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1. Introduction

Skin cancers are among the most common cancers diagnosed in the United States. The incidence of melanoma of the skin has risen in the last three decades and although melanoma accounts for approximately 1% of all skin cancers it causes most of the skin cancer deaths (1). When melanoma is diagnosed in its early stages, surgical resection of the lesion is associated with favorable prognosis. However, for locally advanced and metastatic disease surgery is no longer sufficient. The 5-year survival for localized melanoma is 99%, but it is 20% when distant metastases are present (1).

Melanoma is one of the most sensitive tumors to immune modulation. Several factors may explain melanoma cell susceptibility to immune system activation including high tumor mutational load due to ultraviolet light exposure, expression of cancer testis antigens and mimicry of melanocyte lineage proteins with pathogen-associated antigens (3-5). In this context T-cell response seems to play a central role to keep the melanoma at bay. Tumor infiltrated lymphocytes (TILs) are central to the development of an anti-tumor immune response and a subset of TILs demonstrate cytolytic activity against autologous tumors in melanoma patients (Fig. 1) (6). Their presence also correlates with increased survival and reduced risk of metastasis (6). In the past decades several clinical trials aimed at eliciting T-cell response with local or systemic immunomodulatory drugs such as interferon (IFN)-α (7,8), interleukin (IL)-2 (9,10), cancer vaccines (11,12) and adoptive cell transfer (13). Despite some evidence of activity, these trials failed to demonstrate sustained benefit in metastatic melanoma patients. More recently, immune checkpoint inhibitors (ICIs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) have dramatically changed the management of both unresectable and metastatic melanoma as well as those at high risk for recurrence after resection (Table I) (14-16). Unfortunately, primary and secondary resistance and the absence of predictive markers of response are challenging problems with ICIs therapy (17). Combination of immunotherapy strategies aim to improve response and overcome resistance, while biomarker discovery is fundamental for the optimization of patient selection.

Figure 1 The cancer immunity cycle.

Table I Landmark clinical trials of immunotherapy in locally advanced and metastatic melanoma.

Table I

Landmark clinical trials of immunotherapy in locally advanced and metastatic melanoma.

Trial name	Primary outcome	Treatment arms	ORR (%)	Median PFS (months)	Median OS (months)	1yr-RFS (%)
CA184-002 (20)	OS	gp100 vaccine	1.5	2.8	6.4	-
		gp100 vaccine+ipilimumab	5.7	2.8	10.0	-
		ipilimumab	11.0	2.9	10.1	-
CA184-024	OS	Dacarbazine	10.3	3.0	9.1	-
		Dacarbazine+ipilimumab	15.2	3.0	11.2	-
CheckMate 066 (32)	OS	Dacarbazine	14.4	2.2	11.2	-
		Nivolumab	42.9	5.1	37.5	-
KEYNOTE-006 (28,30)	PFS, OS	Ipilimumab	11.9	3.4	16.0	-
		Pembrolizumab q2w	33.7	5.6	32.7a -
		Pembrolizumab q3w	32.9	4.1
CheckMate 067 (37)	PFS, OS	Ipilimumab	19.0	2.9	19.9	-
		Nivolumab	45.0	6.9	36.9	-
		Nivolumab+ipilimumab	58.0	11.5	NR	-
OPTiM (46)	Durable response lasting ≥6 months	GM-CSF	Not reported	Not reported	18.9	-
		T-VEC	Not reported	Not reported	23.3	-
CheckMate 238 (34)	RFS	Nivolumab	-	-	-	70.5
		Ipilimumab	-	-	-	60.8
EORTC1325/ KEYNOTE-054 (35)	RFS	Pembrolizumab	-	-	-	75.4
		Placebo	-	-	-	61.0

{ label (or @symbol) needed for fn[@id='tfn1-ijo-57-03-0609'] } ORR, overall response rate; OS, overall survival; PFS, progression-free survival; RFS, relapse-free survival; NR, not reached; q2w, every two weeks; q3w, every three weeks.

a Pooled data from the pembrolizumab arms.

Herein, we review and discuss the rational of approved immunotherapy treatments, of preclinical data and ongoing clinical trials of combination strategies for advanced stage and metastatic melanoma. An overview is also provided of biomarker discovery and evidence on the role of gut microbioma in melanoma immunotherapy.

