Non-BRAF-targeted therapy, immunotherapy, and ...

Review

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Non-BRAF-targeted therapy, immunotherapy, and combination therapy for melanoma 1.

Introduction

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FDA-approved treatments for metastatic melanoma

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Targeted therapy

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Immunotherapy

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Combination therapy

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Expert opinion

Sara 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 highthroughput 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]

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Introduction

Cutaneous melanoma is a highly heterogeneous disease characterized by a complex etiology. If diagnosed early, it can be potentially cured by surgical resection; however, once metastasized, it is highly refractory to conventional antineoplastic treatments, urging the identification of novel effective therapeutic solutions. High-throughput experimental strategies, such as gene mutation analysis and comparative genomic hybridization, allowed the identification of crucial signaling pathways essential for melanoma development and progression [1]. The recognition of molecular aberrations occurring during melanoma development facilitated the application of new therapeutic strategies, which have improved the clinical management 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 All rights reserved: reproduction in whole or in part not permitted

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A huge amount of novel therapeutic strategies are being developed for the treatment of metastatic melanoma, including targeted therapies, immunotherapies, and combinatorial strategies. FDA recently approved two new drugs for metastatic melanoma, namely: ipilimumab (anti-cytotoxic T-lymphocyte antigen 4 [anti-CTLA]-4), dabrafenib (BRAF inhibitor), and trametinib (MAPK extracellular signalregulated kinase [MEK] inhibitor). Several additional drugs are being tested and Phase I/II clinical trials are providing encouraging results. MAPK and Phosphatidylinositol 3-Kinase (PI3K) pathways are the most frequently altered pathways in melanoma. Pharmaceutical inhibitors, specifically blocking altered oncogene proteins within these pathways, have been proved to counteract melanoma development (e.g., BRAF inhibitors, MEK inhibitors, AKT inhibitors). Steps forward have been made in the field of melanoma immunotherapy, considering the exceptional immunogenicity of melanoma. These advances refer to high-dose IL-2, vaccination strategies, immune checkpoint blockade, and adoptive T-cell transfer. Albeit targeted therapy and immunotherapy strategies have revolutionized the clinical management of melanoma patients, it is becoming clear that combinatorial strategies are most likely to result in even more significant advances. Examples include BRAF inhibitors plus mTOR inhibitors, BRAF inhibitors plus MEK inhibitors, BRAF inhibitors plus PI3K inhibitor, combination of immune-checkpoint inhibitors and also targeted therapy plus immunotherapy.

This box summarizes key points contained in the article.

it transfers growth signals to the nucleus of the cells. The high success of BRAF inhibitors is mainly due to the fact that melanoma development often relies on the activation of MAPK pathway. BRAF mutations occur in about 50 -- 60% of melanoma [4-7]. More than 90% of BRAF mutations result in a single nucleotide mutation in the 600 codon (V600) [5,6]. Among them, about 90% of mutation are represented by the valine to glutamic acid substitution (V600E) [5,6], associated with a 400-fold increased activity of the protein [8,9]. Overall, approximately 50% of melanoma patients carry the V600E mutation. Vemurafenib, a kinase inhibitor acting by blocking V600E mutated BRAF, have been extensively investigated in Phase II and III trials [4,10]. It has been approved by FDA in August 2011 for the treatment of metastatic or unresectable melanoma patients carrying the V600E mutation. In the interim analysis of the Phase III trial (BRIM-3), vemurafenib has been shown to induce an objective response (OR) in 48% of the patients, significantly higher than that obtained by dacarbazine (5%) [4]. Beyond tumor shrinkage, administration of vemurafenib resulted in a significantly prolonged progression-free survival (PFS; 6.9 vs 1.6 months; vemurafenib vs dacarbazine, respectively) and 2

overall survival (OS; 13.6 vs 9.7 months; vemurafenib vs dacarbazine), according to a recent update of the BRIM-3 trial [11]. In the same setting, similar results in term of OR and PFS have been obtained by the BRAF inhibitor dabrafenib, which has been approved by FDA in May 2013. However, the trial allowed crossover and was not powered enough to detect differences in OS [12]. Even though Phase III trials focused on patients bearing V600E mutation, BRAF inhibitors may be similarly active in patients carrying other 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 vemurafenib and dabrafenib in metastatic or unresectable melanoma patients carrying any kind of BRAF V600 mutation. However, despite BRAF inhibition has shown the most promising results in the field of melanoma targeted therapy, several challenges remain. The first important obstacle is that half of melanoma patients do not carry V600 mutations and therefore cannot be treated with BRAF inhibitors. Moreover, a complete 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 or 2 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 significant advances in the treatment of metastatic melanoma, highlighting the novel therapeutic strategies (currently being tested in clinical trials [15]), which preclude BRAF treatment, encompassing single molecular-targeted therapy, immunotherapy, and combinatorial therapeutic approaches.

FDA-approved treatments for metastatic melanoma

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To date there are four FDA-approved treatments for metastatic melanoma besides the two aforementioned BRAF inhibitors: recombinant IL-2, ipilimumab (approved, respectively, in 1998 and 2011; discussed in the following sections), dacarbazine, and trametinib [4,16-18]. Dacarbazine is an alkylating agent, which was accepted as standard treatment for metastatic melanoma in the 1970s [19,20], and it remains the only cytotoxic chemotherapy drug approved for the treatment of metastatic melanoma. In preliminary studies, OR was reported in up to 25% of patients [21]. However, in more rigorous randomized trials, dacarbazine has shown to induce an OR in about 10% of patients (range 5 -- 12%) [4,12,21,22]. CR is exceptionally rare (1 -- 2%) [4,12,21,22], remission is in general transient [21], and advantage in OS has not been conclusively demonstrated in randomized trials. Although other cytotoxic agents (e.g., fotemustine and temozolomide) have been shown to be at least not inferior to dacarbazine, none of them produces superior benefit to dacarbazine in terms of OS [21]. For this reason, dacarbazine is often chosen as control arm in randomized trials assessing new therapeutics. Trametinib, a MAPK extracellular

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


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

signal–regulated kinase (MEK) inhibitor (see following section), was approved by FDA on 29 May 2013 (concurrently with the approval of dabrafenib) for the treatment of patients with unresectable or metastatic melanoma harboring BRAF V600E or V600K mutation [23]. Currently, several drugs are being tested and several encouraging results have been recently published. Therapeutic strategies for the treatment of melanoma can be systematically divided in three broad categories: targeted therapy, immunotherapy, and combination therapy. Targeted treatments act by directly inhibiting molecules (e.g., proteins encoded by protoncogenes or growth factors), which are essential for the tumor growth. Immunotherapy treatments aim at stimulating the immune system of the host to reject cancer cells. Combination therapy refers to the use of multiple approaches to achieve a stronger anticancer response and overcome potential mechanisms of resistance. Examples of the most effective treatments belonging to each of the above categories will be given in the following sections of this review. 3.

Targeted therapy

Biologic targeted therapy usually refers to a treatment that specifically targets molecules involved in neoplastic process with less harm to normal cells. Targeted treatments act by blocking the activity of specific molecules essential for cancer growth and development. The majority of targeted therapies include either small molecules or monoclonal antibodies. The testing and implementation of possible therapeutic drugs in melanoma is growing up at an exponential rate as our understanding of the genetic heterogeneity of this disease improves. An interesting recent study from Henary et al. [24] showed that the administration of molecularly matched therapies predicted better outcomes, emphasizing the relevance of targeted therapy in the context of personalized medicine. Melanoma cells can acquire a proliferative advantage or protection against cell death by deregulating molecular pathways relevant to cell biology. The understanding of cell growth and molecular signaling pathways in melanoma is essential to develop and implement new therapeutic drugs. In the following, we provide an overview of the most commonly deregulated signaling pathways in melanoma responsible for cell proliferation and cell survival, and the most relevant advance in clinical setting. MAPK pathway MAPK pathway is probably the best-studied signaling pathway in cancer, most likely because of its essential role in melanoma onset and development [25]. A simplified representation of MAPK pathway is given in Figure 1. Signaling through MAPK pathway occurs upon the binding of extracellular growth factors to a variety of tyrosine kinase receptors (TKRs), including c-KIT and c-MER, which in turn lead to the activation of RAS. RAS is a small GTP-binding protein, which can exist in three isoforms: HRAS (Harvey-RAS), KRAS (Kirsten-RAS), and NRAS 3.1

