Non-BRAF-targeted therapy, immunotherapy, and combination therapy for melanoma

1. Introduction

2. FDA-approved treatments for

metastatic melanoma

3. Targeted therapy

4. Immunotherapy

5. Combination therapy

6. Expert opinion

Review

Non-BRAF-targeted therapy,immunotherapy, and combinationtherapy for melanomaSara Tomei†, Ena Wang, Lucia Gemma Delogu, Francesco M Marincola &Davide Bedognetti†National Institutes of Health, Clinical Center and trans-NIH Center for Human Immunology

(CHI), Department of Transfusion Medicine, Infectious Disease and Immunogenetics Section

(IDIS), Bethesda, MD, USA

Introduction: Melanoma is an aggressive disease characterized by a complex

etiology. The discovery of key driving mutations (primarily BRAF mutations)

led to the development of specific molecular inhibitors providing clinical

benefit.

Areas covered: Although BRAF-specific drugs have perhaps yielded the best

results in melanoma-targeted therapy, there still remain several limitations,

mostly due to the emergence of resistance and the lack of efficacy in patients

without BRAF mutation. Novel drugs are currently being tested in clinical

trials and showed encouraging results. Such drugs can specifically target

molecular pathways aberrantly activated or repressed during melanoma

development (targeted therapy) or act in a way to enhance the host immune

system to fight cancer (immunotherapy). Here we provide a detailed overview

of the current clinical strategies, which lay beyond BRAF-targeted therapy,

spanning from molecular-targeted therapy to immunotherapy and to

combination therapy.

Expert opinion: Major advances in our understanding of the mechanisms

behind melanoma development have led to the implementation of novel

therapeutic drugs. Unfortunately, tools allowing prediction of responsiveness

to a given treatment are not available yet. The increasing availability of high-

throughput technologies will allow the elucidation of molecular mechanisms

underlying responsiveness to cancer therapy and unveil an increased number

of potential therapeutic targets.

Keywords: adoptive T-cell transfer, combination therapy, immunotherapy, melanoma,

targeted therapy

Expert Opin. Biol. Ther. [Early Online]

1. Introduction

Cutaneous melanoma is a highly heterogeneous disease characterized by a complexetiology. If diagnosed early, it can be potentially cured by surgical resection;however, once metastasized, it is highly refractory to conventional antineoplastictreatments, urging the identification of novel effective therapeutic solutions.High-throughput experimental strategies, such as gene mutation analysis and com-parative genomic hybridization, allowed the identification of crucial signalingpathways essential for melanoma development and progression [1]. The recognitionof molecular aberrations occurring during melanoma development facilitatedthe application of new therapeutic strategies, which have improved the clinicalmanagement of melanoma patients [2,3].

The best results have been obtained after the introduction of BRAF inhibitors.BRAF is a serine-threonine kinase, member of the MAPK pathway; when activated,

10.1517/14712598.2014.890586 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 1All rights reserved: reproduction in whole or in part not permitted

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http://informahealthcare.com/journal/EBT

it transfers growth signals to the nucleus of the cells. The highsuccess of BRAF inhibitors is mainly due to the fact that mel-anoma development often relies on the activation of MAPKpathway. BRAF mutations occur in about 50 -- 60% ofmelanoma [4-7]. More than 90% of BRAF mutations resultin a single nucleotide mutation in the 600 codon(V600) [5,6]. Among them, about 90% of mutation are repre-sented by the valine to glutamic acid substitution(V600E) [5,6], associated with a 400-fold increased activity ofthe protein [8,9].Overall, approximately 50% of melanoma patients carry

the V600E mutation. Vemurafenib, a kinase inhibitor actingby blocking V600E mutated BRAF, have been extensivelyinvestigated in Phase II and III trials [4,10]. It has beenapproved by FDA in August 2011 for the treatment ofmetastatic or unresectable melanoma patients carrying theV600E mutation. In the interim analysis of the Phase III trial(BRIM-3), vemurafenib has been shown to induce an objec-tive response (OR) in 48% of the patients, significantly higherthan that obtained by dacarbazine (5%) [4]. Beyond tumorshrinkage, administration of vemurafenib resulted in a signif-icantly prolonged progression-free survival (PFS; 6.9 vs1.6 months; vemurafenib vs dacarbazine, respectively) and

overall survival (OS; 13.6 vs 9.7 months; vemurafenib vsdacarbazine), according to a recent update of the BRIM-3trial [11]. In the same setting, similar results in term of ORand PFS have been obtained by the BRAF inhibitor dabrafe-nib, which has been approved by FDA in May 2013. How-ever, the trial allowed crossover and was not poweredenough to detect differences in OS [12]. Even thoughPhase III trials focused on patients bearing V600E mutation,BRAF inhibitors may be similarly active in patients carryingother V600 mutations (i.e., V600K and V600D) [4,10,13,14].These observations led the European Medical Agency(EMA) to express favorable opinion on the use of vemurafe-nib and dabrafenib in metastatic or unresectable melanomapatients carrying any kind of BRAF V600 mutation. How-ever, despite BRAF inhibition has shown the most promisingresults in the field of melanoma targeted therapy, several chal-lenges remain. The first important obstacle is that half of mel-anoma patients do not carry V600 mutations and thereforecannot be treated with BRAF inhibitors. Moreover, a com-plete response (CR) only occurs in a very few cases(3 -- 6%), the duration of response rate is relatively short(5 -- 7 months) and all but few patients relapse within 1 or2 years through developing secondary resistance [4,10,12,13].Thus, much effort needs to be put into developing novel,more effective therapeutic approaches.

The purpose of this review is to discuss the most significantadvances in the treatment of metastatic melanoma, highlight-ing the novel therapeutic strategies (currently being tested inclinical trials [15]), which preclude BRAF treatment, encom-passing single molecular-targeted therapy, immunotherapy,and combinatorial therapeutic approaches.

2. FDA-approved treatments for metastaticmelanoma

To date there are four FDA-approved treatments for metastaticmelanoma besides the two aforementioned BRAF inhibitors:recombinant IL-2, ipilimumab (approved, respectively, in1998 and 2011; discussed in the following sections), dacarba-zine, and trametinib [4,16-18]. Dacarbazine is an alkylating agent,which was accepted as standard treatment for metastatic mela-noma in the 1970s [19,20], and it remains the only cytotoxic che-motherapy drug approved for the treatment of metastaticmelanoma. In preliminary studies, OR was reported in up to25% of patients [21]. However, in more rigorous randomizedtrials, dacarbazine has shown to induce an OR in about 10%of patients (range 5 -- 12%) [4,12,21,22]. CR is exceptionallyrare (1 -- 2%) [4,12,21,22], remission is in general transient [21],and advantage in OS has not been conclusively demonstratedin randomized trials. Although other cytotoxic agents (e.g.,fotemustine and temozolomide) have been shown to be at leastnot inferior to dacarbazine, none of them produces superiorbenefit to dacarbazine in terms of OS [21]. For this reason,dacarbazine is often chosen as control arm in randomized trialsassessing new therapeutics. Trametinib, a MAPK extracellular

Article highlights.

. A huge amount of novel therapeutic strategies arebeing developed for the treatment of metastaticmelanoma, including targeted therapies,immunotherapies, and combinatorial strategies.

. FDA recently approved two new drugs for metastaticmelanoma, namely: ipilimumab (anti-cytotoxicT-lymphocyte antigen 4 [anti-CTLA]-4), dabrafenib (BRAFinhibitor), and trametinib (MAPK extracellular signal-regulated kinase [MEK] inhibitor). Several additionaldrugs are being tested and Phase I/II clinical trials areproviding encouraging results.

. MAPK and Phosphatidylinositol 3-Kinase (PI3K) pathwaysare the most frequently altered pathways in melanoma.Pharmaceutical inhibitors, specifically blocking alteredoncogene proteins within these pathways, have beenproved to counteract melanoma development (e.g.,BRAF inhibitors, MEK inhibitors, AKT inhibitors).

. Steps forward have been made in the field of melanomaimmunotherapy, considering the exceptionalimmunogenicity of melanoma. These advances refer tohigh-dose IL-2, vaccination strategies, immunecheckpoint blockade, and adoptive T-cell transfer.

. Albeit targeted therapy and immunotherapy strategieshave revolutionized the clinical management ofmelanoma patients, it is becoming clear thatcombinatorial strategies are most likely to result in evenmore significant advances. Examples include BRAFinhibitors plus mTOR inhibitors, BRAF inhibitors plusMEK inhibitors, BRAF inhibitors plus PI3K inhibitor,combination of immune-checkpoint inhibitors and alsotargeted therapy plus immunotherapy.

This box summarizes key points contained in the article.

S. Tomei et al.

2 Expert Opin. Biol. Ther. (2014) 14(5)

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signal–regulated kinase (MEK) inhibitor (see following sec-tion), was approved by FDA on 29 May 2013 (concurrentlywith the approval of dabrafenib) for the treatment of patientswith unresectable or metastatic melanoma harboring BRAFV600E or V600K mutation [23].

Currently, several drugs are being tested and severalencouraging results have been recently published. Therapeuticstrategies for the treatment of melanoma can be systematicallydivided in three broad categories: targeted therapy, immuno-therapy, and combination therapy. Targeted treatments act bydirectly inhibiting molecules (e.g., proteins encoded by proto-ncogenes or growth factors), which are essential for the tumorgrowth. Immunotherapy treatments aim at stimulating theimmune system of the host to reject cancer cells. Combina-tion therapy refers to the use of multiple approaches toachieve a stronger anticancer response and overcome potentialmechanisms of resistance. Examples of the most effectivetreatments belonging to each of the above categories will begiven in the following sections of this review.

3. Targeted therapy

Biologic targeted therapy usually refers to a treatment that spe-cifically targets molecules involved in neoplastic process withless harm to normal cells. Targeted treatments act by blockingthe activity of specific molecules essential for cancer growthand development. The majority of targeted therapies includeeither small molecules or monoclonal antibodies. The testingand implementation of possible therapeutic drugs in mela-noma is growing up at an exponential rate as our understand-ing of the genetic heterogeneity of this disease improves.

An interesting recent study from Henary et al. [24] showedthat the administration of molecularly matched therapies pre-dicted better outcomes, emphasizing the relevance of targetedtherapy in the context of personalized medicine. Melanomacells can acquire a proliferative advantage or protectionagainst cell death by deregulating molecular pathways relevantto cell biology. The understanding of cell growth and molec-ular signaling pathways in melanoma is essential to developand implement new therapeutic drugs.

In the following, we provide an overview of the mostcommonly deregulated signaling pathways in melanomaresponsible for cell proliferation and cell survival, and themost relevant advance in clinical setting.

3.1 MAPK pathwayMAPK pathway is probably the best-studied signaling pathwayin cancer, most likely because of its essential role in melanomaonset and development [25]. A simplified representation ofMAPK pathway is given in Figure 1. Signaling through MAPKpathway occurs upon the binding of extracellular growth factorsto a variety of tyrosine kinase receptors (TKRs), including c-KITand c-MER, which in turn lead to the activation of RAS. RAS isa small GTP-binding protein, which can exist in three isoforms:HRAS (Harvey-RAS), KRAS (Kirsten-RAS), and NRAS

(neuroblastoma-RAS). NRAS is the most prevalent form inmelanoma being constitutively activated by point mutation inabout 15% of cases [26]. Once activated, NRAS binds BRAF,which further phosphorylates and activates MEK. MEK finallysignals to ERK, which translocates into the nucleus and enhan-ces the transcription of a bunch of transcription factors resultingin increased cell proliferation and increased cell survival.

Activating mutations within MAPK pathway account for90% of melanomas [27]. Studies have reported a constitutiveactivation of MAPK pathway through mutation and/or geneamplification of c-KIT. C-KIT gene encodes a cell-membranetyrosine kinase receptor, which plays an essential role in cellu-lar differentiation [28]. The role of c-KIT in chronic myeloge-nous leukemia (CML) and gastrointestinal stromal tumors(GIST) has been well established and the use of imatinib, anoral c-KIT inhibitor, has revolutionized the treatment of thesediseases [29].

The use of c-KIT inhibitors in melanoma represents anevolving matter of investigation. C-KIT mutations accountfor about 2% of cutaneous melanoma, albeit mutation and/oramplification of c-KIT are found in about 20 -- 25% in somemelanoma subtypes, such as mucosal melanoma, acral mela-noma, or melanoma arising from chronically sun-damagedskin [30,31]. Consistent with the essential role of c-KIT in mela-nocytes differentiation and migration, several melanomasexpress c-KIT. However, experimental evidence from studiesin thyroid and melanoma showed that the loss of this growthfactor receptor is strangely related to a worse malignantphenotype [32-34]. This may be explained by the acquisition ofa less-differentiated phenotype during tumor progression.A number of drugs targeting c-KIT have been developed andtheir effectiveness has been proved in GISTs. These drugsinclude sunitinib, nilotinib, sorafenib, dasatinib, and imatinib,and their therapeutic activity has been shown to be dependenton the c-KIT specific type of molecular alteration [35-37].

Results of c-KIT inhibitor trials inmetastatic melanomawereinitially disappointing. Three Phase II trials did not show clin-ical activity of imatinib in unselected melanoma patients[38-40]. The scenario substantially changed when the studiesfocused on melanoma patients carrying c-KIT mutations and/or amplifications, therefore highlighting the importance ofpatient selection in early targeted-therapy development.According to two Phase II trials, indeed, an OR is achieved inalmost 25% of patients, and temporary stabilization occurs inabout 50 -- 70% of subjects [31,41]. Although results in termsof OS seem encouraging (median OS from 11 to 14 months),the median PFS appears to be low, ranging from 3 to3.5 months [31,41]. Importantly, responses seem to be restrictedto patients carrying hot spot mutations either in exon 11, 13, ormultiple mutations [31,41]. There is a great interest for c-KITinhibition in mucosal and acral melanoma and other drugs,such as sunitinib, nilotinib, and dasatinib, are currently investi-gated in Phase II trials [42]. In view of the growing enthusiasm inthis field, the Society for Immunotherapy of Cancer (SITC)melanoma expert panels recommend to routine testing of

Non-BRAF-targeted therapy, immunotherapy, and combination therapy for melanoma

Expert Opin. Biol. Ther. (2014) 14 (5) 3

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c-KITmutations in clinical practice [43]. Whether acquisition ofresistances to imatinib in melanoma is due to the acquisition ofadditional c-KIT mutations, as demonstrated in GIST [44],remains to be elucidated [42].Similarly to c-KIT, another important RTK in melanoma

is c-MER tyrosine kinase (MERTK); c-MER is a member ofthe TAM (TYRO, AXL, MERTK) family of RTKs. A recentstudy from Schlegel and colleagues [45] has pointed c-MER asa potential therapeutic target in melanoma. In this work, theywere able to show that treatment of melanoma cell lines withUNC1062, a novel c-MER inhibitor, induced apoptosis andinhibited invasion of melanoma cells, encouraging a deepertesting of such drug.In addition to the deregulation of RTKs, several MAPK

downstream molecules are often altered in melanoma. Thefirst oncogene to be discovered was NRAS [46]. As mentionedearlier, NRAS is a member of the rat sarcoma (RAS) proteinfamily, together with HRAS and KRAS. RAS are GTP-binding proteins, which, once activated by RTKs, can signala complex network of downstream molecular pathways.Although evidence shows KRAS and HRAS to be implicatedin melanoma [47,48], NRAS is the most active form. Mostcommon mutations in NRAS gene have been found locatedat codon 61 [49], causing the impairment of NRAS enzymaticactivity to switch GTP to GDP. Albeit several attempts havebeen made to directly target NRAS, no clinical success hasbeen reached yet. One probable explanation is the low

concentration range (picomolar) required for the affinity ofGTP to bind NRAS, which made the development of GTP-specific antagonists impossible so far [50]. Nevertheless, signif-icant effort has been spent to indirectly inhibit NRAS by tar-geting key post-translational modifications, such asfarnesylation. Farnesylation consists in a lipid modificationnecessary for NRAS membrane association. The inhibitionof farnesylation process results in the blockage of NRAS trans-location to the plasma membrane. A study from Niessner andcolleagues [51] has shown that the farnesyltransferase inhibitor(FTI) lonafarnib was also able to inhibit mTOR signalingpathway enhancing apoptosis induced by sorafenib inmelanoma cell lines. However, testing FTI tipifarnib (alsocalled R115777) in a Phase II clinical trial failed to obtainencouraging results [52], most likely due to aspecific responsesas farnesylation occurs in the maturation process of severalproteins other than RAS. Although in this trial patients wereunselected for NRAS mutation, the intrinsic difficulty todevelop NRAS inhibitors and the scarce activity of thisapproach in other tumors have shifted the efforts to inhibitMAPK pathway toward more effective targets.