2. CTLA-4 blockade

CTLA-4 is an inhibitory checkpoint receptor that blocks T-cell activation and induces immune suppression (Fig. 2) (18). In 1996 Allison and colleagues showed that CTLA-4 blockade could attenuate the growth of several implanted murine tumors (19). In 2011 ipilimumab, a fully human monoclonal antibody (mAb) IgG1 that inhibits the interaction between CTLA-4 and its ligands, was the first ICI approved by the FDA. In previously treated patients with advanced melanoma, ipili-mumab improved median overall survival (OS) compared with gp100 peptide vaccine (10.6 vs. 6.4 months) (20). At 3-years, the survival rate was 22% followed by a plateau of the survival curve for up to 10 years (21). Combination strategies of ipilimumab with IL-2 or Peg-IFN failed to show any improvement over ipilimumab monotherapy (22,23). Tremelimumab, another monoclonal antibody targeting CTLA-4, failed to demonstrate a survival benefit over standard chemotherapy in a phase III clinical trial (24). In the adjuvant setting, a randomized clinical trial in resected stage III patients showed that ipilimumab improves relapse-free survival (RFS) and OS compared to placebo. However, more than 50% or patients experienced grade 3-4 adverse events with ipilimumab and 5 patients (1.1%) died due to immune related adverse events (25).

Figure 2 Immune checkpoints and their inhibitors in advanced melanoma. PD, programmed death; TIM-3, T-cell immunoglobulin and mucin-domain containing-3; LAG3, lymphocyte activation gene 3; TCR, T cell receptor; CTLA-4, cytotoxic T-lymphocyte antigen-4.

3. PD-1 blockade

PD-1 is an immune checkpoint with a central role in immunopathology and tumor immune surveillance through effector T-cell inhibition (Fig. 2) (18). In 2014, two mAbs targeting PD-1 (nivolumab and pembrolizumab) received FDA approval, becoming first line treatment option in metastatic melanoma. Randomized clinical trials have shown that monotherapy with nivolumab or pembrolizumab is superior to ipilimumab alone (26). Pembrolizumab monotherapy in treatment naïve and previously treated patients showed sustained response rates of 30-40% (26-28). In treatment of naïve patients pembrolizumab showed a 3-year OS rate of 51% and a 5-year rate of 41% (29,30). Clinical trials of nivolumab monotherapy provided durable response rate of 32% in untreated patients and 40% in previously treated melanoma (16,31). Three-year survival rate for nivolumab in previously untreated patients is 42% (32) while 5-year survival rate in previously treated patients with nivolumab monotherapy is 35% (15). Cross study comparisons of homogeneous groups of patients treated with pembrolizumab or nivolumab monotherapy have similar results regarding clinical endpoints and adverse event rates (33). At this time, no good predictive biomarkers for anti-PD-1 mAbs are available and clinical benefit is reached regardless of PD-L1 status (32). Since 2017, adjuvant immunotherapy with single agent anti-PD-1 mAb is the first treatment option in patients with resected stage III disease. Nivolumab improved RFS compared to ipilimumab with lower toxicities (34). In a phase 3 double-blind trial pembrolizumab treatment resulted in significantly longer RFS than placebo with no new toxicities compared to other pembrolizumab monotherapy trials (35).