(neuroblastoma-RAS). NRAS is the most prevalent form in melanoma being constitutively activated by point mutation in about 15% of cases [26]. Once activated, NRAS binds BRAF, which further phosphorylates and activates MEK. MEK finally signals to ERK, which translocates into the nucleus and enhances the transcription of a bunch of transcription factors resulting in increased cell proliferation and increased cell survival. Activating mutations within MAPK pathway account for 90% of melanomas [27]. Studies have reported a constitutive activation of MAPK pathway through mutation and/or gene amplification of c-KIT. C-KIT gene encodes a cell-membrane tyrosine kinase receptor, which plays an essential role in cellular differentiation [28]. The role of c-KIT in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST) has been well established and the use of imatinib, an oral c-KIT inhibitor, has revolutionized the treatment of these diseases [29]. The use of c-KIT inhibitors in melanoma represents an evolving matter of investigation. C-KIT mutations account for about 2% of cutaneous melanoma, albeit mutation and/or amplification of c-KIT are found in about 20 -- 25% in some melanoma subtypes, such as mucosal melanoma, acral melanoma, or melanoma arising from chronically sun-damaged skin [30,31]. Consistent with the essential role of c-KIT in melanocytes differentiation and migration, several melanomas express c-KIT. However, experimental evidence from studies in thyroid and melanoma showed that the loss of this growth factor receptor is strangely related to a worse malignant phenotype [32-34]. This may be explained by the acquisition of a less-differentiated phenotype during tumor progression. A number of drugs targeting c-KIT have been developed and their effectiveness has been proved in GISTs. These drugs include sunitinib, nilotinib, sorafenib, dasatinib, and imatinib, and their therapeutic activity has been shown to be dependent on the c-KIT specific type of molecular alteration [35-37]. Results of c-KIT inhibitor trials in metastatic melanoma were initially disappointing. Three Phase II trials did not show clinical activity of imatinib in unselected melanoma patients [38-40]. The scenario substantially changed when the studies focused on melanoma patients carrying c-KIT mutations and/ or amplifications, therefore highlighting the importance of patient selection in early targeted-therapy development. According to two Phase II trials, indeed, an OR is achieved in almost 25% of patients, and temporary stabilization occurs in about 50 -- 70% of subjects [31,41]. Although results in terms of OS seem encouraging (median OS from 11 to 14 months), the median PFS appears to be low, ranging from 3 to 3.5 months [31,41]. Importantly, responses seem to be restricted to patients carrying hot spot mutations either in exon 11, 13, or multiple mutations [31,41]. There is a great interest for c-KIT inhibition in mucosal and acral melanoma and other drugs, such as sunitinib, nilotinib, and dasatinib, are currently investigated in Phase II trials [42]. In view of the growing enthusiasm in this field, the Society for Immunotherapy of Cancer (SITC) melanoma expert panels recommend to routine testing of


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c-KIT

GPCR

Cytosol

PIP2

GNAQ/GNA11

PKC

PI3K

NRAS

PTEN PIP3

PDK1

BRAF


ERK1/2 ELK

MITF

AKT

BAD

MEK1/2

BCL2

JUN

mTOR

BAX

Proliferation, differentiation

IKK

NFκB

MDM2

GSK3β

p53

β-catenin

Figure 1. Simplified representation of MAPK and PI3K pathways. Red bolts indicate targets of molecular alterations in 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 sarcoma virus 17 oncogene homolog; MDM2: Mouse double minute-2; MEK1/2: MAPK extracellular signal–regulated kinase 1 and 2; MITF: Microphthalmia transcription factor; mTOR: Mammalian target of rapamycin; NFkB: nuclear factor-kB; NRAS: Neuroblastoma RAS; p53: Tumor protein 53; PDK1: Phosphoinositidedependent kinase-1; PI3K: Phosphoinositide-3 kinase; PIP: Phosphatidylinositol phosphate; PKC: Protein kinase C; PTEN: Phosphatase and tensin homolog.

c-KIT mutations in clinical practice [43]. Whether acquisition of resistances to imatinib in melanoma is due to the acquisition of additional 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 of the TAM (TYRO, AXL, MERTK) family of RTKs. A recent study from Schlegel and colleagues [45] has pointed c-MER as a potential therapeutic target in melanoma. In this work, they were able to show that treatment of melanoma cell lines with UNC1062, a novel c-MER inhibitor, induced apoptosis and inhibited invasion of melanoma cells, encouraging a deeper testing of such drug. In addition to the deregulation of RTKs, several MAPK downstream molecules are often altered in melanoma. The first oncogene to be discovered was NRAS [46]. As mentioned earlier, NRAS is a member of the rat sarcoma (RAS) protein family, together with HRAS and KRAS. RAS are GTPbinding proteins, which, once activated by RTKs, can signal a complex network of downstream molecular pathways. Although evidence shows KRAS and HRAS to be implicated in melanoma [47,48], NRAS is the most active form. Most common mutations in NRAS gene have been found located at codon 61 [49], causing the impairment of NRAS enzymatic activity to switch GTP to GDP. Albeit several attempts have been made to directly target NRAS, no clinical success has been reached yet. One probable explanation is the low 4

concentration range (picomolar) required for the affinity of GTP to bind NRAS, which made the development of GTPspecific antagonists impossible so far [50]. Nevertheless, significant effort has been spent to indirectly inhibit NRAS by targeting key post-translational modifications, such as farnesylation. Farnesylation consists in a lipid modification necessary for NRAS membrane association. The inhibition of farnesylation process results in the blockage of NRAS translocation to the plasma membrane. A study from Niessner and colleagues [51] has shown that the farnesyltransferase inhibitor (FTI) lonafarnib was also able to inhibit mTOR signaling pathway enhancing apoptosis induced by sorafenib in melanoma cell lines. However, testing FTI tipifarnib (also called R115777) in a Phase II clinical trial failed to obtain encouraging results [52], most likely due to aspecific responses as farnesylation occurs in the maturation process of several proteins other than RAS. Although in this trial patients were unselected for NRAS mutation, the intrinsic difficulty to develop NRAS inhibitors and the scarce activity of this approach in other tumors have shifted the efforts to inhibit MAPK pathway toward more effective targets. Another important protein kinase belonging to MAPK pathway is MEK, and its inhibition is currently under investigation. Considering that MEK activation has been shown to be a resistance mechanism by which melanoma treated with BRAF inhibitors relapse, MEK inhibition was tested in combination with BRAF inhibitors (see combination therapy




section subsequently), yielding extremely promising results. MEK proteins include MEK1 and MEK2, which are the only substrate of BRAF. Mutations on MEK genes are very rare, although they have been reported in a few cases [49]. However, the acquisition of MEK1 mutations P124L and Q56P have been demonstrated to confer resistance to BRAF and MEK inhibitors [53]. A recent review by Salama and Kim [54] lists a broad variety of MEK inhibitors currently under investigation. Worthy of note, selumetinib is an oral potent inhibitor of MEK1 and MEK2 kinases. The efficacy of this drug was evaluated in a large Phase II clinical trial in advanced melanoma patients. Unfortunately, the study failed to show a difference of the OR rate and PFS between the treatment arm and the control group (temozolomide) [55]. Interestingly, five out of six responders were BRAF mutated. Combination of selumetinib and dacarbazine was reported to be clinically active in a Phase II randomized trial (PFS 5.6 vs 3.0 months, selumetinb plus dacarbazine vs dacarbazine alone), although nonsignificant benefit in terms of OS was observed [56]. Several clinical trials are still testing MEK inhibitors in melanoma, given the fact that MAPK reactivation is often driven by MEK signaling. Among the MEK inhibitors tested, trametinib has shown so far the most promising results. In a Phase III study, 322 patients carrying V600E or V600K mutation were randomized to receive either trametinib or chemotherapy (either decarbazine or paclitaxel) [23]. A statistical significant prolongation of PFS was shown for patients treated with trametinib compared to the control group (4.8 vs 1.5 months). OR rate was also higher in trametinib arm than in chemotherapy arm (22 vs 8%). Despite the crossover to trametinib at progression, the 6 months OS was significantly higher in the trametinib arm (81 vs 67%) [23]. On the basis of these results, FDA approved trametinib for the treatment of metastatic melanoma harboring V600E or V600K mutations. Importantly, tremetinib is not active if administered to patients progressing on a BRAF inhibitor, stressing the need to better identify the window of efficacy of trametinib in BRAF mutant melanoma [57]. However, patients carrying NRAS mutations cannot benefit from such treatment, urging the need to find other additional treatment options. Recently, Ascierto and colleagues [58] in an open-label, nonrandomized, Phase II trial assessed the efficacy of MEK162, a potent MEK inhibitor, in patients carrying NRAS or V600E BRAF mutations. In addition to an OR rate of 20% in BRAF-mutated patients, they were able to show 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 observed with the use of pimasertib [59], another MEK inhibitor under clinical development. These proof-of-principle studies pinpoint MEK inhibition as 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 development of resistance. However, while melanoma can take different roads to counteract to MAPK-targeted inhibition, the reactivation of the extracellular-signal-regulated kinases (ERK) appears to be a common feature [60,61]. Two isoforms of ERK, ERK1 and ERK2, transmit proliferative signals once phosphorylated by the upstream MEK proteins. Quite interestingly, Morris and colleagues [62] recently identified a potent ATP-competitive ERK inhibitor by screening a library of 5 million compounds. Such inhibitor, called SCH772984, showed promising clinically attractive results as it was able to inhibit ERK phosphorylation in a dose-dependent manner. Testing this compound in vivo is warranted to prove its efficacy and its potential relevance in clinical settings. Phosphatidylinositol 3-kinase pathway Phosphatidylinositol 3-kinase (PI3K) pathway is the second most frequently deregulated pathway in melanoma after MAPK pathway (Figure 1). Similarly to MAPK, PI3K cascade is triggered by RTKs and RAS protein. Once activated by NRAS, PI3K can catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger, and its production is antagonized by the tumor suppressor phosphatase and tensin homolog (PTEN). PIP3 can activate AKT (through the phosphatidylinositoldependent kinase 1, PDK1), which, in turn, phosphorylates mTOR and multiple additional targets determining increased oncogenic transformation, survival, proliferation, and cellcycle regulation [63]. The deregulation of PI3K pathway in melanoma occurs through activation of PI3K (encoded by PI3KCA gene), PTEN loss, and AKT activation. The independent therapeutic relevance of inhibiting PI3K pathway itself has not been well established yet, however, preclinical evidence suggests that PI3K pathway inhibition may be a relevant adjunct to MAPK-targeted therapy. Several small molecule inhibitors targeting AKT and mTOR activity have been developed and tested in melanoma cell lines and xenografted animal models [64]. However, the number of clinical studies currently evaluating their clinical efficacy is very low. The AKT inhibitors wortmannin and LY294002 have been shown to block AKT activity in a variety of functional studies, but their application in clinical settings is limited by their offtarget activities [63,65]. Several derivatives of rapamycin, such as temsirolimus, have also been used as mTOR inhibitors and have demonstrated clinical activity in other malignancies such as renal cell carcinoma. Nevertheless, the administration of temsirolimus (either used alone or in combination with the multiple kinase inhibitor sorafenib) failed to show significant clinical activity in Phase II trials [66,67]. Even though several additional AKT inhibitors, such as API-2, SR13668, BI-69A11, GSK690693, and MEK-2206 are under investigation, it is widely believed that PI3K pathway inhibition works better when combined to other targeted therapies. An interesting study by Werzowa and colleagues 3.2