Another important protein kinase belonging to MAPKpathway is MEK, and its inhibition is currently under investi-gation. Considering that MEK activation has been shown tobe a resistance mechanism by which melanoma treated withBRAF inhibitors relapse, MEK inhibition was tested in com-bination with BRAF inhibitors (see combination therapy

c-KIT

NRAS

BRAF

MEK1/2

ERK1/2

PI3K PTEN

Cytosol

AKT

mTOR

p53

MDM2

PDK1

GSK3!

!-catenin

IKK NF"BBAD

BCL2

BAX

GPCR

GNAQ/GNA11 PKC

ELK MITF JUN

Proliferation, differentiation

PIP2

PIP3

Figure 1. Simplified representation of MAPK and PI3K pathways. Red bolts indicate targets of molecular alterationsin melanoma.AKT: V-Akt murine thymoma Viral Oncogene Homolog; BAD: BCL2-associated agonist of cell death; BAX: BCL2-associated X protein; BCL2: B cell lymphoma-2;

BRAF: V-raf murine sarcoma viral oncogene homolog B; c-KIT: V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; ELK: ETS-domain protein; ERK1/

2: Extracellular signal--regulated kinase 1 and 2; GPCR: G protein--coupled receptor; GSK3b: Glycogen synthase kinase 3 b; IKK: IkB kinase; JUN: V-Jun avian sar-

coma virus 17 oncogene homolog; MDM2: Mouse double minute-2; MEK1/2: MAPK extracellular signal–regulated kinase 1 and 2; MITF: Microphthalmia transcrip-

tion factor; mTOR: Mammalian target of rapamycin; NFkB: nuclear factor-kB; NRAS: Neuroblastoma RAS; p53: Tumor protein 53; PDK1: Phosphoinositide-

dependent kinase-1; PI3K: Phosphoinositide-3 kinase; PIP: Phosphatidylinositol phosphate; PKC: Protein kinase C; PTEN: Phosphatase and tensin homolog.

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section subsequently), yielding extremely promising results.MEK proteins include MEK1 and MEK2, which are theonly substrate of BRAF. Mutations on MEK genes are veryrare, although they have been reported in a few cases [49].However, the acquisition of MEK1 mutations P124L andQ56P have been demonstrated to confer resistance to BRAFand MEK inhibitors [53]. A recent review by Salama andKim [54] lists a broad variety of MEK inhibitors currentlyunder investigation. Worthy of note, selumetinib is an oralpotent inhibitor of MEK1 and MEK2 kinases. The efficacyof this drug was evaluated in a large Phase II clinical trial inadvanced melanoma patients. Unfortunately, the study failedto show a difference of the OR rate and PFS between thetreatment arm and the control group (temozolomide) [55].Interestingly, five out of six responders were BRAF mutated.Combination of selumetinib and dacarbazine was reportedto be clinically active in a Phase II randomized trial (PFS5.6 vs 3.0 months, selumetinb plus dacarbazine vs dacarbazinealone), although nonsignificant benefit in terms of OS wasobserved [56].

Several clinical trials are still testing MEK inhibitors inmelanoma, given the fact that MAPK reactivation is oftendriven by MEK signaling. Among the MEK inhibitors tested,trametinib has shown so far the most promising results. In aPhase III study, 322 patients carrying V600E or V600K muta-tion were randomized to receive either trametinib or chemo-therapy (either decarbazine or paclitaxel) [23]. A statisticalsignificant prolongation of PFS was shown for patients treatedwith trametinib compared to the control group (4.8 vs1.5 months). OR rate was also higher in trametinib arm thanin chemotherapy arm (22 vs 8%). Despite the crossover to tra-metinib at progression, the 6 months OS was significantlyhigher in the trametinib arm (81 vs 67%) [23]. On the basisof these results, FDA approved trametinib for the treatmentof metastatic melanoma harboring V600E or V600K muta-tions. Importantly, tremetinib is not active if administered topatients progressing on a BRAF inhibitor, stressing the needto better identify the window of efficacy of trametinib inBRAF mutant melanoma [57].

However, patients carrying NRAS mutations cannot bene-fit from such treatment, urging the need to find other addi-tional treatment options. Recently, Ascierto and colleagues[58] in an open-label, nonrandomized, Phase II trial assessedthe efficacy of MEK162, a potent MEK inhibitor, in patientscarrying NRAS or V600E BRAF mutations. In addition to anOR rate of 20% in BRAF-mutated patients, they were able toshow that MEK162 has activity in NRAS mutant patients(20% of OR rate), although no CR was observed. However,all patients relapsed within 9 months. A similar OR rate(18%) in patients bearing NRAS mutation has been observedwith the use of pimasertib [59], another MEK inhibitor underclinical development.

These proof-of-principle studies pinpoint MEK inhibitionas potential new approach for NRAS mutant melanoma,which till now has few treatment options. Once again, the

main limitation of MAPK-targeted therapy is the rapid devel-opment of resistance. However, while melanoma can take dif-ferent roads to counteract to MAPK-targeted inhibition, thereactivation of the extracellular-signal-regulated kinases(ERK) appears to be a common feature [60,61]. Two isoformsof ERK, ERK1 and ERK2, transmit proliferative signalsonce phosphorylated by the upstream MEK proteins. Quiteinterestingly, Morris and colleagues [62] recently identified apotent ATP-competitive ERK inhibitor by screening a libraryof 5 million compounds. Such inhibitor, called SCH772984,showed promising clinically attractive results as it was able toinhibit ERK phosphorylation in a dose-dependent manner.Testing this compound in vivo is warranted to prove itsefficacy and its potential relevance in clinical settings.

3.2 Phosphatidylinositol 3-kinase pathwayPhosphatidylinositol 3-kinase (PI3K) pathway is the secondmost frequently deregulated pathway in melanoma afterMAPK pathway (Figure 1). Similarly to MAPK, PI3K cascadeis triggered by RTKs and RAS protein. Once activated byNRAS, PI3K can catalyze the phosphorylation of phosphati-dylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol3,4,5-trisphosphate (PIP3). PIP3 functions as a second mes-senger, and its production is antagonized by the tumorsuppressor phosphatase and tensin homolog (PTEN).PIP3 can activate AKT (through the phosphatidylinositol-dependent kinase 1, PDK1), which, in turn, phosphorylatesmTOR and multiple additional targets determining increasedoncogenic transformation, survival, proliferation, and cell-cycle regulation [63]. The deregulation of PI3K pathway inmelanoma occurs through activation of PI3K (encoded byPI3KCA gene), PTEN loss, and AKT activation. The inde-pendent therapeutic relevance of inhibiting PI3K pathwayitself has not been well established yet, however, preclinicalevidence suggests that PI3K pathway inhibition may be arelevant adjunct to MAPK-targeted therapy. Several smallmolecule inhibitors targeting AKT and mTOR activity havebeen developed and tested in melanoma cell lines and xeno-grafted animal models [64]. However, the number of clinicalstudies currently evaluating their clinical efficacy is very low.The AKT inhibitors wortmannin and LY294002 have beenshown to block AKT activity in a variety of functional studies,but their application in clinical settings is limited by their off-target activities [63,65]. Several derivatives of rapamycin, suchas temsirolimus, have also been used as mTOR inhibitorsand have demonstrated clinical activity in other malignanciessuch as renal cell carcinoma. Nevertheless, the administrationof temsirolimus (either used alone or in combination with themultiple kinase inhibitor sorafenib) failed to show significantclinical activity in Phase II trials [66,67].

Even though several additional AKT inhibitors, such asAPI-2, SR13668, BI-69A11, GSK690693, and MEK-2206are under investigation, it is widely believed that PI3K path-way inhibition works better when combined to other targetedtherapies. An interesting study by Werzowa and colleagues



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assessed the potential efficacy of PI3K pathway vertical inhibi-tion in melanoma cell lines and xenograft models [68]. Theconcurrent treatment of rapamycin and the mTOR inhibitorPI-103 had a potent antitumor activity both in vitro andin vivo. Although in general PI3K and mTOR inhibitionseems to be a promising strategy against cancer developmentand some drugs have been already approved for the treatmentof renal cancer [69], such drugs did not obtain the same successin melanoma. The reason behind this phenomenon is notclear yet albeit it has been hypothesized that the ability ofrapamycin derivatives to phosphorylate AKT may impedetheir clinical success [70]. Nevertheless, MEK and PI3Kcombination seems promising. An interesting in vitro studyby Greger et al. showed that combinations of BRAF, MEK,and PI3K/mTOR inhibitors overcome acquired resistance todabrafenib, mediated by NRAS or MEK mutations [71].Another study showed that combined MEK and PI3K/mTOR pathway inhibition induces regression of NRAS-mutant melanoma cell lines [72]. An increasing number ofclinical trials combining PI3K and MAPK inhibitions inmelanoma are currently undergoing or in development.As a note, inhibition of Bcl-2, a key anti-apoptotic protein

expressed in cancer cells, through the antisense oligonucleo-tide oblimersen seemed to increase OS in a subset of patients(i.e., those with normal or low level of LDH) [73], but thesefindings were not confirmed in a recent randomizedPhase III trial [74].In light of recent in vitro or in vivo studies, modulation cell-

cycle checkpoints (which are de-regulated in melanoma) andinhibition of G protein-coupled receptor signaling (found tobe activated in up to 80% of uveal melanoma) have recentlygained the attention of clinical investigators [42,72]. Remark-ably, two very recent investigations have found that > 70% ofmelanoma bears somatic mutation of telomerase reverse tran-scriptase (TERT) promoter, therefore highlighting anotherimportant oncogenic mechanism [75,76].

3.3 AngiogenesisBecause melanoma is a highly vascularized cancer, severalattempts to therapeutically target angiogenic mechanisms inmelanoma have been explored. Anti-angiogenic drugs havebeen developed and demonstrated to be useful in differenttypes of cancer. Still, their clinical benefit in melanoma isnot clear. Among the anti-angiogenic drugs, bevacizumab, ahumanized monoclonal antibody targeting the VEGF A(VEGF-A), has been approved for use in certain malignancies,such as colon (in combination with chemotherapy), kidney(in combination with IFN-a), lung (in combination withchemotherapy), and brain cancers.In metastatic melanoma, the use of bevacizumab as single

agent, or in combination with IFN-a failed to show signifi-cant clinical activity [77]. According to a large randomizedPhase II trial (BEAM) enrolling 314 treatment naıve patients,bevacizumab, in combination with chemotherapy enhances,though not significantly, PFS and OR (4.2 vs 5.6 month;

16 vs 26%; PFS and OR, bevacizumab vs bevacizumab pluscarboplatin and paclitaxel). OS advantage was initially signif-icant (8.6 vs 12.3 months; p = 0.04), but it was reduced in asubsequent analysis with longer follow-up, (9.2 vs12.3 months; p = 0.19) [78]. Although it is not clear if thiscombination will move forward, the BEAM study, which isoften considered a positive Phase II trial, still represents oneof the best evidence of the activity of anti-angiogenic therapyin advanced melanoma [79].

Interestingly, Del Vecchio and co-workers [80] evaluated theclinical and biological activity of the use of bevacizumab incombination with fotemustine, a chemotherapeutic drugwidely used in Europe. The authors were able to show thatsuch regimen had important clinical activity as first-line treat-ment of metastatic melanoma, obtaining an encouraging OSof 20.5 months (PFS = 8.8 months, OR 15%), althoughgrade 3 -- 4 hematological toxicity was quite high.

Attempts to use bevacizumab in clinical trials employingother combinatorial setting are currently underway and resultsare awaited. Recently, a preliminary analysis of 20 patientsenrolled in a Phase I study assessing combination of the cyto-toxic T-lymphocyte antigen 4 (CTLA-4) mAb ipilimumaband bevacizumab showed an intriguing OR of 38%; all theresponses lasted > 6 months [81].

Similarly to VEGF-A mAbs, other drugs targeting theVEGF receptors (VEGFRs) have been tested in melanoma.Among them, sorafenib, which inhibits tyrosine kinases asso-ciated with VEGF and plateled-derived growth factor, failedto demonstrate significant clinical activity in combinationwith chemotherapy in Phase III trials [82,83]. The more prom-ising ones as single agents are axitinib and lenvatinib and theirinvestigations alone or in combination are moving forward. Ina multicenter, Phase II study, axitinib monotherapy resultedin a 19% OR rate [84]. Similarly, in a Phase I evaluation ofpatients with solid malignancies, including melanoma, lenva-tinib (also called E7080) demonstrated an OR of 21% [85].

Based on this evidence, it is likely that anti-angiogenictherapies might be helpful in treating melanoma especiallywhen combined with other drugs and much effort should bespent in identifying the more beneficial anti-angiogenic com-bination therapies. Moreover, other novel molecules, as thosemimicking natural anti-angiogenic agent inhibitors (e.g.,endostatin), inhibitors of placental growth factor, and agentsblocking HIFa, a key molecule in hypoxia signaling, arecurrently under development [86].

4. Immunotherapy

Cancer immunotherapy is meant as the treatment aiming atannealing tumors by inducing, enhancing, or suppressing spe-cific immune responses. Aside from the molecular alterationswithin proliferative pathways described earlier, severalimmune alterations have been reported in melanoma, includ-ing decreased number of peripheral B and T cells, increasednumber of regulatory T cells, impaired activation of natural

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killer (NK) cells (due to downregulation of activatingligands), acquisition of tolerance by CD8+ T cells (due toincreased expression of inhibitory receptors), increased levelof pro-tumorigenic cytokines such as TGF-b, TNF-a, IL-6,IL-8, and IL-1, and altered expression of immune-relatedgenes [87,88].

Immunotherapy strategies for melanoma treatment carry ahigh clinical relevance considering the exceptional immuno-genicity of such disease. The immunogenicity of melanomahas been established based on the high number oftumor-infiltrating lymphocytes (TIL) found in resected mela-noma samples, and several strategies have been proposed toconquer melanoma taking advantage of this peculiar feature.A diverse set of melanoma antigens have been identified astarget of cellular immune response, which can be groupedinto three broad categories: tumor differentiation antigens(TDA), including melanoma antigen recognized by T cells 1(MART-1), glycoprotein 100 (gp100), tyrosinase (TYR),tyrosinase-related protein 1 (TYRP1), and 2 (TYRP2), whichare found expressed in normal melanocytes and are overex-pressed in melanoma; tumor-specific antigen (TSA), includ-ing the melanoma antigen (MAGE), the B melanomaantigen (BAGE), and G antigen (GAGE) gene families, theNY-ESO-1 (also known as CTAG1), which are found in nor-mal gametic cells in the testes and in cancers in addition tomelanoma but are not expressed by normal melanocytes;and mutated antigens, including b-catenin, which can befound in different tissues. Additionally, several melanomaantigens have been identified as targets of humoral immuneresponses.

The expression of melanoma antigens is of great impor-tance as it allows the innate and adaptive immune systemto track and destroy nascent tumor cells, through a mech-anism known as ‘immune surveillance’. However, cancercells can escape immune surveillance by becoming poorlyimmunogenic. This phenomenon has been commonlyreferred to as ‘immune editing’ [89]. Several mechanismsof immune editing have been described, including thedownregulation of antigen presentation MHC moleculesby the tumor cells themselves, the secretion of soluble fac-tors and cytokines, such as TGF-b and IL-10, whichinhibit effective immune response, the creation of animmune suppressive environment by recruiting inhibitoryimmune cells, such as T-reg, NKs, and macrophages, andthe expression of negative regulators of the immune system,including programmed death ligand 1 (PD-L1) and theCTLA-4 [90].

We report subsequently the best-characterized immuno-therapy strategies for the treatment of melanoma.