4. Immune checkpoint inhibitor combinations

Monotherapy with ICIs is associated with significant improvement in patient survival, however, response rates are low. With the attempt to increase the number of patients who benefit from ICI therapy, combination of anti-CTLA-4 mAb plus anti-PD-1 mAb have been evaluated in prospective clinical trials. In particular, two randomized trials showed that the combination of anti-CTLA-4 plus anti-PD-1 mAbs results in increased clinical benefit compared to single agent ipilimumab or nivolumab. The Checkmate-067, a phase 3 randomized clinical trial compared ipilimumab plus nivolumab to nivolumab alone and ipilimumab alone in unresectable/metastatic melanoma. Response rates were 57.6, 43.7 and 19%, respectively, and 5-year survival rates were 52% in the combination arm, 44% in the nivolumab group and 26% in the ipilimumab arm (36,37). The combination arm showed increased toxicity compared to each monotherapy treatment arm. Specifically, treatment-related adverse events of any grade occurred in 95% of patients in the combination treatment group compared to 82% in the nivolumab arm and 86% in the ipilimumab group. Grade 3 or 4 treatment related adverse events occurred in 55% of the patients in the nivolumab plus ipilimumab group, 16.3% of the patients in the nivolumab group and 27.3% of those in the ipilimumab group (36). In 2015 the FDA approved the combination ipilimumab plus nivolumab on the basis of overall response rate and progression-free survival (PFS) improvement. In an attempt of reducing the toxicity burden of the combination, different dosing schedule by reducing ipilimumab dose and keep more standard dose anti-PD-1 single agents were investigated. Although the ORR appears to be conserved, the expected difference in terms of efficacy and safety are small and only results from larger trial will be conclusive (38,39). In patients with primary or secondary resistance to single agent PD-1 mAbs, ICIs combination or ipilimumab alone represent potential treatment strategies (40,41). Currently, the respective benefits of combination immunotherapy versus sequential immunotherapy are not yet fully understood and is object of an open debate in the clinical and scientific community. On one hand combination strategies are associated with increased adverse events that can be justified by the intent of gaining long-term disease response. On the other hand, the subset of patients who benefit more from the combination is unknow potentially exposing patients to unnecessary toxicities (42).

Brain metastases are a common cause of disabling neurologic complications and poor prognosis in patients with metastatic melanoma. The phase 2 clinical trial CheckMate-204 enrolled patients with small, untreated and asymptomatic brain metastasis and showed that ipilimumab plus nivolumab have clinically meaningful intracranial efficacy (56% of intracranial response). The safety profile was similar to those reported for the combination in patients without brain metastasis (43). Another phase 2 clinical trial compared the combination of nivolumab plus ipilimumab versus nivolumab alone. Despite the small sample size, ICIs combination was superior to nivolumab monotherapy with a higher proportion of patients achieving intracranial response (44).

No data are yet available on the potential benefit of anti-CTLA-4 plus anti-PD-1 combination in the adjuvant setting. Results from the clinical trial CheckMate-915 (NCT03068455) that compare nivolumab monotherapy to nivolumab plus ipilimumab in resected stage III melanoma are awaited.

5. Talimogene laherparepvec (T-VEC)

T-VEC is a type I herpes simplex virus genetically modified to preferentially replicate in tumor cells, enhance antigen loading of MHC class I and express granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase tumor antigen presentation by dendritic cells (DCs) (45). In 2015 T-VEC received approval by the FDA for advanced melanoma. A phase 3 trial in unresected stage IIIB-IV melanoma showed that intratumoral administration of T-VEC improved response rate compared with GM-CSF (26 vs. 6%). Few responses were obtained in distant non-injected lesions (mainly lung and visceral sites) while the majority of responses were limited to the site of injection and regional non-injected lesions (46). The combination of T-VEC and ICIs has shown interesting results. The phase II study of T-VEC plus ipilimumab versus ipilimumab alone in patients with advanced melanoma showed an improvement in ORR (39 vs. 18%, respectively) (47). In the phase Ib trial evaluating the association of T-VEC plus pembrolizumab, confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria (48). The results of the phase III trial MASTERKEY-265/KEYNOTE-034 of T-VEC plus pembrolizumab compared with pembrolizumab alone are awaited (NCT02263508).

6. Other immunotherapy strategies

Despite the impressive results obtained with currently approved ICI treatments strategies, primary and secondary resistance represent major clinical challenges. Several promising immunomodulatory targets have been included in ongoing clinical trials mainly in association with approved anti-PD-1 and anti-CTLA-4 mAbs (Fig. 2).

Inhibitors of lymphocyte activation gene-3 (LAG-3) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in clinical development

LAG-3 is an immune checkpoint receptor found on the cell surface of effector T cells and regulatory T cells. It is an inhibitor regulator of T cell response, activation and growth (49). Relatlimab, LAG525, and MK-4280 are mAbs that targets LAG-3 and are currently under investigation in combination with pembrolizumab or nivolumab (50). While the inhibition of LAG-3 alone has minor effect on T-cell reactivation, the combination with anti-PD-1 mAb has shown encouraging results (51,52).

TIM-3 is an immune checkpoint expressed on IFN-γ-producing T cells, FoxP3+ Treg cells and macrophages and DCs where it suppresses their responses upon interaction with their ligands. In vivo blockade of TIM-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models (53). INCAGN02390 is a TIM-3 inhibitor that is undergoing trial for the treatment of advanced solid tumor including melanoma (NCT03652077).