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assessed the potential efficacy of PI3K pathway vertical inhibition in melanoma cell lines and xenograft models [68]. The concurrent treatment of rapamycin and the mTOR inhibitor PI-103 had a potent antitumor activity both in vitro and in vivo. Although in general PI3K and mTOR inhibition seems to be a promising strategy against cancer development and some drugs have been already approved for the treatment of renal cancer [69], such drugs did not obtain the same success in melanoma. The reason behind this phenomenon is not clear yet albeit it has been hypothesized that the ability of rapamycin derivatives to phosphorylate AKT may impede their clinical success [70]. Nevertheless, MEK and PI3K combination seems promising. An interesting in vitro study by Greger et al. showed that combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to dabrafenib, mediated by NRAS or MEK mutations [71]. Another study showed that combined MEK and PI3K/ mTOR pathway inhibition induces regression of NRASmutant melanoma cell lines [72]. An increasing number of clinical trials combining PI3K and MAPK inhibitions in melanoma 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 oligonucleotide oblimersen seemed to increase OS in a subset of patients (i.e., those with normal or low level of LDH) [73], but these findings were not confirmed in a recent randomized Phase III trial [74]. In light of recent in vitro or in vivo studies, modulation cellcycle checkpoints (which are de-regulated in melanoma) and inhibition of G protein-coupled receptor signaling (found to be activated in up to 80% of uveal melanoma) have recently gained the attention of clinical investigators [42,72]. Remarkably, two very recent investigations have found that > 70% of melanoma bears somatic mutation of telomerase reverse transcriptase (TERT) promoter, therefore highlighting another important oncogenic mechanism [75,76]. Angiogenesis Because melanoma is a highly vascularized cancer, several attempts to therapeutically target angiogenic mechanisms in melanoma have been explored. Anti-angiogenic drugs have been developed and demonstrated to be useful in different types of cancer. Still, their clinical benefit in melanoma is not clear. Among the anti-angiogenic drugs, bevacizumab, a humanized 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 with chemotherapy), and brain cancers. In metastatic melanoma, the use of bevacizumab as single agent, or in combination with IFN-a failed to show significant clinical activity [77]. According to a large randomized Phase II trial (BEAM) enrolling 314 treatment naı¨ve patients, bevacizumab, in combination with chemotherapy enhances, though not significantly, PFS and OR (4.2 vs 5.6 month; 3.3

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16 vs 26%; PFS and OR, bevacizumab vs bevacizumab plus carboplatin and paclitaxel). OS advantage was initially significant (8.6 vs 12.3 months; p = 0.04), but it was reduced in a subsequent analysis with longer follow-up, (9.2 vs 12.3 months; p = 0.19) [78]. Although it is not clear if this combination will move forward, the BEAM study, which is often considered a positive Phase II trial, still represents one of the best evidence of the activity of anti-angiogenic therapy in advanced melanoma [79]. Interestingly, Del Vecchio and co-workers [80] evaluated the clinical and biological activity of the use of bevacizumab in combination with fotemustine, a chemotherapeutic drug widely used in Europe. The authors were able to show that such regimen had important clinical activity as first-line treatment of metastatic melanoma, obtaining an encouraging OS of 20.5 months (PFS = 8.8 months, OR 15%), although grade 3 -- 4 hematological toxicity was quite high. Attempts to use bevacizumab in clinical trials employing other combinatorial setting are currently underway and results are awaited. Recently, a preliminary analysis of 20 patients enrolled in a Phase I study assessing combination of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) mAb ipilimumab and bevacizumab showed an intriguing OR of 38%; all the responses lasted > 6 months [81]. Similarly to VEGF-A mAbs, other drugs targeting the VEGF receptors (VEGFRs) have been tested in melanoma. Among them, sorafenib, which inhibits tyrosine kinases associated with VEGF and plateled-derived growth factor, failed to demonstrate significant clinical activity in combination with chemotherapy in Phase III trials [82,83]. The more promising ones as single agents are axitinib and lenvatinib and their investigations alone or in combination are moving forward. In a multicenter, Phase II study, axitinib monotherapy resulted in a 19% OR rate [84]. Similarly, in a Phase I evaluation of patients with solid malignancies, including melanoma, lenvatinib (also called E7080) demonstrated an OR of 21% [85]. Based on this evidence, it is likely that anti-angiogenic therapies might be helpful in treating melanoma especially when combined with other drugs and much effort should be spent in identifying the more beneficial anti-angiogenic combination therapies. Moreover, other novel molecules, as those mimicking natural anti-angiogenic agent inhibitors (e.g., endostatin), inhibitors of placental growth factor, and agents blocking HIFa, a key molecule in hypoxia signaling, are currently under development [86]. 4.

Immunotherapy

Cancer immunotherapy is meant as the treatment aiming at annealing tumors by inducing, enhancing, or suppressing specific immune responses. Aside from the molecular alterations within proliferative pathways described earlier, several immune alterations have been reported in melanoma, including decreased number of peripheral B and T cells, increased number of regulatory T cells, impaired activation of natural




killer (NK) cells (due to downregulation of activating ligands), acquisition of tolerance by CD8+ T cells (due to increased expression of inhibitory receptors), increased level of pro-tumorigenic cytokines such as TGF-b, TNF-a, IL-6, IL-8, and IL-1, and altered expression of immune-related genes [87,88]. Immunotherapy strategies for melanoma treatment carry a high clinical relevance considering the exceptional immunogenicity of such disease. The immunogenicity of melanoma has been established based on the high number of tumor-infiltrating lymphocytes (TIL) found in resected melanoma samples, and several strategies have been proposed to conquer melanoma taking advantage of this peculiar feature. A diverse set of melanoma antigens have been identified as target of cellular immune response, which can be grouped into 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), which are found expressed in normal melanocytes and are overexpressed in melanoma; tumor-specific antigen (TSA), including the melanoma antigen (MAGE), the B melanoma antigen (BAGE), and G antigen (GAGE) gene families, the NY-ESO-1 (also known as CTAG1), which are found in normal gametic cells in the testes and in cancers in addition to melanoma but are not expressed by normal melanocytes; and mutated antigens, including b-catenin, which can be found in different tissues. Additionally, several melanoma antigens have been identified as targets of humoral immune responses. The expression of melanoma antigens is of great importance as it allows the innate and adaptive immune system to track and destroy nascent tumor cells, through a mechanism known as ‘immune surveillance’. However, cancer cells can escape immune surveillance by becoming poorly immunogenic. This phenomenon has been commonly referred to as ‘immune editing’ [89]. Several mechanisms of immune editing have been described, including the downregulation of antigen presentation MHC molecules by the tumor cells themselves, the secretion of soluble factors and cytokines, such as TGF-b and IL-10, which inhibit effective immune response, the creation of an immune suppressive environment by recruiting inhibitory immune cells, such as T-reg, NKs, and macrophages, and the expression of negative regulators of the immune system, including programmed death ligand 1 (PD-L1) and the CTLA-4 [90]. We report subsequently the best-characterized immunotherapy strategies for the treatment of melanoma.