4.1 Nonspecific immunotherapy: IL-2IL-2 is a pro-inflammatory cytokine regulating T cells andNK cells homeostasis. Studies in animal models and humanshave shown that IL-2 has strong antitumor activity [91,92]

and high-dose IL-2 (HD-IL-2) was approved by FDA in1998 for the treatment of metastatic melanoma.

HD-IL-2 was initially shown to have dose-dependent anti-cancer effect by the group of Steven Rosenberg at theNational Cancer Institute [93]. FDA approved HD-IL-2 in1998 based on the observation that it can induce long-termremissions. In large retrospective analyses, administration ofHD-IL-2 induce an OR in about 15% of patients (range13 -- 19%) of patients, and CR in 4 -- 6% [92,94-96]. However,in a recent randomized Phase III trial, these percentagesdropped from 10 to 6% for OR and from 2 (two patients)to 1% (one patient) for CR when evaluation was assessed bycentral review. Even though the real rate of OR needs to bedetermined, complete-remission under HD-IL-2 appear tobe extremely durable (median CR duration > 14 years,according to a long follow-up analysis of a retrospectivecasuistic) [95].

However, HD-IL-2-related adverse events are severe (e.g.,hemodynamic shock, respiratory insufficiency, and neurotox-icity) and require intensive inpatient care [21]. The opinions ofEuropean and US specialists about the risk--benefit ratio ofthis treatment largely diverge. In light of the potentiallycurative role of HD-IL-2, the US National ComprehensiveCancer Network US guidelines still list HD-IL-2 as treatmentoption in a subset of (clinically fit) patients, if the center has aconsiderable experience in the management of this regimen.Even more, the (US) melanoma panelists of the Society forImmunotherapy of Cancer (SITC) recommend the adminis-tration of HD-IL-2 as first-line treatment in good perfor-mance status patients without central nervous systemmetastases, even in presence of BRAF mutation [43]. Con-versely, the European Society of Medical Oncology (ESMO)melanoma panelists do not encourage its routine use inclinical practice, in view of the high toxicity and the lack ofrandomized trials demonstrating survival advantage in theoverall population [97].

Considering the relatively low benefit--risk ratio, one of themain limitations of HD-IL-2 is the lack of pretreatment toolsto predict patients who are likely to respond to the treatment.Recently, in a proteomic study, we showed that low levels offibronectin and VEGF, an angiogenetic factor with immuno-suppressive functions, predict response to HD-IL-2 [98] inmetastatic melanoma patients. Although these findings needto be prospectively validated, Hodi and co-workers recentlyreported that low levels of VEGF also predict OS and clinicalresponse to ipilimumab in metastatic melanoma [99] Notewor-thy, an interesting study from Joseph and colleagues hasrecently shown that patients harboring NRAS mutation carrya higher likelihood to respond to therapy [96], linking, for thefirst time, alterations of the MAPK pathway to the responsive-ness to HD-IL-2.

Although IL-2 has been used in oncology since > 20 years, itsmechanism of action is still not completely understood. Onlyfew studies have attempted to comprehensively understandthe mechanisms behind the cancer rejection mediated by IL-2.



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Our group performed a series of proteomic and transcrip-tomic analyses of peripheral mononuclear cells (PBMCs)and pre- and post-melanoma metastasis using fine-needleaspiration biopsies (FNA) obtained from patients undergoingsystemic IL-2 administration [98,100-103]. We observed thatadministration of IL-2 induces a cytokine storm already afterthe first administration, where most of the cytokines investi-gated (e.g., IFN-g , IL-10, and TNF-a) increased significantlycompared with the baseline value. CXCR3 ligands (i.e.,CXCL9, 10 and 11) and CCR5 ligands (i.e., CCL3 andCCL4) were found among the chemokines that significantlyincreased early after treatment [100,101].Even when investigated by whole genome gene-expression

analysis, the effects of HD-IL-2 were dramatic withinPBMCs, whereas effects within the tumor microenvironmentwere lesion-specific and limited [103]. Altogether these findingssuggest that the main effect of IL-2 in the tumor is the activa-tion of innate mechanisms driven by monocytes and eventu-ally sustained by NK, and the tumor infiltrating lymphocytes.These cells in concert can initiate an inflammatory chemo-

tactic cascade, orchestrated by the activation of the IRF-1/IFN-g pathway and inducing release of related specificchemotactic ligands (i.e., CXCR3 and CCR5 ligands) withconsequent polarized (Th1) recruitment of other immunecells later on [104]. Importantly, the degree of inflammationinduced by HD-IL-2 correlates with the development oftumor rejection [102,103].

4.2 Vaccination strategiesCancer vaccines aim at treating cancer by stimulating thehost’s immune reaction against cancer cells. The implementa-tion of therapeutic vaccines to treat melanoma remains achallenge [105-107].Broadly, melanoma vaccines are classified into the follow-

ing main categories: whole-cell vaccines (either autologousor allogeneic, prepared using melanoma cells or subcellularfraction); dendritic-cell (DC) vaccines (which take advantageof the highly specialization of DCs to present antigens andinduce primary cellular immune responses); peptide vaccines(which use melanoma antigens to stimulate cytotoxicT lymphocytes response against tumor cells); ganglioside vac-cines (which take advantage of the high expression of ganglio-sides on the surface of melanoma cells to generate an immuneresponse against them); DNA vaccines (composed of nakedDNA expression plasmids that include a gene encoding thetarget antigens from tumor cells); viral vector vaccines (inwhich a virus is used as vector of melanoma antigens); andoncolytic vaccine (in which an oncolytic virus replicating incancer cells is used to mediate tumor cell lysis) [107-110].The best-characterized vaccines for melanoma treatment

are the peptide-based ones directed to activate T-cell response[111]. The recognition of the critical role of T cells in theimmune-mediated treatment of melanoma dates back to1988, when Rosenberg and colleagues first showed the regres-sion of metastatic melanoma through the autologous transfer

of tumor infiltrating lymphocytes combined with IL-2 [112].In 1991, van der Bruggen et al. described for the first time agene encoding an antigen recognized by T cells (MAGE-1)[113]. This study, followed by the description of the firstcancer-specific epitope HLA-A1 restricted [114], gave molecu-lar accuracy to this rather disregarded field and providedthe opportunity to investigate with scientific precision thephenomenon of immune-mediated cancer rejection. Thisled, in turn, to the characterization of a myriad of tumor anti-gens and their immunodominant peptides, with consequentimplementation of active immunization strategies [115].

The study of the tumor-antigen-specific immunizationrestricted to an individual epitopic determinant offered theopportunity to study the dynamic of the immune responsein humans by reducing the algorithm of tumor--host cross-talk to a specific HLA/epitope interaction with the comple-mentary T-cell receptor [116]. These studies have shown that,in order to develop clinical active immunotherapies, factorsbeyond T-cell/HLA/epitope interactions must be considered.In fact, the large majority of the studies failed to report anassociation between specific response against the administeredantigens and clinical benefit [104]. Humoral and/or cellularimmune response against vaccine antigens are necessary forthe development of an effective antitumor response but theyare certainly not sufficient [117]. Other factors, as the presenceof co-stimulatory or inhibitory stimuli in tumor microenvi-ronment, genetic polymorphisms of the host, genetic of thetumors, and environmental factors (i.e., intestinal micro-biota) [118], can certainly modify the algorithm of immuneresponse, making the evaluation of a single parameter unliketo predict the clinical benefit [119-121].

Besides local toxicity, vaccines are in general extremely welltolerated. However, in melanoma, as well as in many othersolid tumors, vaccination alone has failed to induce significanttumor regression, the OR rate being around 3 -- 4% [122].Nevertheless, recent randomized Phase III trials in othermalignancies (i.e., prostate cancer) have shown that vaccineadministration can prolong survival even in absence of detect-able tumor shrinkage [123]. The convincing detection of aremarkable survival benefit dissociated with measurablechanges in the tumor size forced us to reconsider the mecha-nism of actions of vaccinations. An interesting interpretationof these paradigmatic results comes from the analysis of tumorkinetic following different treatments. By applying a rathernovel mathematical model to Phase II trials in prostate cancerpatients receiving chemotherapy or PROSTVAC vaccine(consisting in recombinant poxviral vectors containing trans-genes for prostate specific antigen (PSA) and the costimula-tory molecules B7), Fojo and Schlom showed thatchemotherapy determines an initial reduction of tumor bur-den followed, at relapse, by a tumor-growth rate similar tothat before chemotherapy [124,125]. Vaccination, however,modifies clinical outcome (OS) by reducing tumor-growthrate. The same mechanisms likely explain differences betweensurvival curves in BRAF and ipilimumab trials (Figure 2).

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Even though several scientific progresses in the field of vac-cinology have been accomplished during the past decade, it isclear that active immunization alone would unlikely representa curative option for patients affected by advanced melanoma.The rather disappointing results of cancer vaccines couldbe due to a mixture of the following factors: i) the presenceof a highly immunosuppressive microenvironment with conse-quent inability of T cells to overcome environmental immuno-suppression in absence of co-stimulatory or anti-inhibitory

stimuli [126]; ii) the scant ability of antitumor T cells to localizeat tumor site [127-129]; iii) the rapid senescence of tumor-specificT cells or T-cell repertoire [122,130]; iv) the highly mutabletumor target capable of immune-evasion and antigenloss [122]; and v) the presence of unfavorable genetic condition(host polymorphisms or cancer mutations) [129,131,132] or unfa-vorable environmental factors (unintact commensale gutmicrobiota) [118] that can negatively switch the balancebetween tolerance and acute (therapeutic) inflammation.

OS

(%)

PFS

(%)

Time (months) Time (months) Time (months)

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0 12 24 36 48 0 12 24 36 48 0 12 24 36 48

Figure 2. Survival curves of BRAF inhibitors, CTLA-4 mAb (ipilimumab) and combination therapy. In each panel blue linerepresents the control treatment (dacarbazine). Orange line represents the alternative treatment (BRAF inhibitors, ipilimumab,or combination therapy), as indicated. The BRAF inhibitor curves were approximated from the vemurafenib and dabrafenibrandomized Phase III trials. Immunotherapy curves were approximated from ipilimumab randomized trials. Dotted linerepresents survival projection when no data are available. PFS of combination therapy was derived from the dabrafenib plustrametinib Phase I/II randomized trial. BRAF inhibition induces response in most of the patients, however the response istransient, and all but few patients relapsewithin 1 or 2 years through developing secondary resistance. The long-termbenefit inOS is expected to be very low. Vice versa, immunotherapy (i.e., ipilimumab) determines a delayed response in a small proportionof patients. Response is however durable, with impact on long-term survival. Combination immuno-targeted therapy could actsinergically therefore switching the curve on the right (targeted therapy) and up (immunotherapy). Although results from suchcombinations are not available yet, certain combination of immunotherapy (i.e., ipilimumab and IL-2 or IL-2 and vaccine) seemsto increase survival, therefore shifting the curve up. However, it seems that combination of BRAF andMAPK extracellular signal-regulated kinase inhibitors as well as certain immunotherapy combination (i.e., ipilimumab and the anti-PD1 nivolumab) areable to shift the curve on right and up by inducing deep, frequent and durable antitumor response. Even though preliminarydata suggest that survival curve of anti-PD1 and anti-PD1 ligand will differ as compared with those of ipilimumab, they are notrepresented here because no formal survival curves of PD-1/PD-1 ligand trials are available yet.BRAFi: BRAF inhibitors; CTLA-4: Cytotoxic T-lymphocyte antigen 4; comb: Combination; IT: Immunotherapy; OS: Overall survival; PFS: Progression-free survival;

TT: Targeted therapy.



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Therefore, novel generation of vaccines [133] could be effectivewhen combined with other therapies (e.g., inflammatory cyto-kines, immune-checkpoint inhibitors, chemotherapy, or radia-tion therapy, see combination therapy section), with the goalto overcome the barriers listed earlier. Vaccination could beeven more successful when used in patients with low tumorburden (i.e., adjuvant setting), or in selected patients particu-larly sensitive to immune-manipulation (i.e., those bearingspecific polymorphisms).Only very few vaccination strategies have shown promising

results in Phase II trials and are currently being tested inPhase III randomized trials (in addition to gp100 vaccineplus IL-2 [134], see combination therapy). In a Phase II trialon stage III/IV M1a melanoma, MAGE-A3 vaccine com-bined with AS15 immunostimulant (a mixture ofQS21 saponin, TLR4, and TLR9 agonist) induced an ORin 11% of patients (including 2% of CR) and a median OSof 33 months [135]. The advantage of vaccination was evenhigher in patients bearing an inflammatory gene-signaturecentered on IRF-1/IFN-g-related transcripts [117,136]. Thisadjuvant--vaccine combination was selected for the Phase IIItrial. Unfortunately, it has been recently announced that thestudy did not meet its first co-primary end point as it didnot significantly extend disease-free survival (DFS) whencompared to placebo [137]. The result of the other co-primaryend point (DFS in patients bearing the inflammatory genesignature identified in Phase II trial) is expected in 2015. Inthe OPTIM Phase III trials by Kaufman et al. T-VEC, anoncolytic immunotherapy derived from herpes simplex virustype-1 designed to selectively replicate within tumors and toproduce GM-CSF to enhance systemic antitumor immuneresponses, induced OR in 26% of patients, including 11%of CR. According to the interim analysis, there was also atrend in OS favoring vaccination (HR: 0.79; vaccination vsGM-CSF alone) [138,139]. Allovectin-7 (a plasmid--lipid com-plex containing the DNA sequences encoding HLA-B7 andb2 microglobulin) in combination with dacarbazine [140],and M-VAX (an autologous hapten-modified vaccine), aloneor in combination with IL-2 (NCT00477906), represent theother two vaccination strategies that are currently being eval-uated in Phase III trials [141]. Notably, there is an increasinginterest for testing nanoparticle as vaccine adjuvant [142].Functionalizing carbon nanotubes are particularly promisingin view of their theranostic (i.e., both diagnostic and thera-peutic) potential. In fact, functionalized carbon nanotubescan stimulate monocytes to release pro-inflammatory cyto-kines and chemokines associated with T helper 1 polarizationand immune-mediated tumor rejection [143]. Moreover,because of their high echogenicity these nanotubes could bepotentially used as ultrasound contrast agents [144].An emerging and promising approach to generate tumor-

specific T cells is represented by chimeric antigen receptor(CAR) or T-cell receptor (TCR) engineered T cells. Studiesin B-cell hematological malignancies have shown that engi-neered T cells against CD19+ can mediate deep tumor

remission [145]. In melanoma, the administration of modifiedT cells expressing high avidity anti-gp100 or anti-MART-1TCR had induced a considerable OR rate (19 -- 30%), withfew complete remissions (one patient in the MART-1 trial)[146]. However, patients exhibited destruction of melanocytesin skin, eye, and ear, which also express, although at lowerlevel, these antigens. Similarly, administration of engineeredT cells against MAGE-A3 is associated with high incidenceof neurotoxicity because of cross reactivity with MAGE pro-teins expressed by brain [146]. Therefore, it is imperative toselect antigens that are not expressed by normal cells and togenerate T cells that do not cross-react with epitope withinself-antigens [146]. The most promising results have beenobtained by targeting NY-ESO (OR in 5/10 melanomapatients, including 2 CR) [146].

4.3 Immune checkpoint blockadeAn extremely promising approach used in clinical settings tocounteract the immune escape mechanisms mounted bycancer cells is the inhibition of the CTLA-4 (Figure 3) [147].CTLA-4 is a key player in establishing immune toleranceand one of the main regulators of T-cell-mediated antitumorimmune responses. The major function of CTLA-4 is tomodulate T cells at the time of their initial response to anti-gen [148]. In fact, although CTLA-4 is expressed by activatedCD8+ effector T cells, its key role relies in the regulation ofT CD4+ populations through down modulation of helperT-cell activity and enhancement of regulatory T-cell immu-nosuppressive function [147,148].

The possible role of CTLA-4 blockage in clinical settingwas proposed in 1995 by Allison and Krummel [149,150]. It isnow clear that antibodies binding the extracellular domainof CTLA-4 can, therefore, block its inhibitory signals andenhance the immune system to fight against cancer cells.Two anti-CTLA-4 monoclonal antibodies, that is, ipilimu-mab and tremelimumab, are currently being applied in meta-static melanoma setting. The former has been approved byFDA in March 2011.

