study con-firmed that vemurafenib conferred a survival advantagecompared with chemotherapy (50, 74). A second, potent,

and specific mutant BRAF inhibitor, dabrafenib, has beenassociated with very similar clinical efficacy as vemurafeniband joined it as the second BRAF inhibitor to achieve FDAapproval (75). A third such BRAF inhibitor, LGX818, hasshown responses at every dose level tested (76).

Although the advantages of BRAF inhibitor therapy arethe rapid onset and high frequency of responses, the dis-advantage is the limited durationof clinical benefit (74, 75).Specifically, BRAF inhibitor treatment is associated with aprogression-free survival (PFS) of only 5 to 7 months as aresult of the development of cellular resistance over thisrelatively short period of time. The mechanisms of thisresistance can be subdivided into those that can be


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Figure 2. Molecular and immunologic signaling in melanoma. Melanoma cells use a diverse set of cell surface receptors and intracellular signalingmolecules to promote growth, cell-cycle progression, cell survival, angiogenesis, and immune evasion. HIF-1a, hypoxia-inducible factor-1a; IGF-1R,insulin-like growth factor receptor.

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predicted on the basis of pretreatment analysis of tumorsand those that clearly were not identified at baseline butrather developed as a result of selective pressure placedupon the tumor cells by BRAF inhibitor treatment.A number of identifiable pretreatment factors have been

described as being associated with either a poorer responseand/or a shorter PFS to BRAF inhibitor treatment. Theseinclude stromal hepatocyte growth factor (HGF) produc-tion, BCL2A1 [an antiapoptotic B-cell leukemia 2 (BCL-2)family member] expression, activation of cyclin D1, andloss of PTEN (77–81). It is interesting that each of theseexamples is associated with critical regulation of growth,survival, or cell-cycle regulation: the activation of the PI3Kpathway (stromal HGF production leads to CMET activa-tion of the pathway; PTEN loss leads to dysregulation of thepathway), resistance to apoptosis (BCL2A1), or cell-cycleprogression (cyclin D1; Fig. 2).Acquired resistance toBRAF inhibitors, definedhere as the

development of cellular resistance by a mechanism notidentified in pretreatment tumors, is associated with reacti-vationof theMAPKpathwayapproximately two thirdsof thetime (82). One of the first described mechanisms of resis-tance (MOR) to BRAF inhibitors was the upregulation ofreceptor tyrosine kinases (RTK) such as insulin-like growthfactor receptor 1 (IGF-R1), platelet-derived growth factor(PDGF), as well as HER3 that can signal through PI3K or theMAPK pathway by activating the C-isoform of RAF (83–86).Although BRAF inhibitors potently inhibit BRAFV600, amutant isoform that signals through a constitutively activekinase in a monomeric form, they paradoxically facilitate

RAFdimerization, thereby leading to activationof theMAPKpathway (87, 88). This so-called "BRAF inhibitor paradox"explains how RTK activation upstream of RAF can reactivatethe MAPK pathway through CRAF homo- or heterodimer-ization and how other MORs to BRAF inhibitor therapyactivate the pathway. For example, concomitantmutationofNRAS and BRAF is seen in more than 20% of the resistancesamples driving MAPK signaling, and an alternative splicevariant of BRAFV600E that can dimerize in the context ofBRAF mutation emerges in 20% to 25% of the resistancesamples; loss of NF1 leading to NRAS activation has alsobeendescribed (86,89, 90). In addition,otherMORs that donot rely on paradoxical activation also are seen and includeincreased expression of BRAFV600, downstream oncogenicmutation of MEK, and alternative MAPK activation (COT)leading to activation of MEK (refs. 91–94; MORs are sum-marized in Fig. 4).