IDO inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme involved in tryptophan catabolism with a central immunosuppressive function within the tumor microenvironment (54). Several IDO inhibitors (indiximod, epacadostat and BMS-986205) are currently evaluated in clinical trials in association with pembrolizumab, nivolumab or ipilimumab (54). Regrettably, the phase III clinical trial ECHO-301/KEYNOTE-252 in advanced melanoma failed to demonstrate PFS benefit in the arm of pembrolizumab with epcadostat compared to pembrolizumab alone (55).

Cytokines

Cytokines are the first class of immunomodula-tory agents that have found clinical application in melanoma. Indeed, IL-2 and IFN-α are both FDA approved for adjuvant treatment in melanoma (7,56). Other cytokines such as IL-12, IL-15, IL-18, IL-21 and GM-CSF have shown interesting results in preclinical and clinical settings. However, single agent cytokine strategy does not appear feasible due to their pleiotropic activity and the critical toxicity profile especially at high dose (56). With this in mind, NTRK-214 is a prodrug of conjugated IL-2, retaining the same amino acid sequence as human recombinant IL-2. The IL-2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains that in vivo slowly release generating active IL-2 conjugates (57). An ongoing phase I/II clinical trial aims to evaluate the tolerability and efficacy of NTRK-214 with nivolumab and ipilimumab plus nivolumab (NCT02983045).

Modulation of the tumor microenvironment and the innate immune system

Tilsotolimod is a synthetic TLR-9 agonist oligonucleotide that acts on macrophages and DCs and can stimulate antigen presentation and T cell activation and proliferation. Intratumoral tilsotolimod in combination with ipilimumab in PD-1 inhibitor refractory metastatic melanoma is well tolerated and shows significant clinical benefit (ORR 38%) and durable response (58). These favorable results have led to an ongoing phase III study of tilsotolimod plus ipilimumab versus ipilimumab alone (NCT03445533). Another intratumoral TLR-9 agonist, SD-101, is in clinical development. The phase Ib/II clinical trial SYNERGY-001/KEYNOTE-184 evaluates the combination of SD-101 and pembrolizumab in patients with unresectable stage IIIC-IV melanoma and naïve to PD-1 axis inhibitors. Preliminary results show that the combination is well-tolerated, with promising high response rates and PFS (59). CD40 is expressed on macrophages and other antigen-presenting cells and its agonists stimulate maturation and increase macrophage killing activity against tumor cells (60). On the other hand, tumor-associated macrophages can be characterized by tumor-promoting phenotype (61). This phenotype is a consequence, among other factors, of the continuous activation of the colony-stimulating factor-1 (CSF-1) axis (62). An ongoing phase I/Ib trial is evaluating the safety and efficacy of the CSF-1 receptor inhibitor, cabiralizumab, combined with the CD40 agonist, APX005M, with or without nivolumab in patients with advanced melanoma (NCT03502330).

Vaccines

Therapeutic cancer vaccines aim at inducing a specific immune response against tumor antigens. In melanoma patients, peptide vaccines have been tested in association with ipilimumab, but failed to demonstrate an advantage compared to ipilimumab alone (20,63). In a phase I clinical trial, tremelimumab plus MART-1 peptide-pulsed DCs resulted in objective and durable tumor responses compared to each agent alone (64). A phase I trial in patients with pretreated advanced melanoma showed that autologous monocyte-derived DCs electroporated with synthetic mRNA (TriMixDC-MEL) are immunogenic and have antitumor activity (65). TriMixDC-MEL combined with ipilimumab has shown 38% of durable tumor responses in a phase II trial (66). The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine uses yeast cell wall particles to load tumor lysate into autologous DCs. The phase IIb trial of TLPLDC vs. placebo in resected stage III/IV patients showed an increased 24-month DFS. The trial showed also a potential synergistic effect of TLPLDC plus ipilimumab to be confirmed in a phase III study evaluating adjuvant TLPLDC plus ipilimumab versus ipilimumab alone in resected stage IV patients (67).