Ipilimumab is a fully human IgG1 monoclonal antibody.Results of ipilimumab trials have boldly increased the enthu-siasm in the field of cancer immunotherapy and deservedetailed description. Two Phase III trials have conclusivelydemonstrated its efficacy in previously treated (ipilimumabmonotherapy, MDX010-020 trial) [17], or naive (ipilimu-mab+dacrabazine, CA184-024 trial) [22] advanced melanomapatients. In the MDX010-20, ipilimumab showed to increaseOS, as compared with gp100 vaccine alone (median OS:10.1 vs 6.4 months, ipilimumab vs gp100 vaccine, p =0.003) [17]. The differences in terms of OS increased withthe time. The 1- and 2-year OS were 46 and 24% in ipilimu-mab arm, and 25 and 14% in the vaccine arm, respectively [17].In the CA184-024 study median OS was 11.1 months in thedacarbazine-ipilimumab arm, and 9.1 months in dacarbazinealone arm [22]. A recent follow-up analysis of this latter trialhighlights the progressive benefit in OS associated with

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ipilimumab. The 1-, 2-, 3- and 4-year OS were, respectively,48, 29, 21, and 19% in the ipilimumab-dacrabazine arm and36, 18, 12 and 10% in the dacarbazine arm [151]. Therefore,ipilimumab almost doubles the 4-year survival rate. Althoughresponses were few in number, they were more likely to occurin patients treated with ipilimumab. As for the MDX010-20-the OR rate was 11% (including 1.5% of CR) in the ipilimu-mab arm, and 1.5% in the gp100 arm. No CRs were observedin the gp100 alone arm [17]. As for CA184-024, the OR ratewas 15% (including 1.5% of CR) in the ipilimumab-dacarbazine arm and 10% (including 1% of CR) in the dacar-bazine alone arm [22]. Even though 15% of OR rate is notparticularly high, the responses were more durable in patientstreated with ipilimumab (OR duration: 19 months vs8 months, dacarbazine plus ipilimumab vs dacarbazine) [22].Similar (although less pronounced) differences were observedin term of PFS in the two studies. The continuous follow-upof patients enrolled in three Phase II trials also confirm thelong-term benefit of ipilimumab, with a 5-year OS rangingfrom 12 to 28% and 38 to 50% for previously treated andnaıve patients [152,153]. These results should, however, be takenwith caution, being derived from early Phase trials. However,the US ipilimumab expanded access program, which prospec-tively surveyed the activity and safety of ipilimumabin > 800 (pre-treated) patients, showed a 2- and 3-year OSrate of 22 and 17%, respectively [154]. Remarkably, theseresults are very close to those obtained in Phase III trials.According to Phase II trials, the combinations of ipilimumabplus fotemustine (NIBIT-M1 trial) [155], and ipilimumab plustemozolomide [156] seem also to be promising. To evaluate theresponse to treatment, the NIBIT-M1 trial was the first toemploy the immune-related criteria, instead than the classicWHO or RECIST criteria for solid tumors. In fact, the onsetof clinical response is often delayed, leading to problems inapplying classic criteria of response. Response can be achievedafter stabilization or even after progression, and CR can occuryears after an initial remission. These observations led to definenew criteria (immune-related response criteria; irRC) forthe definition of response in immunotherapeutic trials [157].

Tremelimumab is a fully human IgG2monoclonal antibody.The G2 isotype was chosen to diminish complement activationand cytokine storm. A recent Phase III trials failed to show a sig-nificant advantage in terms of OS when compared standardchemotherapy (dacarbazine or temozolamide) in treatmentnaıve advanced melanoma patients (median OS: 12.6 vs10.7 months, p = 0.13) [158]. Response rate was also similar inthe two arms (11 vs 10%, tremelimumab vs chemotherapy,respectively) although duration was significantly longer aftertremelimumab (35.8 vs 13.7 months, p = 0.001). However,tremelimumab survival curves are similar to those obtained inthe Phase III ipilimumab trials in the same patient setting.The 2- and 3-year OS were 26 and 21% in patients treatedwith tremelimumab and 23 and 17% in patients treated withchemotherapy. The failure to detect significant differencescould be due, at least in part, to the enrichment in patients

more responsive to chemotherapy or to study contaminations.Moreover, although the trial did not allow crossover, a propor-tion of patients randomized to receive chemotherapy likelyreceived ipilimumab at progression. During trial conduct, infact, ipilimumab became widely available for patients random-ized in the control arm [158]. Dosage and schedules could alsohave influenced the results. Nevertheless, promising results areobtained in Phase I/II trial, in pretreated melanoma [159,160]

and mesothelioma patients [161]. Overall, we believe there is stillroom for this drug in metastatic melanoma setting. One of themain limitations of the CTLA-4 blockades is that immuneresponse takes time to be effective and about 70% of patientsprogress within the first 6 months [17,22]. Common immune-related adverse event include colitis, hepatitis, and endochrino-paties, and can be life threatening in a small but considerableproportion of patients. In view of the toxic profile, anotherlimitation of this approach is that so far there are no toolsallowing to define with confidence the patients who will benefitfrom this approach.

Another critical immune checkpoint is represented byPD-1. Four anti-PD1 and 3 anti-PD1 ligands mAbs arecurrently in clinical development (Figure 3, Table 1).

PD-1 is induced once T cells become activated. Themajor function of PD1 is to repress the activity of effectorT cells in peripheral tissues to limit autoimmunity or collat-eral damages in response to infections [148]. PD1 has twoknown ligands, PD-L1 (B7-H1) and PDL-2 (B7-DC)[162,163], which are expressed by tumor cells to avoid T-cellmediated lysis.

Results of large Phase I/II trials of two anti-PD1 mAbs(nivolumab/BMS-936558 and lambrolizumab/MK-3475) andanti-PD-1 ligand mAbs (BMS-936559 and MPDL3280A)are extremely encouraging. If paradigmatic pattern of responseof CTLA-4 (delayed and slow effect on tumor shrinkageand prolonged OS) in a small proportion of patients haveforced us to define (or redefine) mechanism of action ofimmunotherapy, recent results of Phase I/II anti-PD-1 andanti-PD-1 ligand trials have shown once again that pattern ofresponse among different immune manipulations could beextremely different. Surprisingly, anti-PD-1 or anti-PD-1mAb induce tumor shrinkage in most patients with advancedmelanoma, lung, and kidney cancers [164-166]. OR rate inmelanoma cohorts were 31, 38, 17, and 29% for nivolumab[165,167], lambrolizumab [166], BMS-936559 [164], andMPDL3280A [168], respectively. Tumor shrinkage was alreadyevident at the time of the first assessment (at 6 or 12 weeks),and was extremely durable (1 year or beyond in most cases).Importantly, toxicity (most immunomediated) was muchlower as compared with that observed in ipilimumab trials.Although the magnitude of the clinical benefit needs to beassessed through (undergoing) randomized trials and longerfollow-up, the pattern of response and the (favorable) safetyprofile of anti-PD1/PD-1 ligands appear to be quite unique.Importantly, PD-1/PD-1 blockage was effective in patientsalready previously treated with ipilimumab, therefore,



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confirming that CTLA-4 and PD-1 immune-suppressive path-ways are not redundant.Besides the co-inhibitory interactions described earlier, co-

stimulatory signaling between antigen presenting cells (APC)and T cells could also be targeted to enhance antitumorimmunity. A schematic representation of the clinical develop-ment of immune-checkpoint inhibitors and co-stimulatorymolecules in melanoma is provided in Figure 3. The statusof the corresponding clinical trials (updated as of January2014) is provided in Table 1.Agonist antibodies aimed at enhancing T and NK func-

tions (e.g., CD40, CD137 (4-1BB), anti-CD134 (OX40),and glucocorticoid-induced TNF receptor (GITR)) are beingevaluated in early phase trials [169]. Perhaps, the most clini-cally relevant examples in this regard come from the

interactions between OX40 and OX40L and betweenCD40 and CD40L.

OX40 (also known as CD134) is a TNF-receptor familymember, which is transiently expressed by T cells upon thebinding to the TCR. The engagement of OX40 promotesa vast spectrum of T-cell responses, including increasedproliferation, cytokine production, and increased differentiationand survival. The interaction between OX40 and its ligand,OX40L, occurs in 48 h after antigen recognition by T cells[170]. Targeting of OX40/OX40L has been proposed as a rele-vant approach for autoimmunity and cancer [171]. Preclinicalstudies have shown that ligation of OX40 via agonist anti-OX40 mAB or OX40L-Ig fusion proteins can induce robustT-cell-mediated antitumor immunity [172,173]. In a recentPhase I trial [174], Curti and colleagues showed that the

IpilimumabTremelimumab

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Figure 3. Immune-checkpoints, co-stimulatory signaling, and clinical development of immune-targeted therapy. Stimulatorysignaling is in green, inhibitory signaling is in red. The clinical development status is represented by colored rectangles andrefers to trials that have recruited (or are currently recruiting) patients with advanced or metastatic melanoma. The symbol ‘R’highlights trials currently recruiting melanoma patients. Name of the drug is next to the corresponding targeted molecule(either receptor or ligand). Status of clinical trials is update as of January 2014 (source: clinicaltrials.gov; more detailsin Table 1). Represented expression of ligands and receptors should not be considered exhaustive: for example, PD1L-PD2L arealso expressed by antigen presenting cells as macrophages and dendritic cells (not shown in the figure); MHC-II is alsoexpressed by dendritic cells and can be expressed by melanoma cells as well.CTLA-4: Cytotoxic T-lymphocyte antigen 4.

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Table 1. Agents targeting immune-checkpoints and co-stimulatory signaling*.

Agent Clinical trials

BMS-986016 (anti-LAG3) Phase I trial in solid tumors as monotherapy and in combination with nivolumabrecruiting (NCT01968109)

IMP321 (anti-LAG3) Phase I/II trial in combination with vaccine in melanoma recruiting (NCT01308294)IPILIMUMAB (anti-CTLA-4) FDA approved for melanoma; two positive Phase III trials (published) in previously

treated melanoma patients as monotherapy versus combination with gp100 vaccineversus gp100 vaccine (NCT00094653) and in naıve patients in combination withdacarbazine versus dacarbazine completed (NCT00324155); Phase III trials in melanomaas monotherapy versus combination with nivolumab versus nivolumab (NCT01844505)or versus lambrolizumab (NCT01866319) recruiting; Phase III trials in small-cell lungcancer (NCT0145076) and non-small-cell lung cancer (NCT01285609) in combinationwith chemotherapy versus chemotherapy alone recruiting; enrollment completed in aPhase III trial in prostate cancer versus placebo (NCT01057810); > 60 Phase I/II trialsevaluating combinations with targeted therapy, radiotherapy, or variousimmunotherapies in melanoma and in other cancers are currently recruiting

TREMELIMUMAB (anti-CTLA-4) Negative Phase III trial in melanoma versus chemotherapy published (NCT00257205);Phase I trial in melanoma in combination with the anti-CD40 mAbCP-870,893 (NCT01103635) recruiting; Phase II trial in mesothelioma recruiting(NCT01843374); Phase I trial in non-small-cell lung cancer in combination with gefitinib(NCT02040064) or the anti-PD-L1 mAb MEDI4736 (NCT02000947) and in solid tumorsin combination with MEDI4736 (NCT01975831) recruiting; Phase I trial in combinationwith chemoembolization or ablation in liver cancer recruiting (NCT01853618)

Anti-OX40 (OX40 agonist) Encouraging results from a Phase I trial in multiple cancers recently published(NCT01644968); Phase II trial in melanoma as monotherapy (NCT01416844) withdrawnprior to enrollment; Phase I/II in combination with ipilimumab (NCT01689870) recentlysuspended; Phase I/II trials in breast cancer patients with lung metastases incombination with radiotherapy (NCT01862900) and in prostate cancer in combinationwith radiotherapy and cyclophosphamide recruiting (NCT01303705)

CP-870,893 (CD40 agonist) Phase I trial in melanoma in combination with tremelimumab, recruiting(NCT01103635); Phase I trial in melanoma in combination with vaccination ongoing butnot recruiting (NCT01008527); Phase I trial in pancreatic cancer in combination withchemotherapy recruiting (NCT01456585); two Phase I trials in solid tumors(NCT00607048) and in pancreatic cancer (NCT00711191) in combination withchemotherapy completed and listed as ‘having results’

Dacetuzumab (SGN-40 or huS2C6;anti-CD-40)

Phase I/II trial completed in leukemia patients (NCT00283101); 6 Phase I and 2 Phase IItrials completed in multiple myeloma and non-Hodgkin’s lymphoma

Chi Lob 7/4 (anti-CD40) A Phase I trial in multiple cancers ongoing but not recruiting (NCT01561911)Lucatumumab (HCD122;anti-CD40) Phase I trial for follicular lymphoma patients in combination with bendamustine

completed (NCT01275209)Urelumab (BMS-663513; anti-CD137) Phase II trial in melanoma completed (NCT00612664), Phase I trial in melanoma in

combination with ipilimumab withdrawn prior to enrollment (NCT00803374); Phase Itrials in non-Hodgkin’s lymphoma recruiting (NCT01471210; NCT01775631); Phase Itrials in advanced tumors (NCT00351325, NCT00309023) and non-small-cell lungcancer (NCT00461110) terminated

TRX518 (anti-GITR) Phase I trial in melanoma and other solid tumors recruiting (NCT01239134)MGA271 (anti-BT-H3) Phase I trials in melanoma (NCT01918930) and in melanoma and other solid tumors

(NCT01391143) recruitingNivolumab (BMS-936558/MDX-1106;Anti-PD1)

Positive results of a Phase I trials in patients with melanoma and other solid tumors(NCT00730639) or in melanoma in combination with ipilimumab (NCT01024231)published; Phase I trials in melanoma in combination with vaccination recruiting(NCT01176474, NCT01176461); Phase III trial in melanoma as monotherapy versusdacarbazine (NCT01721772) or versus combination with ipilimumab versus ipilimumab(NCT01844505) recruiting; Phase II trials in melanoma as monotherapy versuscombination with ipilimumab versus ipilimumab (NCT01927419) or given sequentiallywith ipilimumab recruiting (NCT01783938); Phase I trial in solid tumors in combinationwith anti-KIR (NCT01714739), IL-21 (NCT01629758), and the anti-LAG1 mAbBMS-986016 (NCT01968109) recruiting; Phase I trials assessing combination withipilimumab in melanoma (NCT01024231) and other combinations in other solid orliquid tumors recruiting

*Updated as of January 2014 (source: clinicaltrial.gov).

CTLA-4: Cytotoxic T-lymphocyte antigen 4; LAG3: Lymphocyte activation genes 3; PD-L1: Programmed death ligand 1.



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administration of an anti-OX40 mouse mAB in patients withadvanced cancer had an acceptable toxicity profile and wasable to induce regression of at least one metastatic lesion in12 out of 30 patients. The therapy also increased CD8 T-cellIFN-g production in two of three patients with melanoma.Moreover, in another recent study, Redmond and col-leagues [175] demonstrated that the targeting of OX40 in combi-nation with IL2 therapy increased tumor regression inpreclinical tumor model and restored the function of anergictumor-reactive CD8 T cells in mice, leading to enhancedsurvival.CD40 is a cell-surface molecule, member of the TNF-

receptor family, expressed by APC, such as dendritic cells,B cells, and monocytes, but also by many nonimmune cellsand a wide range of tumors [169]. The natural ligand ofCD40 is CD145, which is expressed on T cells. It hasbeen shown that in tumor-bearing hosts, CD40 agonist trig-ger effective immune response against tumor-associated anti-gens [176-178]. Four CD40 agonist mABs have beeninvestigated in clinical trials so far, namely: CP-870,893,dacetuzumab, Chi Lob 7/4, and lucatumumab [169,179].However, these treatments showed wide heterogeneity rang-ing from strong agonism (CP-870,893) to antagonism (luca-tumumab), suggesting that their mechanism of action mightbe different. A study employing CP-870,893 as CD40 ago-nist demonstrated that a single intravenous infusion ofCP-870,893 was well tolerated and an objective PR wasobserved in four patients with metastatic melanoma (14%

of all patients and 27% of melanoma patients) [180]. Addi-tional clinical trials employing CD40 agonists are ongoingand results are strongly awaited.