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

and high-dose IL-2 (HD-IL-2) was approved by FDA in 1998 for the treatment of metastatic melanoma. HD-IL-2 was initially shown to have dose-dependent anticancer effect by the group of Steven Rosenberg at the National Cancer Institute [93]. FDA approved HD-IL-2 in 1998 based on the observation that it can induce long-term remissions. In large retrospective analyses, administration of HD-IL-2 induce an OR in about 15% of patients (range 13 -- 19%) of patients, and CR in 4 -- 6% [92,94-96]. However, in a recent randomized Phase III trial, these percentages dropped from 10 to 6% for OR and from 2 (two patients) to 1% (one patient) for CR when evaluation was assessed by central review. Even though the real rate of OR needs to be determined, complete-remission under HD-IL-2 appear to be extremely durable (median CR duration > 14 years, according to a long follow-up analysis of a retrospective casuistic) [95]. However, HD-IL-2-related adverse events are severe (e.g., hemodynamic shock, respiratory insufficiency, and neurotoxicity) and require intensive inpatient care [21]. The opinions of European and US specialists about the risk--benefit ratio of this treatment largely diverge. In light of the potentially curative role of HD-IL-2, the US National Comprehensive Cancer Network US guidelines still list HD-IL-2 as treatment option in a subset of (clinically fit) patients, if the center has a considerable experience in the management of this regimen. Even more, the (US) melanoma panelists of the Society for Immunotherapy of Cancer (SITC) recommend the administration of HD-IL-2 as first-line treatment in good performance status patients without central nervous system metastases, even in presence of BRAF mutation [43]. Conversely, the European Society of Medical Oncology (ESMO) melanoma panelists do not encourage its routine use in clinical practice, in view of the high toxicity and the lack of randomized trials demonstrating survival advantage in the overall population [97]. Considering the relatively low benefit--risk ratio, one of the main limitations of HD-IL-2 is the lack of pretreatment tools to predict patients who are likely to respond to the treatment. Recently, in a proteomic study, we showed that low levels of fibronectin and VEGF, an angiogenetic factor with immunosuppressive functions, predict response to HD-IL-2 [98] in metastatic melanoma patients. Although these findings need to be prospectively validated, Hodi and co-workers recently reported that low levels of VEGF also predict OS and clinical response to ipilimumab in metastatic melanoma [99] Noteworthy, an interesting study from Joseph and colleagues has recently shown that patients harboring NRAS mutation carry a higher likelihood to respond to therapy [96], linking, for the first time, alterations of the MAPK pathway to the responsiveness to HD-IL-2. Although IL-2 has been used in oncology since > 20 years, its mechanism of action is still not completely understood. Only few studies have attempted to comprehensively understand the mechanisms behind the cancer rejection mediated by IL-2.


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Our group performed a series of proteomic and transcriptomic analyses of peripheral mononuclear cells (PBMCs) and pre- and post-melanoma metastasis using fine-needle aspiration biopsies (FNA) obtained from patients undergoing systemic IL-2 administration [98,100-103]. We observed that administration of IL-2 induces a cytokine storm already after the first administration, where most of the cytokines investigated (e.g., IFN-g, IL-10, and TNF-a) increased significantly compared with the baseline value. CXCR3 ligands (i.e., CXCL9, 10 and 11) and CCR5 ligands (i.e., CCL3 and CCL4) were found among the chemokines that significantly increased early after treatment [100,101]. Even when investigated by whole genome gene-expression analysis, the effects of HD-IL-2 were dramatic within PBMCs, whereas effects within the tumor microenvironment were lesion-specific and limited [103]. Altogether these findings suggest that the main effect of IL-2 in the tumor is the activation of innate mechanisms driven by monocytes and eventually sustained by NK, and the tumor infiltrating lymphocytes. These cells in concert can initiate an inflammatory chemotactic cascade, orchestrated by the activation of the IRF-1/ IFN-g pathway and inducing release of related specific chemotactic ligands (i.e., CXCR3 and CCR5 ligands) with consequent polarized (Th1) recruitment of other immune cells later on [104]. Importantly, the degree of inflammation induced by HD-IL-2 correlates with the development of tumor rejection [102,103]. Vaccination strategies Cancer vaccines aim at treating cancer by stimulating the host’s immune reaction against cancer cells. The implementation of therapeutic vaccines to treat melanoma remains a challenge [105-107]. Broadly, melanoma vaccines are classified into the following main categories: whole-cell vaccines (either autologous or allogeneic, prepared using melanoma cells or subcellular fraction); dendritic-cell (DC) vaccines (which take advantage of the highly specialization of DCs to present antigens and induce primary cellular immune responses); peptide vaccines (which use melanoma antigens to stimulate cytotoxic T lymphocytes response against tumor cells); ganglioside vaccines (which take advantage of the high expression of gangliosides on the surface of melanoma cells to generate an immune response against them); DNA vaccines (composed of naked DNA expression plasmids that include a gene encoding the target antigens from tumor cells); viral vector vaccines (in which a virus is used as vector of melanoma antigens); and oncolytic vaccine (in which an oncolytic virus replicating in cancer 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 the immune-mediated treatment of melanoma dates back to 1988, when Rosenberg and colleagues first showed the regression of metastatic melanoma through the autologous transfer 4.2

8

of tumor infiltrating lymphocytes combined with IL-2 [112]. In 1991, van der Bruggen et al. described for the first time a gene encoding an antigen recognized by T cells (MAGE-1) [113]. This study, followed by the description of the first cancer-specific epitope HLA-A1 restricted [114], gave molecular accuracy to this rather disregarded field and provided the opportunity to investigate with scientific precision the phenomenon of immune-mediated cancer rejection. This led, in turn, to the characterization of a myriad of tumor antigens and their immunodominant peptides, with consequent implementation of active immunization strategies [115]. The study of the tumor-antigen-specific immunization restricted to an individual epitopic determinant offered the opportunity to study the dynamic of the immune response in humans by reducing the algorithm of tumor--host crosstalk to a specific HLA/epitope interaction with the complementary T-cell receptor [116]. These studies have shown that, in order to develop clinical active immunotherapies, factors beyond T-cell/HLA/epitope interactions must be considered. In fact, the large majority of the studies failed to report an association between specific response against the administered antigens and clinical benefit [104]. Humoral and/or cellular immune response against vaccine antigens are necessary for the development of an effective antitumor response but they are certainly not sufficient [117]. Other factors, as the presence of co-stimulatory or inhibitory stimuli in tumor microenvironment, genetic polymorphisms of the host, genetic of the tumors, and environmental factors (i.e., intestinal microbiota) [118], can certainly modify the algorithm of immune response, making the evaluation of a single parameter unlike to predict the clinical benefit [119-121]. Besides local toxicity, vaccines are in general extremely well tolerated. However, in melanoma, as well as in many other solid tumors, vaccination alone has failed to induce significant tumor regression, the OR rate being around 3 -- 4% [122]. Nevertheless, recent randomized Phase III trials in other malignancies (i.e., prostate cancer) have shown that vaccine administration can prolong survival even in absence of detectable tumor shrinkage [123]. The convincing detection of a remarkable survival benefit dissociated with measurable changes in the tumor size forced us to reconsider the mechanism of actions of vaccinations. An interesting interpretation of these paradigmatic results comes from the analysis of tumor kinetic following different treatments. By applying a rather novel mathematical model to Phase II trials in prostate cancer patients receiving chemotherapy or PROSTVAC vaccine (consisting in recombinant poxviral vectors containing transgenes for prostate specific antigen (PSA) and the costimulatory molecules B7), Fojo and Schlom showed that chemotherapy determines an initial reduction of tumor burden followed, at relapse, by a tumor-growth rate similar to that before chemotherapy [124,125]. Vaccination, however, modifies clinical outcome (OS) by reducing tumor-growth rate. The same mechanisms likely explain differences between survival curves in BRAF and ipilimumab trials (Figure 2).



OS (%)

Targeted therapy (BRAFi)

Immunotherapy (anti-CTLA-4 mAb)

100

100

100

80

80

80

60

60

60

40

40

40

20

20

20

0

12

24

36

48

0

PFS (%)

Time (months) Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Cornell University on 03/21/14 For personal use only.

Combination therapy

12

24

36

Time (months)

100

80

80

80

60

60

60

40

40

40

20

20

20

24

Time (months)

36

48

0

12

24

12

24

36

48

Time (months)

100

12

> Comb-TT

0

48

100

0

> Comb-TT

36

Time (months)

48

> Comb-TT > Comb-TT

0

12

24

36

48

Time (months)

Figure 2. Survival curves of BRAF inhibitors, CTLA-4 mAb (ipilimumab) and combination therapy. In each panel blue line represents 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 dabrafenib randomized Phase III trials. Immunotherapy curves were approximated from ipilimumab randomized trials. Dotted line represents survival projection when no data are available. PFS of combination therapy was derived from the dabrafenib plus trametinib Phase I/II randomized trial. BRAF inhibition induces response in most of the patients, however the response is transient, and all but few patients relapse within 1 or 2 years through developing secondary resistance. The long-term benefit in OS is expected to be very low. Vice versa, immunotherapy (i.e., ipilimumab) determines a delayed response in a small proportion of patients. Response is however durable, with impact on long-term survival. Combination immuno-targeted therapy could act sinergically therefore switching the curve on the right (targeted therapy) and up (immunotherapy). Although results from such combinations are not available yet, certain combination of immunotherapy (i.e., ipilimumab and IL-2 or IL-2 and vaccine) seems to increase survival, therefore shifting the curve up. However, it seems that combination of BRAF and MAPK extracellular signalregulated kinase inhibitors as well as certain immunotherapy combination (i.e., ipilimumab and the anti-PD1 nivolumab) are able to shift the curve on right and up by inducing deep, frequent and durable antitumor response. Even though preliminary data suggest that survival curve of anti-PD1 and anti-PD1 ligand will differ as compared with those of ipilimumab, they are not represented 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.