Adoptive T cell transfer

The presence of tumor-reactive T cells has been associated with the success of ICIs (68,69). When patients do not have functional tumor-antigen-specific T cells with high-affinity T cell receptors (TCRs), T cell therapies can transfer such T cell populations by either expanding pre-existing anti-tumor T cells or by using gene-therapy to alter T cells to become melanoma-specific with a high-affinity TCR (70). ACT of autologous tumor infiltrating lymphocytes with high-dose IL-2 was the first clinical trial to show that tumor-reactive T cells could mediate melanoma regression (71). However, TIL has not been approved as anti-cancer treatment yet due to lack of results from sufficiently powered prospective randomized clinical trials (13,72). In order to improve clinical benefit and survival, TIL therapy could be combined with other immunotherapies (70). ACT through chimeric antigen receptor (CAR) T-cell therapy has been successful in hematological malignancies. However, less response was seen in the treatment of solid tumors such as melanoma (73). Combination of CAR-T therapy and immune checkpoint blockade, targeted therapy might induce desired clinical responses (73). The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope is a potentially effective treatment for some refractory metastatic melanoma patients (74).

7. Combination of immune checkpoint inhibitors and targeted therapy

Targeted therapy and ICIs have radically changed the management of different tumor types, including advanced stage melanoma (75). However, both approaches have limitations, including limited duration of response with targeted therapy and low overall response rate without clear predictive biomarkers in patients treated with ICIs. Therefore, great interest has been shown on the possibility of combination strategies that could take advantage from the high response rate of targeted therapy with the long-term disease control of ICIs. Despite some contrasting preclinical results that have been observed on the association of BRAF and MEK inhibitors with immunotherapy, various trials are ongoing investigating the association of MAPK inhibitors with ICIs and other immunotherapy strategies (76). Several trials with CTLA-4 inhibitors and MAPK inhibitors have raised concerns due to the toxicities associated with the combinations that led to early trial termination (77,78). More tolerable and with good disease control rates seem to be the association of PD-1/PD-L1 axis inhibitors with BRAF and MEK inhibitors (79). In this context, study design is fundamental to properly conduct clinical trial of combination strategies without increasing toxicities. The ongoing phase III clinical trial DREAMseq (NCT02224781) studies how well initial treatment works with ipilimumab and nivolumab followed by dabrafenib and trametinib, and compares it to initial treatment with dabrafenib and trametinib followed by ipilimumab and nivolumab in treating patients with stage III-IV BRAF V600 melanoma.

8. Host microbiota and response to immunotherapy

The improvement of clinical efficacy of immunotherapy strategies is a central goal of translational and clinical research in immunooncology. In recent years, several factors have been shown to influence the immune response during ICI treatment (80-83). In this context, the human microbiota seems to be an important modulator of the immune system response in different physiopathological conditions, including cancer development and response to anti-cancer treatment (84). In a melanoma mouse model, different commensal gut microbiota composition was associated with difference in melanoma growth and spontaneous antitumor immunity which were eliminated upon cohousing or after fecal transfer (85). In a RET melanoma mouse model with dysbiosis induced by antibiotic treatment, gut colonization with Akkermansia muciniphila restored responsiveness to PD-1 blockade compared to control and increased the recruitment of CD4+ T lymphocytes into mouse tumor beds (86). In 112 melanoma patients treated with anti-PD-1 mAbs, significant differences were observed in the gut microbiome of responders versus non-responders. Analysis of patient fecal microbiome samples showed significantly higher species diversity and relative abundance of the Ruminococcaceae family in responding patients. Moreover, mice receiving fecal microbiota transplantation (FMT) with stool from responders to PD-1 inhibitors also exhibited improved response to anti-PD-L1 therapy compared to mice transplanted with stool from melanoma patients that did not respond to anti-PD-L1 therapy (87). Several preclinical and clinical works show the relevance of the modulation of the gut microbiota in ICIs efficacy (88-91). Taken together these data sustain potential use of the human microbiome as a predictive biomarker of response to ICIs. Moreover, the modulation of specific component of the human microbiome by FMT or oral supplementation through probiotics may improve response rates and other clinical endpoints of ICIs therapy in cancer patients. Prospective randomized clinical trials are awaited to better understand the impact of such approaches.