Blockage of other inhibitory pathways as the immunosup-pressive enzyme indoleamine 2,3-dioxygenas (IDO) seemspromising in preclinical study [181], and clinical studies areongoing (NCT01961115; NCT00739609). Drugs blockingother immune-checkpoints such as T-cell membrane protein3 (TIM3; inhibitor receptor) and B7-H4 (inhibitory ligand),TGF-b, and KIR inhibitory receptors are under develop-ment [148,169]. Notably, trials evaluating blockage of the inhib-itory receptor lymphocyte activation genes 3 (LAG3), theinhibitory ligand B7-H3 are ongoing [148,182,183] .

Finally, the recent discovery that themanipulation ofBACH2gene can shift the balance between effector and regulatory T-cellresponse also represents a novel starting point for the develop-ment of next-generation immunotherapeutic approaches [184].

4.4 Adoptive cell therapyAdoptive cell therapy (ACT) transfer is a form of passiveimmunotherapy, which consists in the ex vivo expansion ofautologous antitumor reactive lymphocytes and their reinfu-sion into lymphodepleted patients, accompanied by the exog-enous administration of IL-2 to further boost their antitumoractivity. Lymphodepletion, in fact, seems to favor the antitu-mor activity of ACT by suppressing regulatory T cells andlimiting host tumor immunosuppressive factors. ACT wasdeveloped by the group of Steven Rosenberg at the National

Table 1. Agents targeting immune-checkpoints and co-stimulatory signaling* (continued).

Agent Clinical trials

Lambrolizumab (MK-3475 anti-PD1) Phase III trial in melanoma as monotherapy versus ipilimumab (NCT01866319)recruiting; Phase II trial in melanoma ongoing but not recruiting (NCT01704287);Phase I trials in melanoma, solid tumors or non-small-cell lung cancer recruiting(NCT01295827, NCT01848834, NCT01840579); Phase II/III trials in non-small-cell lungcancer versus docetaxel recruiting (NCT01905657); Phase II trial in tumor withmicrosatellite instability recruiting (NCT01876511); positive results of the melanomacohort of the NCT01295827 study recently published

MPDL3280A (anti-PD1) Phase I trial in melanoma or hematologic malignancies (NCT01375842), melanoma(combination with vemurafenib, NCT01656642), or in solid tumors in combination withbevacizumab or chemotherapy (NCT01633970) recruiting; several Phase I-II trials andone Phase III trial in lung cancer recruiting (NCT02008227)

CT-011 (anti-PD1) Phase II trial in melanoma completed (NCT01435369); Phase II trials in prostate, renal,pancreatic cancer and hematological malignancies in combination with chemotherapyor vaccine recruiting; other Phase I and Phase II trials in gastrointestinal tumors andhematological malignancies recently concluded

BMS-936559 (MDX-1105; anti-PD-L1) Phase I trials in melanoma (NCT01455103) and hematological malignancies(NCT01452334) withdrawn before enrollment; a Phase I trial in solid cancer is ongoingbut not recruiting (NCT00729664)

AMP-224 (anti-PD-L2) Phase I trial in solid cancers ongoing but not recruiting (NCT01352884)MEDI4736 (anti-PD-L1) Phase I/II trial in melanoma in combination with dabrafenib or dabrafenib and

trametinib recruiting (NCT02027961); Phase I trial in japanese patients with solid tumorsrecruiting (NCT01938612); Phase I trial in solid tumors recruiting (NCT01693562);Phase I trial in combination with tremelimumab in solid tumors (NCT01975831) or innon-small-lung cancer recruiting (NCT02000947)

*Updated as of January 2014 (source: clinicaltrial.gov).

CTLA-4: Cytotoxic T-lymphocyte antigen 4; LAG3: Lymphocyte activation genes 3; PD-L1: Programmed death ligand 1.

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Cancer Institute and has shown encouraging results inPhase II clinical trials. In a recent analysis evaluating93 heavily pretreated metastatic melanoma patients enrolledin three Phase II trials, ACT was able to induce OR in 56%of patients. Complete remission was obtained in 20 patients(21.5%), with 19 patients potentially cured as they had ongo-ing regression beyond 3 years [185]. A similar OR rate (48%,including 6.5% of CR), measured using immune-relatedresponse criteria (irRC), had been obtained by Radvanyiet al. in a Phase II trial enrolling 31 patients [186].

Beyond the toxicity related to HD-IL-2 administration, themain limitation associated to ACT is that TIL expansionrequires highly specialized personnel. Using the traditionalapproach, TILs are generally successfully grown from only50 to 60% of the patients and require a long period of time(about 5 weeks). Simplified protocols using short-term culturesderive TILs from up to 90% of patients in a shorter period oftime (10 -- 18 days) [187]. Rosenberg’s group, however, recentlyreported a global OR of 28% (CR rate 7%), in randomizedPhase II trials comparing two different TIL protocols (youngTILs vs CD8 enriched TILs) [188]. By using young TILsBesser et al., in a Phase II trial enrolling 70 patients, reportedan encouragingOR of 40% in the evaluated population, includ-ing 5 CR [187]. In all the ACT trials responses were durable andpatients in CR very rarely relapsed. The results of these Phase IItrials need to be taken with caution, and the true response rateneeds to be evaluated in randomized Phase III trials. Neverthe-less, ACT seems to represent a potential curative therapy forpatients withmetastatic melanoma [189]. Randomizedmulticen-ter trials aimed at evaluating the true response rate and OSadvantage of ACT represent the reasonable next step.

Despite these encouraging results, the dynamic of TILmigration at tumor site is still not completely clear.

Studies where TILs were labeled with Indium 111 showedthat TILs migration in the tumor site does not follow alinear kinetic. After re-infusion, which is followed by IL-2administration, TIL localize in lung, spleen, and liver butnot at tumor sites [190,191]. The migration of TILs at tumorsite is detectable only after 24 to 48 h. It is possible that thisearly compartmentalization is mediated by the release(following HD-IL-2 administration) of specific chemokines(i.e., CXCR3 and CCR5 ligands) from resident immunecells and stromal cell in peripheral oragans (e.g., spleen,lung, and liver) [129,192]. Intriguingly, by analyzing a largecohort of patients treated with adoptive therapy and HD-IL-2, we recently showed that the downregulation ofCXCR3 and CCR5 receptors in TILs correlates with thefrequency and the degree of response [129]. This receptordown-modulation is due to the transcript downregulationand/or to the presence of common polymorphism (CCR5D32, which codify for a nonfunctioning receptor) [129]. Itis tempting to hypothesize that a reduced, but not absent,expression of these receptors might prevent the earliersequestration of TIL by extra-tumoral sites and paradoxi-cally allow their subsequent localization to the tumor

when the cytokine storm has subsided and the tumor repre-sents the only tissue maintaining chemokine secretion [129].

5. Combination therapy

Combination therapy is intended as the use of multiple ther-apies to treat a single disease with the aim to overcome thehurdles of mono-therapeutic strategies [193]. Combining che-motherapy agents represents a pillar of the medical treatmentof virtually all the major liquid and solid malignancies, withexception of melanoma and renal cell carcinoma. In thesetwo tumors combination of cytotoxic agents failed to showincrease efficacy as compared with monotherapy. Inmelanoma, > 40 randomized trials have evaluated combina-tion of chemotherapy +/- cytokines (IFN-a and/or IL-2)[21]. It is now clear that although polychemotherapy +/-IFN-a and/or IL-2 in general enhance the OR rate, they donot increase the OS [21]. The failure of polychemotherapy isin general attributed to the intrinsic chemo-resistance ofmelanoma. As for first generation immunochemotherapy, itshould be noted that, because of safety issues, IFN-a and orIL-2 were administered at low dose, while their maximalantitumor effect is in general achieved using high dosages.Nevertheless, prospective trials have shown that combinationof HD-IL-2 and IFN-a does not increase OS, as comparedwith HD-IL-2 alone [194,195].

This depressing scenario dramatically changed after adventof targeted therapy immune checkpoint inhibitors. Althoughthe mono-targeted therapy and immunotherapy strategieshave revolutionized the clinical management of melanomapatients, it is becoming clear that combination strategiesare most likely to result in even more significant advances.Building upon the early success of mono-therapy agents inmelanoma, trials involving new combination therapy areunderway. It has been thought that combining vaccineswith anti-CTLA-4 could produce a synergic effect. Whenipilimumab was administered together with peptide vaccina-tion (gp100), the combined treatment was more effective(both in term of PFS and OS) than gp100 alone [17]. How-ever, there were no significant differences in both PFS andOS between ipilimumab alone and ipilimumab combinedwith vaccine [30]. Conversely, gp100 vaccine and IL-2 canact in a synergistic way. In a Phase III randomized trials inmetastatic melanoma patients, Schwartzentruber et al.showed that the addition of the gp100 vaccine to HD-IL-2 can significantly increase the number of responses(CR: 7 vs 1%, and OR: 16 vs 6%, IL-2 + gp100 vs IL-2,respectively, p = 0.03) and lengthen PFS survival (medianPFS 2.2 vs 1.6 months, p = 0.008), with similar trend inOS [134]. Two recent studies showed that efficacy ofCTLA-4 can be enhanced by the concomitant administrationof immune-stimulant. In a recent randomized Phase II trialHodi et al. showed that the addition of GM-CSF to ipilimu-mab increase OS (1 year OS: 68 vs 51%, GM-CSF plus ipi-limumab vs ipilimumab alone, p = 0.03) and was also



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associate with less toxicity [196]. Remarkably, long-term fol-low-up Phase I/II trials in metastatic melanoma patientshave suggested that the addition of IL-2 to ipilimumab coulddramatically improve the rate of durable CR (CR rate: 17 vs7%, ipilimumab + IL-2 vs ipilimumab +/- gp 100 vaccine)and extend five overall survival up to 25% [197].Overall, these apparently clashing results underline once

again the need to understand the mechanisms of action ofsimultaneous administration by designing clinical or transla-tion trial aimed at evaluating the effect of immunotherapyin vivo in humans [198]. However, in view of the huge numberof potential combinations, novel generations of clinical trialsusing factorial design are needed [199] to select the morepromising combinations.Another possible combination approach is the use of tar-

geted therapy together with immunotherapy [200]. The ratio-nale supporting this type of combination approach relies onthe possibility to achieve a deeper and long lasting antitumoreffect (Figure 2C), as pointed out by Ribas et al. [201].BRAF inhibitors, for example, induce almost immediate

tumor shrinkage in most patients (median time to response1.5 months). Responses are, however, transient, and the effectof long-term survival is predicted to be very limited(Figure 2A). Conversely, CTLA-4 blockades induce less num-ber of clinical responses (usually delayed), but they are muchmore durable (Figure 2B).A number of observations support the target-immunother-

apy combination. Several cytotoxic agents have been shown toinduce antitumor immune responses by promoting cell deathwith consequent release of tumor-associated antigens [202]. Inmelanoma, this could be achieved through targeted therapy [203],rather than with conventional chemotherapy. Moreover, con-stitutive activation of oncogenic pathways, as the BRAF-MAPK pathway, can induce several immunosuppressive mech-anisms [204,205], including promotion of stromal cell-mediatedimmunosuppression via induction of IL-1 [7,206]. In addition,BRAF inhibition can enhance melanoma antigens expressionand create a more favorable immune environment, facilitatingthe effectiveness of immunotherapy [89,207-209].A study evaluating combination of ipilimumab and vemura-

fenib was object of a Phase I study by Ribas et al. [210]. Althoughthis combination is supported by a strong rationale, investiga-tors noticed an unacceptable hepatotoxicity, which promptedthem to close prematurely the study. This example reinforcesthe need to carefully evaluate the safety of combinationapproach in appropriate early trials [210].Very recently, two additional prospective studies in

metastatic melanoma have highlighted the potentiality of com-bining highly effective agents. In a first Phase I/II randomizedtrial, combination of BRAF inhibitor dabrafenib andtrametinib resulted superior to dabrafenib alone [23]. The twodrugs combined at full monotherapy dose resulted in 76% ofOR (including 9% of CR), while 54% of patients achievedOR in the dabrafenib group (including 4% of CR; p = 0.03).Progression-free survival in the combination was 9.4 months,

as compared with 5.8 months in the monotherapy group(p < 0.001). Responses were also more durable with the combi-nation (10.2 vs 5.6). Importantly, > 40% of patients were aliveand progression-free at 1 year in the combination arm, as com-pared with 10% in the monotherapy arm. Combination wasalso feasible in term of safety [211]. This landmark study demon-strated that the inhibition of the same pathways through highlyeffective targeted therapy agents (vertical inhibition) coulddramatically impact on the course of this aggressive disease,overcoming (at least in part) mechanisms of resistance occur-ring during monotherapy. Several trials also employing thera-pies targeting different pathways are underway; examplesinclude vemurafenib (BRAF inhibitor) plus everolimus or tem-sirolmus (mTOR inhibitors, identifier NCT01596140),MEK162 plus AMG479 (insulin-like growth factor1 receptorinhibitor, identifier NCT015 62899), vemurafenib plusBKM120 (a PI3K inhibitor, identifier NCT01512251).

In a second Phase I trial, combination of PD-1 andCTLA-4 blockers (nivolumab and ipilimumab) resulted, atthe maximum tolerated dose, in 53% of OR rate, with tumorregression of 80% or more in every patient who had response[212]. The profound effect of concurrent combination wasalready evident at the first imaging assessment (3 months aftertreatment initiation). Responses were extremely durable andongoing in 90% patients who had a response (duration rang-ing from 6 to 72 weeks at the time of data analysis). Thispattern of response (rapid, deep, and durable) appeared tobe distinct of that observed in previous immune-checkpointblockade trials. Importantly, toxicity was qualitatively similarto that of monotherapy [212].

6. Expert opinion

Although major advances have been made for the treatment ofmetastatic melanoma, with combination therapies paving theroad toward clinical success, much effort needs to be spent inidentifying patients who are more likely to respond to a giventreatment.

It is becoming now clear that patients who are most likelyto reject tumors following immunotherapy are the ones whodisplay a constitutive activation of the IFN-g/signal trans-ducers and activators of transcription (STAT)-1/IRF-1 axis(Th1 phenotype) [81,117,121] with IRF-1 playing a central rolein this regard [1,117,121,192,213]. Gene signatures predictive ofresponse to immunotherapy (i.e., IL-2-based therapy, vac-cines) invariably included genes belonging to the followingfunctional modules:

1) IFN-g/T helper 1 module (STAT-1/IRF-1/IFN-g-stimulated genes pathway);

2) The specific cytotoxic recruitment module (CXCR3/CXCR3 ligand and CCR5/CCR5 ligand pathway);

3) The immuno-effector function module (Granzyme/Perforin/Granulisin/TIA-1 pathway).

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This phenotype is also characterized by the counter-activation of suppressive mechanisms. Therefore, it is not sur-prising that high expression of IDO and PD-1 ligands, whichare both IFN-g inducible genes, have been found to be asso-ciated with responsiveness to ipilimumab [214] and anti-PD1treatment [165]. The determinants of this responsive immunephenotype are object of investigations.

Coordinate enhancement of these modules following treat-ment is associated with responsiveness to IL-2 [103], ipilimu-mab [215], and other immunotherapies, including vaccines[216] and Toll-like receptor agonists [217]. These observationssuggest that, although distinct immunotherapies clearly actin very different ways, their final therapeutic relies on the acti-vation of similar and redundant downstream mechanisms, asrecently reviewed elsewhere [121].

It is currently becoming accepted that immune-mediatedtumor rejection is mediated by both the genetic backgroundof the host and the genetic make-up of individual cancer cellscarrying their own specific mutations [119,120,218]. Yet, the rel-ative weight played by each of those factors is not completelyunderstood as well as their contribution to mediate respon-siveness to a given cancer therapy. The relevance of the geneticbackground of the host in mediating tumor rejection has beenrecently witnessed by two studies from our group. In the firstone, we hypothesized that polymorphisms of IRF5 gene,known to be implicated in autoimmune diseases and in par-ticular to systemic lupus erythematosus, might play a role inthe immune-mediated rejection of the tumor. Concordantto our hypothesis, we were able to show that the IRF5 geneticvariants protecting against the development of autoimmunitywere highly predictive of nonresponsiveness to adoptive ther-apy in melanoma [131]. These results support the link betweenautoimmunity and immune responsiveness. In the secondstudy, we assessed the prognostic relevance of genetic varia-tions of CTLA-4 and HLA polymorphisms in melanomapatients who received adjuvant IFN-a [132]. Multivariate anal-ysis revealed that a five-marker genotyping signature based onHLA-B38, HLA-C15, HLA-C3 DRB1*15, and CTLA-4CT60* polymorphisms, was predictive of OS in 284 mela-noma patients. The findings of these two studies strengthenthe relevance of inherited genetic variations in mediatingtumor rejection induced by immunotherapy regimens.