Even though several scientific progresses in the field of vaccinology have been accomplished during the past decade, it is clear that active immunization alone would unlikely represent a curative option for patients affected by advanced melanoma. The rather disappointing results of cancer vaccines could be due to a mixture of the following factors: i) the presence of a highly immunosuppressive microenvironment with consequent inability of T cells to overcome environmental immunosuppression in absence of co-stimulatory or anti-inhibitory

stimuli [126]; ii) the scant ability of antitumor T cells to localize at tumor site [127-129]; iii) the rapid senescence of tumor-specific T cells or T-cell repertoire [122,130]; iv) the highly mutable tumor target capable of immune-evasion and antigen loss [122]; and v) the presence of unfavorable genetic condition (host polymorphisms or cancer mutations) [129,131,132] or unfavorable environmental factors (unintact commensale gut microbiota) [118] that can negatively switch the balance between tolerance and acute (therapeutic) inflammation.


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Therefore, novel generation of vaccines [133] could be effective when combined with other therapies (e.g., inflammatory cytokines, immune-checkpoint inhibitors, chemotherapy, or radiation therapy, see combination therapy section), with the goal to overcome the barriers listed earlier. Vaccination could be even more successful when used in patients with low tumor burden (i.e., adjuvant setting), or in selected patients particularly sensitive to immune-manipulation (i.e., those bearing specific polymorphisms). Only very few vaccination strategies have shown promising results in Phase II trials and are currently being tested in Phase III randomized trials (in addition to gp100 vaccine plus IL-2 [134], see combination therapy). In a Phase II trial on stage III/IV M1a melanoma, MAGE-A3 vaccine combined with AS15 immunostimulant (a mixture of QS21 saponin, TLR4, and TLR9 agonist) induced an OR in 11% of patients (including 2% of CR) and a median OS of 33 months [135]. The advantage of vaccination was even higher in patients bearing an inflammatory gene-signature centered on IRF-1/IFN-g-related transcripts [117,136]. This adjuvant--vaccine combination was selected for the Phase III trial. Unfortunately, it has been recently announced that the study did not meet its first co-primary end point as it did not significantly extend disease-free survival (DFS) when compared to placebo [137]. The result of the other co-primary end point (DFS in patients bearing the inflammatory gene signature identified in Phase II trial) is expected in 2015. In the OPTIM Phase III trials by Kaufman et al. T-VEC, an oncolytic immunotherapy derived from herpes simplex virus type-1 designed to selectively replicate within tumors and to produce GM-CSF to enhance systemic antitumor immune responses, induced OR in 26% of patients, including 11% of CR. According to the interim analysis, there was also a trend in OS favoring vaccination (HR: 0.79; vaccination vs GM-CSF alone) [138,139]. Allovectin-7 (a plasmid--lipid complex containing the DNA sequences encoding HLA-B7 and b2 microglobulin) in combination with dacarbazine [140], and M-VAX (an autologous hapten-modified vaccine), alone or in combination with IL-2 (NCT00477906), represent the other two vaccination strategies that are currently being evaluated in Phase III trials [141]. Notably, there is an increasing interest for testing nanoparticle as vaccine adjuvant [142]. Functionalizing carbon nanotubes are particularly promising in view of their theranostic (i.e., both diagnostic and therapeutic) potential. In fact, functionalized carbon nanotubes can stimulate monocytes to release pro-inflammatory cytokines and chemokines associated with T helper 1 polarization and immune-mediated tumor rejection [143]. Moreover, because of their high echogenicity these nanotubes could be potentially used as ultrasound contrast agents [144]. An emerging and promising approach to generate tumorspecific T cells is represented by chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T cells. Studies in B-cell hematological malignancies have shown that engineered T cells against CD19+ can mediate deep tumor 10

remission [145]. In melanoma, the administration of modified T cells expressing high avidity anti-gp100 or anti-MART-1 TCR had induced a considerable OR rate (19 -- 30%), with few complete remissions (one patient in the MART-1 trial) [146]. However, patients exhibited destruction of melanocytes in skin, eye, and ear, which also express, although at lower level, these antigens. Similarly, administration of engineered T cells against MAGE-A3 is associated with high incidence of neurotoxicity because of cross reactivity with MAGE proteins expressed by brain [146]. Therefore, it is imperative to select antigens that are not expressed by normal cells and to generate T cells that do not cross-react with epitope within self-antigens [146]. The most promising results have been obtained by targeting NY-ESO (OR in 5/10 melanoma patients, including 2 CR) [146]. Immune checkpoint blockade An extremely promising approach used in clinical settings to counteract the immune escape mechanisms mounted by cancer cells is the inhibition of the CTLA-4 (Figure 3) [147]. CTLA-4 is a key player in establishing immune tolerance and one of the main regulators of T-cell-mediated antitumor immune responses. The major function of CTLA-4 is to modulate T cells at the time of their initial response to antigen [148]. In fact, although CTLA-4 is expressed by activated CD8+ effector T cells, its key role relies in the regulation of T CD4+ populations through down modulation of helper T-cell activity and enhancement of regulatory T-cell immunosuppressive function [147,148]. The possible role of CTLA-4 blockage in clinical setting was proposed in 1995 by Allison and Krummel [149,150]. It is now clear that antibodies binding the extracellular domain of CTLA-4 can, therefore, block its inhibitory signals and enhance the immune system to fight against cancer cells. Two anti-CTLA-4 monoclonal antibodies, that is, ipilimumab and tremelimumab, are currently being applied in metastatic melanoma setting. The former has been approved by FDA in March 2011. Ipilimumab is a fully human IgG1 monoclonal antibody. Results of ipilimumab trials have boldly increased the enthusiasm in the field of cancer immunotherapy and deserve detailed description. Two Phase III trials have conclusively demonstrated its efficacy in previously treated (ipilimumab monotherapy, MDX010-020 trial) [17], or naive (ipilimumab+dacrabazine, CA184-024 trial) [22] advanced melanoma patients. In the MDX010-20, ipilimumab showed to increase OS, 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 with the time. The 1- and 2-year OS were 46 and 24% in ipilimumab arm, and 25 and 14% in the vaccine arm, respectively [17]. In the CA184-024 study median OS was 11.1 months in the dacarbazine-ipilimumab arm, and 9.1 months in dacarbazine alone arm [22]. A recent follow-up analysis of this latter trial highlights the progressive benefit in OS associated with 4.3




ipilimumab. The 1-, 2-, 3- and 4-year OS were, respectively, 48, 29, 21, and 19% in the ipilimumab-dacrabazine arm and 36, 18, 12 and 10% in the dacarbazine arm [151]. Therefore, ipilimumab almost doubles the 4-year survival rate. Although responses were few in number, they were more likely to occur in patients treated with ipilimumab. As for the MDX010-20the OR rate was 11% (including 1.5% of CR) in the ipilimumab arm, and 1.5% in the gp100 arm. No CRs were observed in the gp100 alone arm [17]. As for CA184-024, the OR rate was 15% (including 1.5% of CR) in the ipilimumabdacarbazine arm and 10% (including 1% of CR) in the dacarbazine alone arm [22]. Even though 15% of OR rate is not particularly high, the responses were more durable in patients treated with ipilimumab (OR duration: 19 months vs 8 months, dacarbazine plus ipilimumab vs dacarbazine) [22]. Similar (although less pronounced) differences were observed in term of PFS in the two studies. The continuous follow-up of patients enrolled in three Phase II trials also confirm the long-term benefit of ipilimumab, with a 5-year OS ranging from 12 to 28% and 38 to 50% for previously treated and naı¨ve patients [152,153]. These results should, however, be taken with caution, being derived from early Phase trials. However, the US ipilimumab expanded access program, which prospectively surveyed the activity and safety of ipilimumab in > 800 (pre-treated) patients, showed a 2- and 3-year OS rate of 22 and 17%, respectively [154]. Remarkably, these results are very close to those obtained in Phase III trials. According to Phase II trials, the combinations of ipilimumab plus fotemustine (NIBIT-M1 trial) [155], and ipilimumab plus temozolomide [156] seem also to be promising. To evaluate the response to treatment, the NIBIT-M1 trial was the first to employ the immune-related criteria, instead than the classic WHO or RECIST criteria for solid tumors. In fact, the onset of clinical response is often delayed, leading to problems in applying classic criteria of response. Response can be achieved after stabilization or even after progression, and CR can occur years after an initial remission. These observations led to define new criteria (immune-related response criteria; irRC) for the definition of response in immunotherapeutic trials [157]. Tremelimumab is a fully human IgG2 monoclonal antibody. The G2 isotype was chosen to diminish complement activation and cytokine storm. A recent Phase III trials failed to show a significant advantage in terms of OS when compared standard chemotherapy (dacarbazine or temozolamide) in treatment naı¨ve advanced melanoma patients (median OS: 12.6 vs 10.7 months, p = 0.13) [158]. Response rate was also similar in the two arms (11 vs 10%, tremelimumab vs chemotherapy, respectively) although duration was significantly longer after tremelimumab (35.8 vs 13.7 months, p = 0.001). However, tremelimumab survival curves are similar to those obtained in the Phase III ipilimumab trials in the same patient setting. The 2- and 3-year OS were 26 and 21% in patients treated with tremelimumab and 23 and 17% in patients treated with chemotherapy. The failure to detect significant differences could 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 proportion of patients randomized to receive chemotherapy likely received ipilimumab at progression. During trial conduct, in fact, ipilimumab became widely available for patients randomized in the control arm [158]. Dosage and schedules could also have influenced the results. Nevertheless, promising results are obtained in Phase I/II trial, in pretreated melanoma [159,160] and mesothelioma patients [161]. Overall, we believe there is still room for this drug in metastatic melanoma setting. One of the main limitations of the CTLA-4 blockades is that immune response takes time to be effective and about 70% of patients progress within the first 6 months [17,22]. Common immunerelated adverse event include colitis, hepatitis, and endochrinopaties, and can be life threatening in a small but considerable proportion of patients. In view of the toxic profile, another limitation of this approach is that so far there are no tools allowing to define with confidence the patients who will benefit from this approach. Another critical immune checkpoint is represented by PD-1. Four anti-PD1 and 3 anti-PD1 ligands mAbs are currently in clinical development (Figure 3, Table 1). PD-1 is induced once T cells become activated. The major function of PD1 is to repress the activity of effector T cells in peripheral tissues to limit autoimmunity or collateral damages in response to infections [148]. PD1 has two known ligands, PD-L1 (B7-H1) and PDL-2 (B7-DC) [162,163], which are expressed by tumor cells to avoid T-cell mediated lysis. Results of large Phase I/II trials of two anti-PD1 mAbs (nivolumab/BMS-936558 and lambrolizumab/MK-3475) and anti-PD-1 ligand mAbs (BMS-936559 and MPDL3280A) are extremely encouraging. If paradigmatic pattern of response of CTLA-4 (delayed and slow effect on tumor shrinkage and prolonged OS) in a small proportion of patients have forced us to define (or redefine) mechanism of action of immunotherapy, recent results of Phase I/II anti-PD-1 and anti-PD-1 ligand trials have shown once again that pattern of response among different immune manipulations could be extremely different. Surprisingly, anti-PD-1 or anti-PD-1 mAb induce tumor shrinkage in most patients with advanced melanoma, lung, and kidney cancers [164-166]. OR rate in melanoma cohorts were 31, 38, 17, and 29% for nivolumab [165,167], lambrolizumab [166], BMS-936559 [164], and MPDL3280A [168], respectively. Tumor shrinkage was already evident 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 much lower as compared with that observed in ipilimumab trials. Although the magnitude of the clinical benefit needs to be assessed through (undergoing) randomized trials and longer follow-up, the pattern of response and the (favorable) safety profile of anti-PD1/PD-1 ligands appear to be quite unique. Importantly, PD-1/PD-1 blockage was effective in patients already previously treated with ipilimumab, therefore,