9. Immunotherapy biomarkers

Targeted therapy derives its efficacy from the presence of a specific tumor feature, such as the BRAF V600 mutation, that drives tumor growth and that represent a specific biomarker of response to the drug targeting the aberrant pathway. In melanoma, primary and secondary resistance to targeted therapy are challenging problems and several studies have tried to improve BRAF V600 detection of prognostic and predictive markers (92-94). Despite great efforts, melanoma immunotherapy and especially ICIs, which are already approved for clinical use, lack biomarker response. This is especially urgent due to the relatively low response rate of immunotherapy. Regarding inhibitors of PD-1/PD-L1 axis, PD-L1 expression assessed by immunohistochemistry (IHC) staining has been used as biomarkers in several clinical trials. Different PD-L1 IHC antibodies with non-homogeneous cut-off values among studies have generated contrasting results on the role of PD-L1 for patient stratification (26,31,36). Although PD-L1 status is not currently considered a valid stratification marker, it warrants further analysis since it may indicate underlying biological insights (95). More comprehensive models are under investigation to better characterize the tumor microenvironment and define predictive biomarkers for immunotherapy. In this context an important study by Tumeh and collaborators (96) has shown that the presence of CD8+ T cells that cause upregulation of PD-L1 expression on melanoma cells at the invasive tumor margin might more clearly explain primary resistance or responsiveness to anti-PD-1 mAbs. Gene-expression profile has been suggested as a marker of response (97). IFNγ secreted by CD8+ T cells is the principal mediator of intratumoural antitumor inflammation, a gene expression profile termed 'T-cell-inflamed tumor' has been associated with response to diverse immunotherapies including IL-2, vaccines, ICIs and cancer vaccine (98,99). Moreover, primary and secondary resistance to PD-1/PD-L1 pathway inhibitors is associated with a low IFNγ gene expression signature that can be mediated by activation of PTEN and WNT/β-catenin pathway, impairment of JAK2 signaling or alteration of antigen presentation through structural or functional impairment of MHC class I mediated antigen presentation (100-103). A study with the largest whole exome sequencing and transcriptome sequencing analysis of tumor material from patients with metastatic melanoma receiving immune-checkpoint inhibitors has been published (104). The study supports the correlations between baseline immune infiltrate and treatment response, but also show inconsistent associations of tumor mutational burden, and prove that multiple novel genomic and transcriptomic features predict selective response, including features associated with MHC-I and MHC-II antigen presentation (104). Moreover, the authors constructed predictive models integrating clinical, genomic and transcriptomic characteristics to identify patients with melanoma with intrinsic resistance to anti-PD1 mAb (104). Recently, a growing body of evidence is trying to correlate ICI efficacy to the expression levels of PD-L1 detected in circulating tumor DNA (105,106). Finally, studies have proposed the analysis of matrix metalloproteinase (MMPs), known to be strictly involved in melanoma growth (107,108), as good indicator of response to immunotherapy. Moogk and colleagues (109) showed an inverse association between anti-tumor T-cell response and MMP-23 expression in primary melanoma tumors treated with adjuvant immunotherapy. The authors concluded that MMP-23 expression is associated with shorter periods of PFS and therefore may represent a potential therapeutic target in melanoma, as well as a possible biomarker for evaluating response of melanoma patients to immunotherapies.

10. Conclusions

The number of effective treatments for patients with metastatic melanoma have increased rapidly in recent few years. In this scenario, immunotherapy combinations and immunotherapy plus targeted therapy hold great expectation. Moreover, results from the clinical trial DREAMseq are eagerly awaited and will shed light on the best sequential treatment option in metastatic melanoma patients with BRAF V600E. Given the complexity of the antitumor immune response, the identification of biomarkers of response and the mechanisms associated with primary and secondary resistance are of utmost importance. These insights will allow the next generation of immunotherapy approaches that will be tailored on specific melanoma genomic features and its tumor microenvironment characteristics.

Acknowledgments

Not applicable.

Funding

This study was partially supported by the Italian League against Cancer (LILT).

Availability of data and materials

Not applicable

Authors' contributions

GCL conceived the work, performed bibliographic research and wrote the manuscript, SC prepared the figures and provided critical revisions, LF performed bibliographic research and provided critical revisions, DAS, ML contributed to the conception of the work and provided critical revisions. All authors agreed on the final version of the manuscript.

Ethics approval and consent to participate

Not applicable

Patient consent for publication

Not applicable

Competing interest

GCL, SC, LF, ML no competing interest to declare, DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article.

References