Aside from inherited genetic variations, somatic mutationsacquired during tumor development are likely to influencetumor rejection. The most evident proof that the geneticbackground of individual cancer cells plays a role in mediatingcancer rejection comes from the observation of mixedresponses, a rare phenomenon where multiple metastasesfrom the same patient respond differently to a given therapy.By comparing mixed metastases from two melanoma patientswho received immunotherapy, we were able to show thatmetastases who responded to the treatment were the oneswho had upregulated genes associated with antigen presenta-tion and IFN-mediated rejection [216]. These findings demon-strate that tumors sharing the same host’s genetic backgroundcan behave differently depending on the differential expres-sion of genes involved in triggering an IFN-mediated immunesignaling. It should be also noted that cancer cells share thesame genetic background of the host, thus, tumor behavioris concurrently influenced by inherited variations other thansomatic mutations.

Environmental factors also play a role in this regard, andevidence accumulated in the recent years show that it is acombination of all the three categories that affect tumorrejection rather than each single one alone [218]. Understand-ing the mechanisms underlying tumor rejection is essentialto implement personalized therapies.

We believe that this goal is reachable only by understand-ing the complex interplay between the genetic backgroundof the host, the genetic make-up of individual cancer cells,and environmental factors. The availability of high-throughput technologies allowed the investigators to startthe race toward cancer-personalized medicine. We believethat, in the future, the integration of the ‘omic’ researchapproaches will allow a more potent understanding of thealgorithm governing tumor rejection and responsiveness toa given therapy and lead to improved clinical managementof melanoma patients.

Declaration of interest

The authors have no competing interests to declare and havereceived no funding in preparation of the manuscript.



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BibliographyPapers of special note have been highlighted as

either of interest (!) or of considerable interest(!!) to readers.

1. Spivey TL, De Giorgi V, Zhao Y, et al.

The stable traits of melanoma genetics:

an alternate approach to target discovery.

BMC Genomics 2012;13:156

2. Ibrahim N, Haluska FG. Molecular

pathogenesis of cutaneous melanocytic

neoplasms. Annu Rev Pathol

2009;4:551-79

3. Palmieri G, Capone M, Ascierto ML,

et al. Main roads to melanoma.

J Transl Med 2009;7:86

4. Chapman PB, Hauschild A, Robert C,

et al. Improved survival with

vemurafenib in melanoma with BRAF

V600E mutation. N Engl J Med

2011;364:2507-16

5. Davies H, Bignell GR, Cox C, et al.

Mutations of the BRAF gene in human

cancer. Nature 2002;417:949-54

6. Colombino M, Lissia A, Capone M,

et al. Heterogeneous distribution of

BRAF/NRAS mutations among Italian

patients with advanced melanoma.


7. Tomei S, Civini S, Bedognetti D, et al.

The immune-related role of BRAF

mutation in melanoma [abstract p788].

J Immuno Therapy of Cancer 2012;23:9

8. Pollock PM, Harper UL, Hansen KS,

et al. High frequency of BRAF mutations

in nevi. Nat Genet 2003;33:19-20

9. Kumar R, Angelini S, Snellman E,

Hemminki K. BRAF mutations are

common somatic events in melanocytic

nevi. J Invest Dermatol 2004;122:342-8

10. Sosman JA, Kim KB, Schuchter L, et al.

Survival in BRAF V600-mutant

advanced melanoma treated with

vemurafenib. N Engl J Med

2012;366:707-14

11. Chapman PB, Hauschild A, Robert C,

et al. Updated overall survival (OS)

results for BRIM-3, a phase III

randomized, open-label, multicenter trial

comparing BRAF inhibitor vemurafenib

(vem) with dacarbazine (DTIC) in

previously untreated patients with

BRAFV600E-mutated melanoma.

J Clin Oncol 2012:30(Suppl):

abstract 8502

12. Hauschild A, Grob JJ, Demidov LV,

et al. Dabrafenib in BRAF-mutated

metastatic melanoma: a multicentre,

open-label, phase 3 randomised

controlled trial. Lancet 2012;380:358-65

13. Ascierto PA, Minor D, Ribas A, et al.

Phase II trial (BREAK-2) of the BRAF

inhibitor dabrafenib (GSK2118436) in

patients with metastatic melanoma.

J Clin Oncol 2013;31:3205-11

14. Ascierto PA, Kirkwood JM, Grob JJ,

et al. The role of BRAF V600 mutation

in melanoma. J Transl Med 2012;10:85

15. Clinical Trials. Avaialable from http://

clinicaltrial.gov/)

16. Tsao H, Atkins MB, Sober AJ.

Management of cutaneous melanoma.

N Engl J Med 2004;351:998-1012

17. Hodi FS, O’Day SJ, McDermott DF,

et al. Improved survival with ipilimumab

in patients with metastatic melanoma.

N Engl J Med 2010;363:711-23.. First Phase III study showing an

advantage of overall survival in

metastatic melanoma

using immunotherapy.

18. Eigentler TK, Meier F, Garbe C. Protein

kinase inhibitors in melanoma.

Expert Opin Pharmacother

2013;14(16):2195-201

19. Benjamin RS. Chemotherapy of

malignant melanoma. World J Surg

1979;3:321-8

20. Luce JK. Chemotherapy of malignant

melanoma. Cancer 1972;30:1604-15

21. Garbe C, Eigentler TK, Keilholz U,

et al. Systematic review of medical

treatment in melanoma: current status

and future prospects. Oncologist

2011;16:5-24

22. Robert C, Thomas L, Bondarenko I,

et al. Ipilimumab plus dacarbazine for

previously untreated metastatic

melanoma. N Engl J Med

2011;364:2517-26

23. Flaherty KT, Robert C, Hersey P, et al.

Improved survival with MEK inhibition

in BRAF-mutated melanoma. N Engl

J Med 2012;367:107-14.. First Phase III study showing an

advantage of overall survival in

metastatic melanoma using non-BRAf

target therapy MEK inhibitor.

24. Henary H, Hong DS, Falchook GS,

et al. Melanoma patients in a phase I

clinic: molecular aberrations, targeted

therapy and outcomes. Ann Oncol

2013;24(8):2158-65

25. Smalley KS. A pivotal role for ERK in

the oncogenic behaviour of malignant

melanoma? Int J Cancer

2003;104:527-32

26. Colombino M, Capone M, Lissia A,

et al. BRAF/NRAS mutation frequencies

among primary tumors and metastases in

patients with melanoma. J Clin Oncol

2012;30:2522-9

27. Nikolaou VA, Stratigos AJ, Flaherty KT,

Tsao H. Melanoma: new insights and

new therapies. J Invest Dermatol

2012;132:854-63

28. Yoshida H, Kunisada T, Grimm T, et al.

Review: melanocyte migration and

survival controlled by SCF/c-kit

expression. J Investig Dermatol

Symp Proc 2001;6:1-5

29. Hochhaus A. Imatinib mesylate (Glivec,

Gleevec) in the treatment of chronic

myelogenous leukemia (CML) and

gastrointestinal stromal tumors (GIST).

Ann Hematol 2004;83(Suppl 1):S65-6

30. Beadling C, Jacobson-Dunlop E,

Hodi FS, et al. KIT gene mutations and

copy number in melanoma subtypes.

Clin Cancer Res 2008;14:6821-8

31. Carvajal RD, Antonescu CR,

Wolchok JD, et al. KIT as a therapeutic

target in metastatic melanoma. JAMA

2011;305:2327-34. First study showing that imatinib

mesylate is active in a subset of

metastatic melanoma patients carrying

c-KIT mutations and amplification.

32. Natali PG, Nicotra MR, Winkler AB,

et al. Progression of human cutaneous

melanoma is associated with loss of

expression of c-kit proto-oncogene

receptor. Int J Cancer 1992;52:197-201

33. Shen SS, Zhang PS, Eton O, Prieto VG.

Analysis of protein tyrosine kinase

expression in melanocytic lesions by

tissue array. J Cutan Pathol

2003;30:539-47

34. Tomei S, Mazzanti C, Marchetti I, et al.

c-KIT receptor expression is strictly

associated with the biological behaviour

of thyroid nodules. J Transl Med

2012;10:7

35. Guo T, Agaram NP, Wong GC, et al.

Sorafenib inhibits the imatinib-resistant

KITT670I gatekeeper mutation in

S. Tomei et al.


Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.

www.ncbi.nlm.nih.gov/pubmed/22537248?dopt=Abstract






































http://clinicaltrial.gov/)

http://clinicaltrial.gov/)





















































gastrointestinal stromal tumor.


36. Prenen H, Cools J, Mentens N, et al.

Efficacy of the kinase inhibitor

SU11248 against gastrointestinal stromal

tumor mutants refractory to imatinib

mesylate. Clin Cancer Res

2006;12:2622-7

37. Schittenhelm MM, Shiraga S,

Schroeder A, et al. Dasatinib

(BMS-354825), a dual SRC/ABL kinase

inhibitor, inhibits the kinase activity of

wild-type, juxtamembrane, and activation

loop mutant KIT isoforms associated

with human malignancies. Cancer Res

2006;66:473-81

38. Ugurel S, Hildenbrand R, Zimpfer A,

et al. Lack of clinical efficacy of imatinib

in metastatic melanoma. Br J Cancer

2005;92:1398-405

39. Kim KB, Eton O, Davis DW, et al.

Phase II trial of imatinib mesylate in


Br J Cancer 2008;99:734-40

40. Wyman K, Atkins MB, Prieto V, et al.

Multicenter Phase II trial of high-dose

imatinib mesylate in metastatic

melanoma: significant toxicity with no

clinical efficacy. Cancer

2006;106:2005-11

41. Guo J, Si L, Kong Y, et al. Phase II,

open-label, single-arm trial of imatinib

mesylate in patients with metastatic

melanoma harboring c-Kit mutation or

amplification. J Clin Oncol

2011;29:2904-9

42. Flaherty KT, Hodi FS, Fisher DE. From

genes to drugs: targeted strategies for

melanoma. Nat Rev Cancer

2012;12:349-61

43. Kaufman HL, Kirkwood JM, Hodi FS,

et al. The Society for Immunotherapy of

Cancer consensus statement on tumour

immunotherapy for the treatment of

cutaneous melanoma. Nat Rev

Clin Oncol 2013;10:588-98.. This manuscript provides the first

guidelines on the use of

immunotherapy in melanoma patients.

44. Antonescu CR. The GIST paradigm:

lessons for other kinase-driven cancers.

J Pathol 2011;223:251-61

45. Schlegel J, Sambade MJ, Sather S, et al.

MERTK receptor tyrosine kinase is a

therapeutic target in melanoma.

J Clin Invest 2013;123:2257-67

46. Albino AP, Le Strange R, Oliff AI, et al.

Transforming ras genes from human

melanoma: a manifestation of tumour

heterogeneity? Nature 1984;308:69-72

47. Milagre C, Dhomen N, Geyer FC, et al.

A mouse model of melanoma driven by

oncogenic KRAS. Cancer Res

2010;70:5549-57

48. Tomei S, Adams S, Uccellini L, et al.

Association between HRAS rs12628 and

rs112587690 polymorphisms with the

risk of melanoma in the North American

population. Med Oncol

2012;29:3456-61

49. COSMIC (Catalogue Of Somatic

Mutations In Cancer. Available from:

http://cancer.sanger.ac.uk/cancergenome/

projects/cosmic

50. Posch C, Ortiz-Urda S. NRAS mutant

melanoma--undrugable? Oncotarget

2013;4:494-5

51. Niessner H, Beck D, Sinnberg T, et al.

The farnesyl transferase inhibitor

lonafarnib inhibits mTOR signaling and

enforces sorafenib-induced apoptosis in

melanoma cells. J Invest Dermatol

2011;131:468-79

52. Gajewski TF, Salama AK,

Niedzwiecki D, et al. Phase II study of

the farnesyltransferase inhibitor

R115777 in advanced melanoma

(CALGB 500104). J Transl Med

2012;10:246

53. Emery CM, Vijayendran KG,

Zipser MC, et al. MEK1 mutations

confer resistance to MEK and B-RAF

inhibition. Proc Natl Acad Sci USA

2009;106:20411-16

54. Salama AK, Kim KB. MEK Inhibition in

the treatment of advanced melanoma.

Curr Oncol Rep 2013;15:473-82

55. Kirkwood JM, Bastholt L, Robert C,

et al. Phase II, open-label, randomized

trial of the MEK1/2 inhibitor

selumetinib as monotherapy versus

temozolomide in patients with advanced

melanoma. Clin Cancer Res

2012;18:555-67

56. Robert C, Dummer R, Gutzmer R, et al.

Selumetinib plus dacarbazine versus

placebo plus dacarbazine as first-line

treatment for BRAF-mutant metastatic

melanoma: a phase 2 double-blind

randomised study. Lancet Oncol

2013;14:733-40

57. Kim KB, Kefford R, Pavlick AC, et al.

Phase II study of the MEK1/

MEK2 inhibitor Trametinib in patients

with metastatic BRAF-mutant cutaneous

melanoma previously treated with or

without a BRAF inhibitor. J Clin Oncol

2013;31:482-9

58. Ascierto PA, Schadendorf D, Berking C,

et al. MEK162 for patients with

advanced melanoma harbouring NRAS

or Val600 BRAF mutations: a non-

randomised, open-label phase 2 study.

Lancet Oncol 2013;14:249-56

59. Lebbe C, Lesimple T, Raymond E, et al.

Pimasertib (MSC1936369B/AS703026),

a selective oral MEK1/2 inhibitor, shows

clinical activity in cutaneous and uveal

metastatic melanoma in the phase I

program [abstract C07]. EADO 2012

Annual Meeting; 2012

60. Basile KJ, Abel EV, Dadpey N, et al.

In vivo MAPK reporting reveals the

heterogeneity in tumoral selection of

resistance to RAF inhibitors. Cancer Res

2013;73(23):7101-10

61. Nazarian R, Shi H, Wang Q, et al.

Melanomas acquire resistance to B-RAF

(V600E) inhibition by RTK or N-RAS

upregulation. Nature 2010;468:973-7

62. Morris EJ, Jha S, Restaino CR, et al.

Discovery of a novel ERK inhibitor with

activity in models of acquired resistance

to BRAF and MEK inhibitors.

Cancer Discov 2013;3:742-50

63. Wangari-Talbot J, Chen S. Genetics of

melanoma. Front Genet 2012;3:330

64. Madhunapantula SV, Robertson GP.

Therapeutic Implications of Targeting

AKT Signaling in Melanoma.

Enzyme Res 2011;2011:327923

65. Knight ZA, Shokat KM. Chemically

targeting the PI3K family.

Biochem Soc Trans 2007;35:245-9

66. Margolin K, Longmate J, Baratta T,

et al. CCI-779 in metastatic melanoma:

a phase II trial of the California Cancer

Consortium. Cancer 2005;104:1045-8

67. Margolin KA, Moon J, Flaherty LE,

et al. Randomized phase II trial of

sorafenib with temsirolimus or tipifarnib

in untreated metastatic melanoma

(S0438). Clin Cancer Res

2012;18:1129-37

68. Werzowa J, Koehrer S, Strommer S,

et al. Vertical inhibition of the

mTORC1/mTORC2/PI3K pathway

shows synergistic effects against

melanoma in vitro and in vivo.

J Invest Dermatol 2011;131:495-503



Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.













































http://cancer.sanger.ac.uk/cancergenome/projects/cosmic)

http://cancer.sanger.ac.uk/cancergenome/projects/cosmic)































































69. Hudes G, Carducci M, Tomczak P,

et al. Temsirolimus, interferon alfa, or

both for advanced renal-cell carcinoma.