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Macrophage

Clinical development status T cell

Dendritic cell MHC-II

BMS-986016 LAG-3 IMP-321 Ipilimumab CTLA-4 Tremelimumab

CD80/CD86


R R

Phase III trials

CD40L

Phase II trials R

CD137 Urelumab

CD137L

R

R

FDA approved

R CP-870,893 Dacetuzumab CD40 Chi Lob 7/4 Lucatumumab

GITRL

GITR

B7-H3

?

BMS-936559 AMP-224 R MEDI4736

R

R R

OX40 Anti-OX40

OX40L

R MGA271

R

TRX518

Nivolumab PD-1 Lambrolizumab MPDL3280A CT-011

PD-L1/PD-L2

R

R R

R

Phase I trials Recruiting trials

Stimulatory signal Inhibitory signal

R R R

Tumor

Figure 3. Immune-checkpoints, co-stimulatory signaling, and clinical development of immune-targeted therapy. Stimulatory signaling is in green, inhibitory signaling is in red. The clinical development status is represented by colored rectangles and refers 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 details in Table 1). Represented expression of ligands and receptors should not be considered exhaustive: for example, PD1L-PD2L are also expressed by antigen presenting cells as macrophages and dendritic cells (not shown in the figure); MHC-II is also expressed by dendritic cells and can be expressed by melanoma cells as well. CTLA-4: Cytotoxic T-lymphocyte antigen 4.

confirming that CTLA-4 and PD-1 immune-suppressive pathways are not redundant. Besides the co-inhibitory interactions described earlier, costimulatory signaling between antigen presenting cells (APC) and T cells could also be targeted to enhance antitumor immunity. A schematic representation of the clinical development of immune-checkpoint inhibitors and co-stimulatory molecules in melanoma is provided in Figure 3. The status of the corresponding clinical trials (updated as of January 2014) is provided in Table 1. Agonist antibodies aimed at enhancing T and NK functions (e.g., CD40, CD137 (4-1BB), anti-CD134 (OX40), and glucocorticoid-induced TNF receptor (GITR)) are being evaluated in early phase trials [169]. Perhaps, the most clinically relevant examples in this regard come from the 12

interactions between OX40 and OX40L and between CD40 and CD40L. OX40 (also known as CD134) is a TNF-receptor family member, which is transiently expressed by T cells upon the binding to the TCR. The engagement of OX40 promotes a vast spectrum of T-cell responses, including increased proliferation, cytokine production, and increased differentiation and 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 relevant approach for autoimmunity and cancer [171]. Preclinical studies have shown that ligation of OX40 via agonist antiOX40 mAB or OX40L-Ig fusion proteins can induce robust T-cell-mediated antitumor immunity [172,173]. In a recent Phase I trial [174], Curti and colleagues showed that the



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 nivolumab recruiting (NCT01968109) Phase I/II trial in combination with vaccine in melanoma recruiting (NCT01308294) FDA approved for melanoma; two positive Phase III trials (published) in previously treated melanoma patients as monotherapy versus combination with gp100 vaccine versus gp100 vaccine (NCT00094653) and in naı¨ve patients in combination with dacarbazine versus dacarbazine completed (NCT00324155); Phase III trials in melanoma as monotherapy versus combination with nivolumab versus nivolumab (NCT01844505) or versus lambrolizumab (NCT01866319) recruiting; Phase III trials in small-cell lung cancer (NCT0145076) and non-small-cell lung cancer (NCT01285609) in combination with chemotherapy versus chemotherapy alone recruiting; enrollment completed in a Phase III trial in prostate cancer versus placebo (NCT01057810); > 60 Phase I/II trials evaluating combinations with targeted therapy, radiotherapy, or various immunotherapies in melanoma and in other cancers are currently recruiting Negative Phase III trial in melanoma versus chemotherapy published (NCT00257205); Phase I trial in melanoma in combination with the anti-CD40 mAb CP-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 tumors in combination with MEDI4736 (NCT01975831) recruiting; Phase I trial in combination with chemoembolization or ablation in liver cancer recruiting (NCT01853618) Encouraging results from a Phase I trial in multiple cancers recently published (NCT01644968); Phase II trial in melanoma as monotherapy (NCT01416844) withdrawn prior to enrollment; Phase I/II in combination with ipilimumab (NCT01689870) recently suspended; Phase I/II trials in breast cancer patients with lung metastases in combination with radiotherapy (NCT01862900) and in prostate cancer in combination with radiotherapy and cyclophosphamide recruiting (NCT01303705) Phase I trial in melanoma in combination with tremelimumab, recruiting (NCT01103635); Phase I trial in melanoma in combination with vaccination ongoing but not recruiting (NCT01008527); Phase I trial in pancreatic cancer in combination with chemotherapy recruiting (NCT01456585); two Phase I trials in solid tumors (NCT00607048) and in pancreatic cancer (NCT00711191) in combination with chemotherapy completed and listed as ‘having results’ Phase I/II trial completed in leukemia patients (NCT00283101); 6 Phase I and 2 Phase II trials completed in multiple myeloma and non-Hodgkin’s lymphoma A Phase I trial in multiple cancers ongoing but not recruiting (NCT01561911) Phase I trial for follicular lymphoma patients in combination with bendamustine completed (NCT01275209) Phase II trial in melanoma completed (NCT00612664), Phase I trial in melanoma in combination with ipilimumab withdrawn prior to enrollment (NCT00803374); Phase I trials in non-Hodgkin’s lymphoma recruiting (NCT01471210; NCT01775631); Phase I trials in advanced tumors (NCT00351325, NCT00309023) and non-small-cell lung cancer (NCT00461110) terminated Phase I trial in melanoma and other solid tumors recruiting (NCT01239134) Phase I trials in melanoma (NCT01918930) and in melanoma and other solid tumors (NCT01391143) recruiting 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 versus dacarbazine (NCT01721772) or versus combination with ipilimumab versus ipilimumab (NCT01844505) recruiting; Phase II trials in melanoma as monotherapy versus combination with ipilimumab versus ipilimumab (NCT01927419) or given sequentially with ipilimumab recruiting (NCT01783938); Phase I trial in solid tumors in combination with anti-KIR (NCT01714739), IL-21 (NCT01629758), and the anti-LAG1 mAb BMS-986016 (NCT01968109) recruiting; Phase I trials assessing combination with ipilimumab in melanoma (NCT01024231) and other combinations in other solid or liquid tumors recruiting


IMP321 (anti-LAG3) IPILIMUMAB (anti-CTLA-4)

TREMELIMUMAB (anti-CTLA-4)

Anti-OX40 (OX40 agonist)

CP-870,893 (CD40 agonist)