MEK-directed therapy. The clinical development ofmore selective MEK1/2 inhibitors, such as trametinib andMEK162, has led to the proof of principle that MEK is alegitimate target in melanoma, both in BRAF-mutant mel-anoma and, to a lesser degree, in BRAFwild-typemelanoma(harboring NRAS or NF1 mutations; refs. 95, 96). In fact,trametinib has recently been FDA approved for the treat-ment of BRAF-mutant metastatic melanoma based on arandomized phase III study showing that treatment withtremetinib is associatedwith a survival advantage comparedwith conventional chemotherapy [response rate (RR) 22%,PFS 4.8months, 6-monthOS 81% for trametinib vs. RR 8%,PFS 1.5 months, 6-month OS 67% for chemo; ref. 96).MEK162 has also shown clinical efficacy in both BRAF- andNRAS-mutantmelanoma in a phase II study (BRAFmutants:RR 20%, PFS 3.6 months; NRAS mutants: RR 20%, PFS 3.7months; ref. 97). A phase III study is under way to explore


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Figure 3. Oncogenic mutations in melanoma. Oncogenic mutations andmolecular aberrations are regularly present in the MAPK pathway inmelanoma and include, in order of frequency, BRAF, NRAS, PTEN loss,NF1 loss, CKIT mutation or amplification, and GNAQ/GNA11 (<1% of allmelanoma, >80% of ocular melanoma).

CCR Focus© 2013 American Association for Cancer Research

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Figure 4. Mechanisms of acquired resistance to BRAF inhibitors: 1, RTKactivation that can signal either through CRAF (1A) or the PI3K pathway(1B); 2, concomitant NRAS mutation; 3, emergence of a truncatedBRAFV600E variant from alternative splicing; 4, concomitant MEKmutation; 5, BRAFV600 overexpression; 6, loss of NF1; 7, COT, analternative MAPK activation. ERK, extracellular signal-regulated kinase;FGFR3, fibroblast growth factor receptor 3.





whether MEK162 is more effective than chemotherapy inNRAS-mutantmelanoma (NCT01763164). Finally, selume-tinib was shown to have modest efficacy in patients withuvealmelanoma. In a randomized, phase II study, treatmentwith selumetinib was associated with a two-fold improve-ment in PFS compared with patients who received chemo-therapy, though notably, OS was not different in the twotreatment groups (98). On the basis of the results of all ofthese studies, it seems clear thatMEK inhibition is associatedwithmodest benefit (20%RR,PFS4–5months) in subsets ofpatientswithmelanoma andwill have a role as a single agentin the treatment of this disease. The MORs of single-agentMEK inhibitor therapy have not been well elucidated.

The future of targeted therapy in melanomaIt is important to acknowledge that targeted therapy in

melanoma remains in its infancy. Only 4 years have passedsince initial clinical data with vemurafenib were presented.During this time, the collective knowledge about bothmechanisms of action and resistance of BRAF and MEKinhibitor therapy has grown nearly exponentially. As thesedata have emerged, so toohave clinical trial ideas using BRAFandMEK inhibitor therapy as the backbone to combinatorialregimens that have been rationally designed from our scien-tific understanding of how these agents change tumor cells.

The first example of this second wave of trials focusing oncombination regimens is a phase I/II combination of dabra-feniband trametinib (99). Itwaspredicted that reactivationofthe MAPK pathway would occur in the setting of BRAFinhibitors (82). Therefore, inhibition of the pathway down-streamofBRAFby targeting eitherMEKor extracellular signal-regulated kinase (ERK) was considered as an approach thatmight lead to further clinical benefit, and perhaps moreremarkably, improvement in severityof toxicity. Interestingly,the sequential administrationof aBRAF inhibitor followedbya MEK inhibitor is ineffective and exposes patients to thepotential toxicities seenwith each single agent, yet concurrenttreatment with both agents is associated with an improve-ment in RR, response depth (i.e., greater maximal response),response duration, and PFS, and a reduction in toxicityseverity (99, 100). This peculiar safety signal is based on thefact that BRAF inhibitors paradoxically activate the MAPKpathway through facilitationofRAFdimerization (87, 88). Asdiscussed above, manyMORs to BRAF inhibitors emerge as aresult of this phenomenon, however, the toxicity of BRAFinhibitors is also likely explained by this. Namely, BRAFinhibitor toxicity is likely a result of upregulation of theMAPK in nonmelanoma cells; the best-described example isthe development of squamous cell carcinomas of the skinsecondary to RAS mutations in skin cells (101). This phe-nomenon is seen at a much lower frequency with the treat-ment of BRAF-mutant melanoma with MEK inhibitors (99).Therefore, in BRAF-mutant cells, BRAF and MEK inhibitorsboth inhibit the pathway leading to augmented inhibitionbut exert differential effects on the MAPK pathway in non-BRAF–mutant cells suchassquamouscells of the skin, leadingto an attenuation of toxicity. There are now two additionalphase I combinations of BRAF plus MEK inhibitors showing