N Engl J Med 2007;356:2271-81

70. Hay N. The Akt-mTOR tango and its

relevance to cancer. Cancer Cell

2005;8:179-83

71. Greger JG, Eastman SD, Zhang V, et al.

Combinations of BRAF, MEK, and

PI3K/mTOR inhibitors overcome

acquired resistance to the BRAF inhibitor

GSK2118436 dabrafenib, mediated by

NRAS or MEK mutations.

Mol Cancer Ther 2012;11:909-20

72. Posch C, Moslehi H, Feeney L, et al.

Combined targeting of MEK and PI3K/

mTOR effector pathways is necessary to

effectively inhibit NRAS mutant

melanoma in vitro and in vivo.

Proc Natl Acad Sci USA

2013;110:4015-20

73. Bedikian AY, Millward M,

Pehamberger H, et al. Bcl-2 antisense

(oblimersen sodium) plus dacarbazine in

patients with advanced melanoma: the

Oblimersen Melanoma Study Group.

J Clin Oncol 2006;24:4738-45

74. Bedikian AY, Lebbe C, Robert C, et al.

Survival in a phase III, randomized,

double-blind study of dacarbazine with

or without oblimersen (Bcl-2 antisense)

in patients with advanced melanoma and

low-normal serum lactate dehydrogenase

(LDH; AGENDA). J Clin Oncol

2011;29(Suppl):abstract 8531

75. Horn S, Figl A, Rachakonda PS, et al.

TERT promoter mutations in familial

and sporadic melanoma. Science

2013;339:959-61

76. Huang FW, Hodis E, Xu MJ, et al.

Highly recurrent TERT promoter

mutations in human melanoma. Science

2013;339:957-9

77. Varker KA, Biber JE, Kefauver C, et al.

A randomized phase 2 trial of

bevacizumab with or without daily low-

dose interferon alfa-2b in metastatic

malignant melanoma. Ann Surg Oncol

2007;14:2367-76

78. Kim KB, Sosman JA, Fruehauf JP, et al.

BEAM: a randomized phase II study

evaluating the activity of bevacizumab in

combination with carboplatin plus

paclitaxel in patients with previously

untreated advanced melanoma.

J Clin Oncol 2012;30:34-41

79. Minor DR. Bevacizumab advanced

melanoma (BEAM) was a positive trial.

J Clin Oncol 2012;30:2023;

author reply 2023-2024. This is an interesting comment on the

BEAM trial, describing why inhibiting

angiogenesis represents an approach

that should not be dismissed.

80. Del Vecchio M, Mortarini R, Canova S,

et al. Bevacizumab plus fotemustine as

first-line treatment in metastatic

melanoma patients: clinical activity and

modulation of angiogenesis and

lymphangiogenesis factors.

Clin Cancer Res 2010;16:5862-72.. An interesting Phase II study

evaluating combination of

bevacizumab and chemotherapy and

assessing biological correlates.

81. Hodi FS, Friedlander PA, Atkins MB,

et al. A phase I trial of ipilimumab plus

bevacizumab in patients with

unresectable stage III or stage IV

melanoma. J Clin Oncol


82. Flaherty KT, Lee SJ, Zhao F, et al.

Phase III trial of carboplatin and

paclitaxel with or without sorafenib in

metastatic melanoma. J Clin Oncol

2013;31:373-9

83. Hauschild A, Agarwala SS, Trefzer U,

et al. Results of a phase III, randomized,

placebo-controlled study of sorafenib in

combination with carboplatin and

paclitaxel as second-line treatment in

patients with unresectable stage III or

stage IV melanoma. J Clin Oncol

2009;27:2823-30

84. Fruehauf J, Lutzky J, McDermott D,

et al. Multicenter, phase II study of

axitinib, a selective second-generation

inhibitor of vascular endothelial growth

factor receptors 1, 2, and 3, in patients

with metastatic melanoma.


85. Hong DS, Koetz BS, Kurzrock R, et al.

Phase I dose-escalation study of E7080, a

selective tyrosine kinase inhibitor,

administered orally to patients with solid

tumors. J Clin Oncol

2010;28:15s(Suppl):abstract 2540

86. Corrie PG, Basu B, Zaki KA. Targeting

angiogenesis in melanoma: prospects for

the future. Ther Adv Med Oncol

2010;2:367-80

87. Ascierto ML, De Giorgi V, Liu Q, et al.

An immunologic portrait of cancer.

J Transl Med 2011;9:146. A comprehensive review proposing an

immunological interpretation

of cancer.

88. Shimanovsky A, Jethava A, Dasanu CA.

Immune alterations in malignant

melanoma and current immunotherapy

concepts. Expert Opin Biol Ther

2013;13:1413-27

89. Wilmott JS, Long GV, Howle JR, et al.

Selective BRAF inhibitors induce marked

T-cell infiltration into human metastatic

melanoma. Clin Cancer Res

2012;18:1386-94

90. Gajewski TF, Meng Y, Harlin H.

Immune Suppression in the tumor

microenvironment. J Immunother

2006;29:233-40

91. Krieg C, Letourneau S, Pantaleo G,

Boyman O. Improved IL-2

immunotherapy by selective stimulation

of IL-2 receptors on lymphocytes and

endothelial cells. Proc Natl Acad

Sci USA 2010;107:11906-11

92. Atkins MB, Lotze MT, Dutcher JP, et al.

High-dose recombinant interleukin

2 therapy for patients with metastatic

melanoma: analysis of 270 patients

treated between 1985 and 1993.

J Clin Oncol 1999;17:2105-16

93. Rosenberg SA, Mule JJ, Spiess PJ, et al.

Regression of established pulmonary

metastases and subcutaneous tumor

mediated by the systemic administration

of high-dose recombinant interleukin 2.

J Exp Med 1985;161:1169-88

94. Phan GQ, Attia P, Steinberg SM, et al.

Factors associated with response to

high-dose interleukin-2 in patients with


2001;19:3477-82

95. Smith FO, Downey SG, Klapper JA,

et al. Treatment of metastatic melanoma

using interleukin-2 alone or in

conjunction with vaccines.


96. Joseph RW, Sullivan RJ, Harrell R, et al.

Correlation of NRAS mutations with

clinical response to high-dose IL-2 in


J Immunother 2012;35:66-72

97. Dummer R, Hauschild A,

Guggenheim M, et al. Melanoma:

ESMO Clinical Practice Guidelines for

S. Tomei et al.


Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.






































































































diagnosis, treatment and follow-up.

Ann Oncol 2010;21(Suppl 5):v194-7

98. Sabatino M, Kim-Schulze S, Panelli MC,

et al. Serum vascular endothelial growth

factor and fibronectin predict clinical

response to high-dose interleukin-

2 therapy. J Clin Oncol

2009;27:2645-52

99. Yuan J, Zhou J, Dong Z, et al.

Pre-treatment serum vascular endothelial

growth factor is associated with clinical

response and overall survival in advanced

melanoma patients treated with

ipilimumab. J Immuno Ther Cancer

2013;1(Suppl 1):P247

100. Panelli MC, Wang E, Phan G, et al.

Gene-expression profiling of the response

of peripheral blood mononuclear cells

and melanoma metastases to systemic

IL-2 administration. Genome Biol

2002;3:RESEARCH0035

101. Panelli MC, White R, Foster M, et al.

Forecasting the cytokine storm following

systemic interleukin (IL)-

2 administration. J Transl Med

2004;2:17

102. Wang E, Miller LD, Ohnmacht GA,

et al. Prospective molecular profiling of

melanoma metastases suggests classifiers

of immune responsiveness. Cancer Res

2002;62:3581-6

103. Weiss GR, Grosh WW,

Chianese-Bullock KA, et al. Molecular

insights on the peripheral and

intratumoral effects of systemic high-dose

rIL-2 (aldesleukin) administration for the

treatment of metastatic melanoma.

Clin Cancer Res 2011;17:7440-50. An interesting prospective study on

gene expression in tumor and PBMC

following IL-2 administration.

104. Bedognetti D, Wang E, Sertoli MR,

Marincola FM. Gene-expression profiling

in vaccine therapy and immunotherapy

for cancer. Expert Rev Vaccines

2010;9:555-65

105. Blanchard T, Srivastava PK, Duan F.

Vaccines against advanced melanoma.

Clin Dermatol 2013;31:179-90

106. Mocellin S, Rossi CR, Nitti D, et al.

Dissecting tumor responsiveness to

immunotherapy: the experience of

peptide-based melanoma vaccines.

Biochim Biophys Acta 2003;1653:61-71

107. Lens M. The role of vaccine therapy in

the treatment of melanoma. Expert Opin

Biol Ther 2008;8:315-23

108. Terando AM, Faries MB, Morton DL.

Vaccine therapy for melanoma: current

status and future directions. Vaccine

2007;25(Suppl 2):B4-16

109. Ascierto ML, Worschech A, Yu Z, et al.

Permissivity of the NCI-60 cancer cell

lines to oncolytic Vaccinia Virus

GLV-1h68. BMC Cancer 2011;11:451

110. Reinboth J, Ascierto ML, Chen NG,

et al. Correlates between host and viral

transcriptional program associated with

different oncolytic vaccinia virus isolates.

Hum Gene Ther Methods

2012;23:285-96

111. Wang E, Phan GQ, Marincola FM.

T-cell-directed cancer vaccines: the

melanoma model. Expert Opin

Biol Ther 2001;1:277-90

112. Rosenberg SA, Packard BS,

Aebersold PM, et al. Use of tumor-

infiltrating lymphocytes and interleukin-2

in the immunotherapy of patients with

metastatic melanoma. A preliminary

report. N Engl J Med 1988;319:1676-80

113. van der Bruggen P, Traversari C,

Chomez P, et al. A gene encoding an

antigen recognized by cytolytic T

lymphocytes on a human melanoma.

J Immunol 2007;178:2617-21

114. Traversari C, van der Bruggen P,

Luescher IF, et al. A nonapeptide

encoded by human gene MAGE-1 is

recognized on HLA-A1 by cytolytic

T lymphocytes directed against tumor

antigen MZ2-E. J Exp Med

1992;176:1453-7

115. Alexandrescu DT, Ichim TE,

Riordan NH, et al. Immunotherapy for

melanoma: current status and

perspectives. J Immunother

2010;33:570-90

116. Wang E, Selleri S, Sabatino M, et al.

Spontaneous and treatment-induced

cancer rejection in humans. Expert Opin

Biol Ther 2008;8:337-49

117. Wang E, Bedognetti D, Marincola FM.

Prediction of response to anticancer

immunotherapy using gene signatures.

J Clin Oncol 2013;31:2369-71. An interesting mini-review on the role

of gene-expression in predicting

outcome to immunotherapy.

118. Iida N, Dzutsev A, Stewart CA, et al.

Commensal bacteria control cancer

response to therapy by modulating the

tumor microenvironment. Science

2013;342:967-70. An interesting study showing the role of

gut microbiota in modulating the tumor

microenvironment and thereby

influencing response to immunotherapy.

119. Wang E, Bedognetti D, Tomei S,

Marincola FM. Common pathways to

tumor rejection. Ann NY Acad Sci

2013;1284:75-9

120. Wang E, Tomei S, Marincola FM.

Reflections upon human cancer immune

responsiveness to T cell-based therapy.

Cancer Immunol Immunother

2012;61:761-70. This manuscript reflects on the

mechanisms behind patients’

individual tumor rejection and

immune responsiveness.

121. Galon J, Angell HK, Bedognetti D,

Marincola FM. The continuum of cancer

immunosurveillance: prognostic,

predictive, and mechanistic signatures.

Immunity 2013;39:11-26.. An updated review summarizing and

explaining striking analogies between

prognostic and predictive

gene-expression signatures.

122. Klebanoff CA, Acquavella N, Yu Z,

Restifo NP. Therapeutic cancer vaccines:

are we there yet? Immunol Rev

2011;239:27-44

123. Kantoff PW, Higano CS, Shore ND,

et al. Sipuleucel-T immunotherapy for

castration-resistant prostate cancer.

N Engl J Med 2010;363:411-22

124. Stein WD, Gulley JL, Schlom J, et al.

Tumor regression and growth rates

determined in five intramural NCI

prostate cancer trials: the growth rate

constant as an indicator of therapeutic

efficacy. Clin Cancer Res

2011;17:907-17

125. Schlom J. Therapeutic cancer vaccines:

current status and moving forward.

J Natl Cancer Inst 2012;104:599-613

126. Motz GT, Coukos G. Deciphering and

reversing tumor immune Suppression.

Immunity 2013;39:61-73

127. Salerno EP, Shea SM, Olson WC, et al.

Activation, dysfunction and retention of

T cells in vaccine sites after injection of

incomplete Freund’s adjuvant, with or

without peptide.


2013;62:1149-59



Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.
























































































128. Hailemichael Y, Dai Z, Jaffarzad N,

et al. Persistent antigen at vaccination

sites induces tumor-specific CD8(+) T

cell sequestration, dysfunction and

deletion. Nat Med 2013;19:465-72

129. Bedognetti D, Spivey TL, Zhao Y, et al.

CXCR3/CCR5 pathways in metastatic

melanoma patients treated with adoptive

therapy and interleukin-2. Br J Cancer

2013;109:2412-23. An interesting study evaluating a

combinatorial approach to biomarker

discovery in cancer immunotherapy.

130. Gattinoni L, Lugli E, Ji Y, et al.

A human memory T cell subset with

stem cell-like properties. Nat Med

2011;17:1290-7

131. Uccellini L, De Giorgi V, Zhao Y, et al.

IRF5 gene polymorphisms in melanoma.


132. Wang E, Zhao Y, Monaco A, et al.

A multi-factorial genetic model for

prognostic assessment of high risk

melanoma patients receiving adjuvant

interferon. PLoS One 2012;7:e40805

133. Parmiani G, Cimminiello C, Maccalli C.

Increasing immunogenicity of cancer

vaccines to improve their clinical

outcome. Expert Rev Vaccines

2013;12:1111-13

134. Schwartzentruber DJ, Lawson DH,

Richards JM, et al. gp100 peptide

vaccine and interleukin-2 in patients with

advanced melanoma. N Engl J Med

2011;364:2119-27.. The first randomized trial showing

that vaccination could be effective in

metastatic melanoma when combined

to other modalities.

135. Kruit WH, Suciu S, Dreno B, et al.

Selection of immunostimulant AS15 for

active immunization with MAGE-A3

protein: results of a randomized phase II

study of the European Organisation for

Research and Treatment of Cancer

Melanoma Group in Metastatic

Melanoma. J Clin Oncol

2013;31:2413-20

136. Ulloa-Montoya F, Louahed J, Dizier B,

et al. Predictive gene signature in

MAGE-A3 antigen-specific cancer

immunotherapy. J Clin Oncol

2013;31:2388-95

137. Brichard VG. Selection of patients for

cancer immunotherapy. Society for

immunotherapy of cancer

2013 Annual Meeting, oral presentation

138. Kaufman HL. Vaccines for melanoma

and renal cell carcinoma. Semin Oncol

2012;39:263-75

139. Andtbacka R, Collichio FA, Amatruda T,

et al. OPTiM: a randomized phase III

trial of talimogene laherparepvec

(T-VEC) versus subcutaneous (SC)

granulocyte-macrophage colony-

stimulating factor (GM-CSF) for the

treatment (tx) of unresected stage IIIB/C

and IV melanoma. J Clin Oncol

2013;31(Suppl):abstract LBA9008

140. Chowdhery R, Gonzalez R. Immunologic

therapy targeting metastatic melanoma:

allovectin-7. Immunotherapy

2011;3:17-21

141. Berd D, Sato T, Maguire HC Jr, et al.

Immunopharmacologic analysis of an

autologous, hapten-modified human

melanoma vaccine. J Clin Oncol

2004;22:403-15

142. Gottardi R, Douradinha B. Carbon

nanotubes as a novel tool for vaccination

against infectious diseases and cancer.

J Nanobiotechnology 2013;11:30

143. Pescatori M, Bedognetti D, Venturelli E,

et al. Functionalized carbon nanotubes as

immunomodulator systems. Biomaterials

2013;34:4395-403

144. Delogu LG, Vidili G, Venturelli E, et al.

Functionalized multiwalled carbon

nanotubes as ultrasound contrast agents.