Dacetuzumab (SGN-40 or huS2C6; anti-CD-40) Chi Lob 7/4 (anti-CD40) Lucatumumab (HCD122;anti-CD40) Urelumab (BMS-663513; anti-CD137)

TRX518 (anti-GITR) MGA271 (anti-BT-H3) Nivolumab (BMS-936558/MDX-1106; Anti-PD1)

*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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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 lung cancer versus docetaxel recruiting (NCT01905657); Phase II trial in tumor with microsatellite instability recruiting (NCT01876511); positive results of the melanoma cohort of the NCT01295827 study recently published Phase I trial in melanoma or hematologic malignancies (NCT01375842), melanoma (combination with vemurafenib, NCT01656642), or in solid tumors in combination with bevacizumab or chemotherapy (NCT01633970) recruiting; several Phase I-II trials and one Phase III trial in lung cancer recruiting (NCT02008227) Phase II trial in melanoma completed (NCT01435369); Phase II trials in prostate, renal, pancreatic cancer and hematological malignancies in combination with chemotherapy or vaccine recruiting; other Phase I and Phase II trials in gastrointestinal tumors and hematological malignancies recently concluded Phase I trials in melanoma (NCT01455103) and hematological malignancies (NCT01452334) withdrawn before enrollment; a Phase I trial in solid cancer is ongoing but not recruiting (NCT00729664) Phase I trial in solid cancers ongoing but not recruiting (NCT01352884) Phase I/II trial in melanoma in combination with dabrafenib or dabrafenib and trametinib recruiting (NCT02027961); Phase I trial in japanese patients with solid tumors recruiting (NCT01938612); Phase I trial in solid tumors recruiting (NCT01693562); Phase I trial in combination with tremelimumab in solid tumors (NCT01975831) or in non-small-lung cancer recruiting (NCT02000947)


MPDL3280A (anti-PD1)

CT-011 (anti-PD1)

BMS-936559 (MDX-1105; anti-PD-L1)

AMP-224 (anti-PD-L2) MEDI4736 (anti-PD-L1)

*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.

administration of an anti-OX40 mouse mAB in patients with advanced cancer had an acceptable toxicity profile and was able to induce regression of at least one metastatic lesion in 12 out of 30 patients. The therapy also increased CD8 T-cell IFN-g production in two of three patients with melanoma. Moreover, in another recent study, Redmond and colleagues [175] demonstrated that the targeting of OX40 in combination with IL2 therapy increased tumor regression in preclinical tumor model and restored the function of anergic tumor-reactive CD8 T cells in mice, leading to enhanced survival. CD40 is a cell-surface molecule, member of the TNFreceptor family, expressed by APC, such as dendritic cells, B cells, and monocytes, but also by many nonimmune cells and a wide range of tumors [169]. The natural ligand of CD40 is CD145, which is expressed on T cells. It has been shown that in tumor-bearing hosts, CD40 agonist trigger effective immune response against tumor-associated antigens [176-178]. Four CD40 agonist mABs have been investigated in clinical trials so far, namely: CP-870,893, dacetuzumab, Chi Lob 7/4, and lucatumumab [169,179]. However, these treatments showed wide heterogeneity ranging from strong agonism (CP-870,893) to antagonism (lucatumumab), suggesting that their mechanism of action might be different. A study employing CP-870,893 as CD40 agonist demonstrated that a single intravenous infusion of CP-870,893 was well tolerated and an objective PR was observed in four patients with metastatic melanoma (14% 14

of all patients and 27% of melanoma patients) [180]. Additional clinical trials employing CD40 agonists are ongoing and results are strongly awaited. Blockage of other inhibitory pathways as the immunosuppressive enzyme indoleamine 2,3-dioxygenas (IDO) seems promising in preclinical study [181], and clinical studies are ongoing (NCT01961115; NCT00739609). Drugs blocking other immune-checkpoints such as T-cell membrane protein 3 (TIM3; inhibitor receptor) and B7-H4 (inhibitory ligand), TGF-b, and KIR inhibitory receptors are under development [148,169]. Notably, trials evaluating blockage of the inhibitory receptor lymphocyte activation genes 3 (LAG3), the inhibitory ligand B7-H3 are ongoing [148,182,183] . Finally, the recent discovery that the manipulation of BACH2 gene can shift the balance between effector and regulatory T-cell response also represents a novel starting point for the development of next-generation immunotherapeutic approaches [184]. Adoptive cell therapy Adoptive cell therapy (ACT) transfer is a form of passive immunotherapy, which consists in the ex vivo expansion of autologous antitumor reactive lymphocytes and their reinfusion into lymphodepleted patients, accompanied by the exogenous administration of IL-2 to further boost their antitumor activity. Lymphodepletion, in fact, seems to favor the antitumor activity of ACT by suppressing regulatory T cells and limiting host tumor immunosuppressive factors. ACT was developed by the group of Steven Rosenberg at the National 4.4




Cancer Institute and has shown encouraging results in Phase II clinical trials. In a recent analysis evaluating 93 heavily pretreated metastatic melanoma patients enrolled in 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 ongoing regression beyond 3 years [185]. A similar OR rate (48%, including 6.5% of CR), measured using immune-related response criteria (irRC), had been obtained by Radvanyi et al. in a Phase II trial enrolling 31 patients [186]. Beyond the toxicity related to HD-IL-2 administration, the main limitation associated to ACT is that TIL expansion requires highly specialized personnel. Using the traditional approach, TILs are generally successfully grown from only 50 to 60% of the patients and require a long period of time (about 5 weeks). Simplified protocols using short-term cultures derive TILs from up to 90% of patients in a shorter period of time (10 -- 18 days) [187]. Rosenberg’s group, however, recently reported a global OR of 28% (CR rate 7%), in randomized Phase II trials comparing two different TIL protocols (young TILs vs CD8 enriched TILs) [188]. By using young TILs Besser et al., in a Phase II trial enrolling 70 patients, reported an encouraging OR of 40% in the evaluated population, including 5 CR [187]. In all the ACT trials responses were durable and patients in CR very rarely relapsed. The results of these Phase II trials need to be taken with caution, and the true response rate needs to be evaluated in randomized Phase III trials. Nevertheless, ACT seems to represent a potential curative therapy for patients with metastatic melanoma [189]. Randomized multicenter trials aimed at evaluating the true response rate and OS advantage of ACT represent the reasonable next step. Despite these encouraging results, the dynamic of TIL migration at tumor site is still not completely clear. Studies where TILs were labeled with Indium 111 showed that TILs migration in the tumor site does not follow a linear kinetic. After re-infusion, which is followed by IL-2 administration, TIL localize in lung, spleen, and liver but not at tumor sites [190,191]. The migration of TILs at tumor site is detectable only after 24 to 48 h. It is possible that this early compartmentalization is mediated by the release (following HD-IL-2 administration) of specific chemokines (i.e., CXCR3 and CCR5 ligands) from resident immune cells and stromal cell in peripheral oragans (e.g., spleen, lung, and liver) [129,192]. Intriguingly, by analyzing a large cohort of patients treated with adoptive therapy and HDIL-2, we recently showed that the downregulation of CXCR3 and CCR5 receptors in TILs correlates with the frequency and the degree of response [129]. This receptor down-modulation is due to the transcript downregulation and/or to the presence of common polymorphism (CCR5 D32, which codify for a nonfunctioning receptor) [129]. It is tempting to hypothesize that a reduced, but not absent, expression of these receptors might prevent the earlier sequestration of TIL by extra-tumoral sites and paradoxically allow their subsequent localization to the tumor

when the cytokine storm has subsided and the tumor represents the only tissue maintaining chemokine secretion [129]. 5.

Combination therapy

Combination therapy is intended as the use of multiple therapies to treat a single disease with the aim to overcome the hurdles of mono-therapeutic strategies [193]. Combining chemotherapy agents represents a pillar of the medical treatment of virtually all the major liquid and solid malignancies, with exception of melanoma and renal cell carcinoma. In these two tumors combination of cytotoxic agents failed to show increase efficacy as compared with monotherapy. In melanoma, > 40 randomized trials have evaluated combination 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 do not increase the OS [21]. The failure of polychemotherapy is in general attributed to the intrinsic chemo-resistance of melanoma. As for first generation immunochemotherapy, it should be noted that, because of safety issues, IFN-a and or IL-2 were administered at low dose, while their maximal antitumor effect is in general achieved using high dosages. Nevertheless, prospective trials have shown that combination of HD-IL-2 and IFN-a does not increase OS, as compared with HD-IL-2 alone [194,195]. This depressing scenario dramatically changed after advent of targeted therapy immune checkpoint inhibitors. Although the mono-targeted therapy and immunotherapy strategies have revolutionized the clinical management of melanoma patients, it is becoming clear that combination strategies are most likely to result in even more significant advances. Building upon the early success of mono-therapy agents in melanoma, trials involving new combination therapy are underway. It has been thought that combining vaccines with anti-CTLA-4 could produce a synergic effect. When ipilimumab was administered together with peptide vaccination (gp100), the combined treatment was more effective (both in term of PFS and OS) than gp100 alone [17]. However, there were no significant differences in both PFS and OS between ipilimumab alone and ipilimumab combined with vaccine [30]. Conversely, gp100 vaccine and IL-2 can act in a synergistic way. In a Phase III randomized trials in metastatic melanoma patients, Schwartzentruber et al. showed that the addition of the gp100 vaccine to HDIL-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 (median PFS 2.2 vs 1.6 months, p = 0.008), with similar trend in OS [134]. Two recent studies showed that efficacy of CTLA-4 can be enhanced by the concomitant administration of immune-stimulant. In a recent randomized Phase II trial Hodi et al. showed that the addition of GM-CSF to ipilimumab increase OS (1 year OS: 68 vs 51%, GM-CSF plus ipilimumab vs ipilimumab alone, p = 0.03) and was also