similar improvements in efficacy and abrogation of toxicityseverity (102, 103). Phase III studies of each of these combi-nations are under way (NCT01689519; NCT01597908;NCT01584648) or being planned. It is expected that overthe coming 5 years, triple and quadruple drug regimens willbe studied in the clinic to treat BRAF-mutantmelanomawiththe BRAF and MEK inhibitor combination at the core.

In NRAS-mutant melanoma, it is anticipated that twoevents will occur in the near future that will hopefully leadto the dramatic improvement in how these patients aretreated. First, a phase III study of MEK162 (NCT01763164)has been launched to determine the efficacy of this agentcompared with chemotherapy. If successful, regulatoryapproval would be expected. Second, a number ofcombination regimens are expected on the basis of preclin-ical data showing that a number of agents may augmentMEK inhibitor toxicity in patients with NRAS-mutant mel-anoma (104, 105). Two examples are the combination of aMEK inhibitor with a CDK4/6 inhibitor (NCT01781572)and a combination of a MEK inhibitor with an HDM2antagonist, though many more doublet and triplet combi-nations are expected in the near future.

Grand unification: the intersection of MAPK inhibitionand immunotherapy

When ipilimumab and vemurafenib were approved bythe FDAwithinmonths of eachother in 2011, a great deal ofadvocacy was directed toward the makers of each drug tosupport a combination trial. It turns out that there is actuallya compelling rationale to combine BRAF inhibitors withimmunotherapies that goes beyond the fact that they areboth effective in patients with melanoma. In particular,emerging evidence suggests that oncogenic BRAF is immu-nosuppressive (106, 107). Furthermore, treatment withMAPK inhibitors is associated with enhanced expressionof melanocytic antigens, antigen recognition by T cells, andan influx of CTLs (108–112). These findings offer compel-ling evidence for the development of combined targetedand immune therapies, although based on the earlyattempts at combining BRAF inhibitors with checkpointinhibitors, clinical trials may not be so simple. As anexample, the phase I trial of vemurafenib plus ipilimumabwas closed because of toxicity concerns, namely a high rateof severe hepatic toxicity (113). Still, a number of trials haveopened exploring various BRAF-directed therapies (singleagent, BRAF inhibitor plus MEK inhibitor combinations,etc.) with checkpoint inhibitors and cytokines alike. Thegreat hope is that the ideal combination will be identifiedthat will be associated with a very high rate of durableclinical response and no untoward toxicity.

Conclusions/Future DirectionsFrom the bleakness of the recent past to the great promise

of the near future, the development of melanoma thera-peutics has always relied on a strong connection with hard-core molecular biology and immunology laboratories todrive the clinical progress. This is more important than everas critical issues remain about the ideal sequences and

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combinations of the various agents that have proven pre-clinical and clinical efficacy.

Disclosure of Potential Conflicts of InterestK.T. Flaherty is a consultant/advisory boardmember of GlaxoSmithKline,

Roche/Genentech, and Novartis. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: R.J. Sullivan, K.T. FlahertyDevelopment of methodology: R.J. Sullivan

Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R.J. SullivanAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R.J. SullivanWriting, review, and/or revision of the manuscript: R.J. Sullivan, P.M.LoRusso, K.T. FlahertyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.J. SullivanStudy supervision: P.M. LoRusso

Received August 4, 2013; accepted August 6, 2013; published onlineOctober 2, 2013.

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