Proc Natl Acad Sci USA

2012;109:16612-17

145. Restifo NP, Dudley ME, Rosenberg SA.

Adoptive immunotherapy for cancer:

harnessing the T cell response.

Nat Rev Immunol 2012;12:269-81.. A comprehensive review on the

advances in adoptive therapy.

146. Hinrichs CS, Restifo NP. Reassessing

target antigens for adoptive T-cell

therapy. Nat Biotechnol

2013;31:999-1008

147. Wang XY, Zuo D, Sarkar D, Fisher PB.

Blockade of cytotoxic T-lymphocyte

antigen-4 as a new therapeutic approach

for advanced melanoma.

Expert Opin Pharmacother

2011;12:2695-706

148. Pardoll DM. The blockade of immune

checkpoints in cancer immunotherapy.

Nat Rev Cancer 2012;12:252-64

149. Allison JP, Krummel MF. The Yin and

Yang of T cell costimulation. Science

1995;270:932-3

150. Krummel MF, Allison JP. CD28 and

CTLA-4 have opposing effects on the

response of T cells to stimulation.

J Exp Med 1995;182:459-65

151. Maio M, Bondarenko I, Robert C, et al.

Four-year survival update for metastatic

melanoma (MM) patients (pts) treated

with ipilimumab (IPI) + dacarbazine

(DTIC) on Phase 3 study CA184-024.

Ann Oncol 23(9s Suppl):

abstract 1127P

152. Wolchok JD, Weber JS, Maio M, et al.

Four-year survival rates for patients with

metastatic melanoma who received

ipilimumab in phase II clinical trials.

Ann Oncol 2013;24:2174-80

153. Maio M, Di Giacomo AM, Robert C,

Eggermont AM. Update on the role of

ipilimumab in melanoma and first data

on new combination therapies.

Curr Opin Oncol 2013;25:166-72

154. Hamid O, Hwu W, Richards J, et al.

Ipilimumab (Ipi) expanded access

program (EAP) for patients (pts) with

stage III/IV melanoma: 10 mg/kg cohort

interim results. J Clin Oncol


155. Di Giacomo AM, Ascierto PA, Pilla L,

et al. Ipilimumab and fotemustine in

patients with advanced melanoma

(NIBIT-M1): an open-label, single-arm

phase 2 trial. Lancet Oncol

2012;13:879-86

156. Patel SP, Hwu W, Kim KB, et al. Phase

II study of the frontline combination of

ipilimumab and temozolomide in


J Clin Oncol 2012;30(Suppl):

abstract 8514

157. Wolchok JD, Hoos A, O’Day S, et al.

Guidelines for the evaluation of immune

therapy activity in solid tumors:

immune-related response criteria.


158. Ribas A, Kefford R, Marshall MA, et al.

Phase III randomized clinical trial

comparing tremelimumab with

standard-of-care chemotherapy in


J Clin Oncol 2013;31:616-22

159. Ribas A. Clinical development of the

anti-CTLA-4 antibody tremelimumab.

Semin Oncol 2010;37:450-4

160. Tarhini AA, Cherian J, Moschos SJ,

et al. Safety and efficacy of combination

immunotherapy with interferon alfa-2b

and tremelimumab in patients with stage

S. Tomei et al.


Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.





































































































IV melanoma. J Clin Oncol

2012;30:322-8

161. Calabro L, Morra A, Fonsatti E, et al.

Tremelimumab for patients with

chemotherapy-resistant advanced

malignant mesothelioma: an open-label,

single-arm, phase 2 trial. Lancet Oncol

2013;14:1104-11

162. Freeman GJ, Long AJ, Iwai Y, et al.

Engagement of the PD-1

immunoinhibitory receptor by a novel

B7 family member leads to negative

regulation of lymphocyte activation.

J Exp Med 2000;192:1027-34

163. Latchman Y, Wood CR, Chernova T,

et al. PD-L2 is a second ligand for PD-1

and inhibits T cell activation.

Nat Immunol 2001;2:261-8

164. Brahmer JR, Tykodi SS, Chow LQ,

et al. Safety and activity of

anti-PD-L1 antibody in patients with

advanced cancer. N Engl J Med

2012;366:2455-65

165. Topalian SL, Hodi FS, Brahmer JR,

et al. Safety, activity, and immune

correlates of anti-PD-1 antibody in

cancer. N Engl J Med 2012;366:2443-54

166. Hamid O, Robert C, Daud A, et al.

Safety and tumor responses with

lambrolizumab (anti-PD-1) in

melanoma. N Engl J Med

2013;369:134-44

167. Sznol M, Kluger H, Hodi FS, et al.

Survival and long-term follow-up of

safety and response in patients (pts) with

advanced melanoma (MEL) in a phase I

trial of nivolumab (anti-PD-1; BMS-

936558; ONO-4538). J Clin Oncol

2013;31(Suppl):abstract CRA9006

168. Hamid O, Sosman J, Lawrence D, et al.

Clinical activity, safety, and biomarkers of

MPDL3280A, an engineered PD-L1

antibody in patients with locally advanced

or metastatic melanoma (mM).


abstract 9010

169. Melero I, Grimaldi AM, Perez-Gracia JL,

Ascierto PA. Clinical development of

immunostimulatory monoclonal

antibodies and opportunities for

combination. Clin Cancer Res

2013;19:997-1008

170. Sugamura K, Ishii N, Weinberg AD.

Therapeutic targeting of the effector

T-cell co-stimulatory molecule OX40.

Nat Rev Immunol 2004;4:420-31

171. Redmond WL, Weinberg AD. Targeting

OX40 and OX40L for the treatment of

autoimmunity and cancer.

Crit Rev Immunol 2007;27:415-36

172. Watts TH. TNF/TNFR family members

in costimulation of T cell responses.

Annu Rev Immunol 2005;23:23-68

173. Croft M. Control of immunity by the

TNFR-related molecule OX40 (CD134).

Annu Rev Immunol 2010;28:57-78

174. Curti BD, Kovacsovics-Bankowski M,

Morris N, et al. OX40 Is a Potent

Immune-Stimulating Target in Late-

Stage Cancer Patients. Cancer Res

2013;73:7189-98

175. Redmond WL, Triplett T, Floyd K,

Weinberg AD. Dual anti-OX40/IL-

2 therapy augments tumor

immunotherapy via IL-2R-mediated

regulation of OX40 expression.

PLoS One 2012;7:e34467

176. French RR, Chan HT, Tutt AL,

Glennie MJ. CD40 antibody evokes a

cytotoxic T-cell response that eradicates

lymphoma and bypasses T-cell help.

Nat Med 1999;5:548-53

177. Diehl L, den Boer AT, Schoenberger SP,

et al. CD40 activation in vivo overcomes

peptide-induced peripheral cytotoxic

T-lymphocyte tolerance and augments

anti-tumor vaccine efficacy. Nat Med

1999;5:774-9

178. Sotomayor EM, Borrello I, Tubb E,

et al. Conversion of tumor-specific

CD4+ T-cell tolerance to T-cell priming

through in vivo ligation of CD40.

Nat Med 1999;5:780-7

179. Vonderheide RH, Glennie MJ. Agonistic

CD40 antibodies and cancer therapy.


180. Vonderheide RH, Flaherty KT,

Khalil M, et al. Clinical activity and

immune modulation in cancer patients

treated with CP-870,893, a novel

CD40 agonist monoclonal antibody.

J Clin Oncol 2007;25:876-83

181. Spranger S, Gajewski T. Therapeutic

efficacy of combined blockade of CTLA-

4 +/- PD-L1 +/- IDO is associated with

re-activation of T cells directly within the

tumor microenvironment. J Immuno

Ther Cancer 2013;1(Suppl 1):O8

182. Brignone C, Gutierrez M, Mefti F, et al.

First-line chemoimmunotherapy in

metastatic breast carcinoma: combination

of paclitaxel and IMP321 (LAG-3Ig)

enhances immune responses and

antitumor activity. J Transl Med

2010;8:71

183. Poirier N, Haudebourg T, Brignone C,

et al. Antibody-mediated depletion of

lymphocyte-activation gene-3 (LAG-3(+))

-activated T lymphocytes prevents

delayed-type hypersensitivity in non-

human primates. Clin Exp Immunol

2011;164:265-74

184. Roychoudhuri R, Hirahara K,

Mousavi K, et al. BACH2 represses

effector programs to stabilize T(reg)-

mediated immune homeostasis. Nature

2013;498:506-10

185. Rosenberg SA, Yang JC, Sherry RM,

et al. Durable complete responses in

heavily pretreated patients with

metastatic melanoma using T-cell transfer

immunotherapy. Clin Cancer Res

2011;17:4550-7

186. Radvanyi LG, Bernatchez C, Zhang M,

et al. Specific lymphocyte subsets predict

response to adoptive cell therapy using

expanded autologous tumor-infiltrating

lymphocytes in metastatic melanoma

patients. Clin Cancer Res

2012;18:6758-70

187. Besser MJ, Shapira-Frommer R,

Itzhaki O, et al. Adoptive transfer of

tumor-infiltrating lymphocytes in

patients with metastatic melanoma:

intent-to-treat analysis and efficacy after

failure to prior immunotherapies.


188. Dudley ME, Gross CA, Somerville RP,

et al. Randomized selection design trial

evaluating CD8+-enriched versus

unselected tumor-infiltrating lymphocytes

for adoptive cell therapy for patients with

melanoma. J Clin Oncol 2013;31:2152-9

189. Phan GQ, Rosenberg SA. Adoptive cell

transfer for patients with metastatic

melanoma: the potential and promise of

cancer immunotherapy. Cancer Control

2013;20:289-97

190. Bedognetti D, Wang E,

Sato-Matsushita M, et al. Molecular

profiling of immunotherapeutic

resistance. In: Prendergsat GC,

Jaffee EM, editors, Cancer

immunotherapy, immune suppression

and tumor growth. 2nd edition. Elsevier,

2013.

191. Fisher B, Packard BS, Read EJ, et al.

Tumor localization of adoptively

transferred indium-111 labeled tumor

infiltrating lymphocytes in patients with



Expe

rt O

pin.

Bio

l. Th

er. D

ownl

oade

d fro

m in

form

ahea

lthca

re.c

om b

y Co

rnel

l Uni

vers

ity o

n 03

/21/

14Fo

r per

sona

l use

onl

y.









































































































1989;7:250-61

192. Spivey TL, Uccellini L, Ascierto ML,

et al. Gene expression profiling in acute

allograft rejection: challenging the

immunologic constant of rejection

hypothesis. J Transl Med 2011;9:174

193. Ascierto PA, Marincola FM.

Combination therapy: the next

opportunity and challenge of medicine.


194. Marincola FM, White DE, Wise AP,

Rosenberg SA. Combination therapy

with interferon alfa-2a and interleukin-2

for the treatment of metastatic cancer.

J Clin Oncol 1995;13:1110-22

195. Daponte A, Signoriello S, Maiorino L,

et al. Phase III randomized study of

fotemustine and dacarbazine versus

dacarbazine with or without interferon-

alpha in advanced malignant melanoma.


196. Hodi FS, Lee SJ, McDermott DF, et al.

Multicenter, randomized phase II trial of

GM-CSF plus ipilimumab versus ipi

alone in metastatic melanoma.


abstract CRA9007

197. Prieto PA, Yang JC, Sherry RM, et al.

CTLA-4 blockade with ipilimumab:

long-term follow-up of 177 patients with

metastatic melanoma. Clin Cancer Res

2012;18:2039-47

198. Marincola FM. The trouble with

translational medicine. J Intern Med

2011;270:123-7

199. Simon R. Novel clinical trial designs

for development of immunotherapy

combinations. SITC Cancer

Immunotherapy Clinical Trials:

Concepts and Challenges Meeting

2013, oral presentation

200. Yang Y, Lizee G, Hwu P. Strong

emerging rationale for combining

oncogene-targeted agents with

immunotherapy. Oncoimmunology

2013;2:e22730

201. Ribas A, Hersey P, Middleton MR, et al.

New challenges in endpoints for drug

development in advanced melanoma.


202. Zitvogel L, Galluzzi L, Smyth MJ,

Kroemer G. Mechanism of action of

conventional and targeted anticancer

therapies: reinstating immunosurveillance.

Immunity 2013;39:74-88

203. Ribas A, Wolchok JD. Combining

cancer immunotherapy and targeted

therapy. Curr Opin Immunol

2013;25:291-6

204. Castelli C, Sensi M, Lupetti R, et al.

Expression of interleukin 1 alpha,

interleukin 6, and tumor necrosis factor

alpha genes in human melanoma clones

is associated with that of mutated N-RAS

oncogene. Cancer Res 1994;54:4785-90

205. Sumimoto H, Imabayashi F, Iwata T,

Kawakami Y. The BRAF-MAPK

signaling pathway is essential for cancer-

immune evasion in human melanoma

cells. J Exp Med 2006;203:1651-6

206. Khalili JS, Liu S, Rodriguez-Cruz TG,

et al. Oncogenic BRAF(V600E)

promotes stromal cell-mediated

immunosuppression via induction of

interleukin-1 in melanoma.


207. Frederick DT, Piris A, Cogdill AP, et al.

BRAF inhibition is associated with

enhanced melanoma antigen expression

and a more favorable tumor

microenvironment in patients with

metastatic melanoma. Clin Cancer Res

2013;19:1225-31

208. Liu C, Peng W, Xu C, et al. BRAF

inhibition increases tumor infiltration by

T cells and enhances the antitumor

activity of adoptive immunotherapy in

mice. Clin Cancer Res 2013;19:393-403

209. Boni A, Cogdill AP, Dang P, et al.

Selective BRAFV600E inhibition

enhances T-cell recognition of melanoma

without affecting lymphocyte function.

Cancer Res 2010;70:5213-19

210. Ribas A, Hodi FS, Callahan M, et al.

Hepatotoxicity with combination of

vemurafenib and ipilimumab. N Engl

J Med 2013;368:1365-6

211. Flaherty KT, Infante JR, Daud A, et al.

Combined BRAF and MEK inhibition in

melanoma with BRAF V600 mutations.

N Engl J Med 2012;367:1694-703

212. Wolchok JD, Kluger H, Callahan MK,

et al. Nivolumab plus ipilimumab in

advanced melanoma. N Engl J Med

2013;369:122-33

213. Murtas D, Maric D, De Giorgi V, et al.

IRF-1 responsiveness to IFN-gamma

predicts different cancer immune

phenotypes. Br J Cancer 2013;109:76-82

214. Hamid O, Schmidt H, Nissan A, et al.

A prospective phase II trial exploring the

association between tumor

microenvironment biomarkers and

clinical activity of ipilimumab in

advanced melanoma. J Transl Med

2011;9:204

215. Ji RR, Chasalow SD, Wang L, et al. An

immune-active tumor microenvironment

favors clinical response to ipilimumab.


2012;61:1019-31

216. Carretero R, Wang E, Rodriguez AI,

et al. Regression of melanoma metastases

after immunotherapy is associated with

activation of antigen presentation and

interferon-mediated rejection genes.

Int J Cancer 2012;131:387-95

217. Panelli MC, Stashower ME, Slade HB,

et al. Sequential gene profiling of basal

cell carcinomas treated with imiquimod

in a placebo-controlled study defines the

requirements for tissue rejection.

Genome Biol 2007;8:R8

218. Wang E, Uccellini L, Marincola FM.

A genetic inference on cancer immune

responsiveness. Oncoimmunology

2012;1:520-5

AffiliationSara Tomei†1,2, Ena Wang1,3,

Lucia Gemma Delogu4,

Francesco M Marincola1,3 &

Davide Bedognetti*1

†,*Authors for correspondence1National Institutes of Health, Clinical Center

and trans-NIH Center for Human Immunology

(CHI), Department of Transfusion Medicine,

Infectious Disease and Immunogenetics Section

(IDIS), Bethesda, MD 20892, USA

E-mail: [email protected] Cornell Medical College in Qatar,

Department of Genetic Medicine,

P.O. Box 24144, Doha, Qatar

E-mail: [email protected] Medical and Research Center,

Research Branch, P.O. Box 26999, Doha, Qatar4Universita degli Studi di Sassari,

Dipartimento di Chimica e Farmacia,

07100 Sassari, Italy

S. Tomei et al.


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Non-BRAF-targeted therapy, immunotherapy, and combination therapy for melanoma

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