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associate with less toxicity [196]. Remarkably, long-term follow-up Phase I/II trials in metastatic melanoma patients have suggested that the addition of IL-2 to ipilimumab could dramatically improve the rate of durable CR (CR rate: 17 vs 7%, 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 of simultaneous administration by designing clinical or translation trial aimed at evaluating the effect of immunotherapy in vivo in humans [198]. However, in view of the huge number of potential combinations, novel generations of clinical trials using factorial design are needed [199] to select the more promising combinations. Another possible combination approach is the use of targeted therapy together with immunotherapy [200]. The rationale supporting this type of combination approach relies on the possibility to achieve a deeper and long lasting antitumor effect (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 response 1.5 months). Responses are, however, transient, and the effect of long-term survival is predicted to be very limited (Figure 2A). Conversely, CTLA-4 blockades induce less number of clinical responses (usually delayed), but they are much more durable (Figure 2B). A number of observations support the target-immunotherapy combination. Several cytotoxic agents have been shown to induce antitumor immune responses by promoting cell death with consequent release of tumor-associated antigens [202]. In melanoma, this could be achieved through targeted therapy [203], rather than with conventional chemotherapy. Moreover, constitutive activation of oncogenic pathways, as the BRAFMAPK pathway, can induce several immunosuppressive mechanisms [204,205], including promotion of stromal cell-mediated immunosuppression via induction of IL-1 [7,206]. In addition, BRAF inhibition can enhance melanoma antigens expression and create a more favorable immune environment, facilitating the effectiveness of immunotherapy [89,207-209]. A study evaluating combination of ipilimumab and vemurafenib was object of a Phase I study by Ribas et al. [210]. Although this combination is supported by a strong rationale, investigators noticed an unacceptable hepatotoxicity, which prompted them to close prematurely the study. This example reinforces the need to carefully evaluate the safety of combination approach in appropriate early trials [210]. Very recently, two additional prospective studies in metastatic melanoma have highlighted the potentiality of combining highly effective agents. In a first Phase I/II randomized trial, combination of BRAF inhibitor dabrafenib and trametinib resulted superior to dabrafenib alone [23]. The two drugs combined at full monotherapy dose resulted in 76% of OR (including 9% of CR), while 54% of patients achieved OR in the dabrafenib group (including 4% of CR; p = 0.03). Progression-free survival in the combination was 9.4 months, 16

as compared with 5.8 months in the monotherapy group (p < 0.001). Responses were also more durable with the combination (10.2 vs 5.6). Importantly, > 40% of patients were alive and progression-free at 1 year in the combination arm, as compared with 10% in the monotherapy arm. Combination was also feasible in term of safety [211]. This landmark study demonstrated that the inhibition of the same pathways through highly effective targeted therapy agents (vertical inhibition) could dramatically impact on the course of this aggressive disease, overcoming (at least in part) mechanisms of resistance occurring during monotherapy. Several trials also employing therapies targeting different pathways are underway; examples include vemurafenib (BRAF inhibitor) plus everolimus or temsirolmus (mTOR inhibitors, identifier NCT01596140), MEK162 plus AMG479 (insulin-like growth factor1 receptor inhibitor, identifier NCT015 62899), vemurafenib plus BKM120 (a PI3K inhibitor, identifier NCT01512251). In a second Phase I trial, combination of PD-1 and CTLA-4 blockers (nivolumab and ipilimumab) resulted, at the maximum tolerated dose, in 53% of OR rate, with tumor regression of 80% or more in every patient who had response [212]. The profound effect of concurrent combination was already evident at the first imaging assessment (3 months after treatment initiation). Responses were extremely durable and ongoing in 90% patients who had a response (duration ranging from 6 to 72 weeks at the time of data analysis). This pattern of response (rapid, deep, and durable) appeared to be distinct of that observed in previous immune-checkpoint blockade trials. Importantly, toxicity was qualitatively similar to that of monotherapy [212].

6.

Expert opinion

Although major advances have been made for the treatment of metastatic melanoma, with combination therapies paving the road toward clinical success, much effort needs to be spent in identifying patients who are more likely to respond to a given treatment. It is becoming now clear that patients who are most likely to reject tumors following immunotherapy are the ones who display a constitutive activation of the IFN-g/signal transducers and activators of transcription (STAT)-1/IRF-1 axis (Th1 phenotype) [81,117,121] with IRF-1 playing a central role in this regard [1,117,121,192,213]. Gene signatures predictive of response to immunotherapy (i.e., IL-2-based therapy, vaccines) invariably included genes belonging to the following functional modules: 1) IFN-g/T helper 1 module (STAT-1/IRF-1/IFN-gstimulated 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).




This phenotype is also characterized by the counteractivation of suppressive mechanisms. Therefore, it is not surprising that high expression of IDO and PD-1 ligands, which are both IFN-g inducible genes, have been found to be associated with responsiveness to ipilimumab [214] and anti-PD1 treatment [165]. The determinants of this responsive immune phenotype are object of investigations. Coordinate enhancement of these modules following treatment is associated with responsiveness to IL-2 [103], ipilimumab [215], and other immunotherapies, including vaccines [216] and Toll-like receptor agonists [217]. These observations suggest that, although distinct immunotherapies clearly act in very different ways, their final therapeutic relies on the activation of similar and redundant downstream mechanisms, as recently reviewed elsewhere [121]. It is currently becoming accepted that immune-mediated tumor rejection is mediated by both the genetic background of the host and the genetic make-up of individual cancer cells carrying their own specific mutations [119,120,218]. Yet, the relative weight played by each of those factors is not completely understood as well as their contribution to mediate responsiveness to a given cancer therapy. The relevance of the genetic background of the host in mediating tumor rejection has been recently witnessed by two studies from our group. In the first one, we hypothesized that polymorphisms of IRF5 gene, known to be implicated in autoimmune diseases and in particular to systemic lupus erythematosus, might play a role in the immune-mediated rejection of the tumor. Concordant to our hypothesis, we were able to show that the IRF5 genetic variants protecting against the development of autoimmunity were highly predictive of nonresponsiveness to adoptive therapy in melanoma [131]. These results support the link between autoimmunity and immune responsiveness. In the second study, we assessed the prognostic relevance of genetic variations of CTLA-4 and HLA polymorphisms in melanoma patients who received adjuvant IFN-a [132]. Multivariate analysis revealed that a five-marker genotyping signature based on HLA-B38, HLA-C15, HLA-C3 DRB1*15, and CTLA-4 CT60* polymorphisms, was predictive of OS in 284 melanoma patients. The findings of these two studies strengthen the relevance of inherited genetic variations in mediating tumor rejection induced by immunotherapy regimens.

Aside from inherited genetic variations, somatic mutations acquired during tumor development are likely to influence tumor rejection. The most evident proof that the genetic background of individual cancer cells plays a role in mediating cancer rejection comes from the observation of mixed responses, a rare phenomenon where multiple metastases from the same patient respond differently to a given therapy. By comparing mixed metastases from two melanoma patients who received immunotherapy, we were able to show that metastases who responded to the treatment were the ones who had upregulated genes associated with antigen presentation and IFN-mediated rejection [216]. These findings demonstrate that tumors sharing the same host’s genetic background can behave differently depending on the differential expression of genes involved in triggering an IFN-mediated immune signaling. It should be also noted that cancer cells share the same genetic background of the host, thus, tumor behavior is concurrently influenced by inherited variations other than somatic mutations. Environmental factors also play a role in this regard, and evidence accumulated in the recent years show that it is a combination of all the three categories that affect tumor rejection rather than each single one alone [218]. Understanding the mechanisms underlying tumor rejection is essential to implement personalized therapies. We believe that this goal is reachable only by understanding the complex interplay between the genetic background of the host, the genetic make-up of individual cancer cells, and environmental factors. The availability of highthroughput technologies allowed the investigators to start the race toward cancer-personalized medicine. We believe that, in the future, the integration of the ‘omic’ research approaches will allow a more potent understanding of the algorithm governing tumor rejection and responsiveness to a given therapy and lead to improved clinical management of melanoma patients.

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


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Affiliation Sara Tomei†1,2, Ena Wang1,3, Lucia Gemma Delogu4, Francesco M Marincola1,3 & Davide Bedognetti*1 †,* Authors for correspondence 1 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 20892, USA E-mail: [email protected] 2 Weill Cornell Medical College in Qatar, Department of Genetic Medicine, P.O. Box 24144, Doha, Qatar E-mail: [email protected] 3 Sidra Medical and Research Center, Research Branch, P.O. Box 26999, Doha, Qatar 4 Universita` degli Studi di Sassari, Dipartimento di Chimica e Farmacia, 07100 Sassari, Italy