FormalPara Key Points

Drugs that can be administered locally via injection into skin cancers have shown promise biochemically and clinically, especially considering the drastic reduction in side effects when compared to systemic treatment.

Many studies suggest that to achieve a lasting, effective response, these treatments should be used in concert with other anti-cancer therapies such as systemic immunotherapy.

Additional clinical and pre-clinical studies are needed to build an appropriate treatment algorithm that addresses patients’ specific tumor microenvironments.

1 Introduction

The last 2 decades have seen astonishing progress in effective treatment for skin cancers. While 20 years ago, chemotherapy was the mainstay of treatment, systemic immune and targeted therapies have transformed the treatment landscape in melanoma and in other skin cancers. Many patients who receive these therapies experience a marked response, but there are still patients who do not benefit or who develop serious immune side effects. An emerging alternative is the use of intralesional therapy as it opens the possibility for immune microenvironment manipulation leading to a local and, theoretically, systemic response. Further, promise may lie in complementing established drugs rather than finding new first-line treatments. Understanding and harnessing abscopal effects of locally administered drugs may present the next leap in the field. In order to reach that jumping point, much more research is needed in the realm of intratumoral therapy. Such research will likely uncover intricacies of the tumor microenvironment and present possibilities for reliable systemic activity following local intervention.

The initial rationale for intratumoral use revolved around the reduced toxicities observed when compared to using the same agents systemically [1]. While surgery alone is considered the standard of care for primary melanoma and for other skin cancers, it can be morbid and ineffective on a patient with undiscovered in-transit or metastatic disease. Currently, intralesional therapy is used on occasion for patients with unresectable disease when accessible lesions are available. However, some data suggest that indications exist for these therapies in a first-line setting in selected patients as well where toxicity concerns may be present [2].

2 Intralesional Therapy Trends Over Time

2.1 Intralesional Chemotherapy

The accessibility of skin cancer primaries and metastases on the skin naturally led to many attempts to treat these by directly applying antineoplastic agents. Isolated limb perfusion created a bridge between traditional chemotherapy and intratumoral therapy, and the goal was straightforward: destroy tumor cells while minimizing side effects. Approaches have since become increasingly local, abandoning the reliance on the venous system for drug delivery in favor of intralesional injection. Antineoplastic or cytotoxic drugs were default treatments for any cancer to begin with, and as such were the default for studying intratumoral therapy. An agent still under study is 10% Rose bengal, or PV-10. Initial experiments in melanoma mouse models with injected PV-10 demonstrated an immune-recruiting response, accumulating in lysosomes of nearby tissues and leading to enzymatic cell death [3, 4]. The exact mechanism behind the uptake of PV-10 by tumor cells is not well understood, and more research is needed to determine its full potential. Meanwhile, PV-10 has since been studied intralesionally in vivo in clinical trials and demonstrates dose-dependent responses and modest bystander effects, showing fidelity to the murine and in vitro models thus far [5, 6]. However, the quality of this response is unclear; for example, a phase II intralesional PV-10 study in metastatic melanoma encountered a median lesion-based response duration of 4.0 months [6]. For now, long-term follow-up data on clinical trial patients is limited, but such short-lived responses call into question the utility of pursuing further study of this agent.

For agents which do not efficiently affect tumor cells by injection alone, electrochemotherapy (ECT) was developed. ECT is a delivery method in which the patient’s tumors are injected with cytotoxic agents, such as bleomycin or cisplatin, and shock is applied intralesionally to force permeability of local cell membranes, allowing drug uptake and subsequent cell death [7, 8]. Results have even been demonstrated in cutaneous metastases of breast cancer and melanoma using calcium flux [9]. A meta-analysis of ECT studies in melanoma completed to date demonstrates that ECT generated an objective response rate (ORR) of 74% from 502 patients [10]. While this result is impressive, it is based on twelve studies with no staging stratification, and six of the 12 studies had no intratumoral injection component. The authors aptly conclude that further investigation is needed to establish more robust data. However, a case can certainly be made for diverting such efforts away from chemotherapy. Upon reviewing trends in skin cancer patient responses to chemotherapy compared to immunotherapy, immunotherapy is an improvement to chemotherapy in every comparison [11]. This observation calls into question the reason for continuing to study and develop chemotherapy-based treatment approaches, especially in the context of metastatic disease.

2.2 Oncolytic Virotherapy

The next step in increasing the tumor-targeting potential of local therapy is using oncoloytic viruses. Viruses have proved useful in local cancer therapy for three important reasons: specificity, proliferation, and cargo carrying capacity. Once the tumor is targeted, viral proliferation lyses the tumor cells, and DNA transduced by the virus further impacts the surrounding microenvironment. Modified viruses are capable of delivering a two-pronged attack to cancer cells and their environment. Replication-competent viruses rapidly proliferate in tumor cells until the cells lyse. At the same time, these viruses can carry additional genetic material, upregulating proteins to either inhibit tumor growth or recruit surrounding immune cells. Using a viral vector to deliver DNA into tumor cells has arguably been the most successful example to date, as evidenced by the Food and Drug Administration (FDA) approval of talimogene laherparepvec (T-VEC), which uses a modified herpes simplex virus (HSV) as a vector to induce cell lysis and deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) in melanoma (Fig. 1). Modification to HSV prevents it from leading to HSV outbreaks. Still, there was initially concern regarding whether this drug’s administration posed danger to medical practitioners and surrounding patients via exposure to HSV. A phase II clinical trial, NCT02014441, analyzed blood and urine samples from melanoma patients undergoing T-VEC therapy in order to elucidate the shedding patterns [12]. The results showed T-VEC activity in treated patients and included a plethora of secondary outcome results, which taken together with results from a phase I trial of intratumoral T-VEC in melanoma patients helped demonstrate the safety of both treatment with and administration of the drug [12, 13].

Fig. 1
figure 1

Key concepts. Upper half: No-treatment scenario. In the lymph node (left), T cells encounter antigen-presenting cells, such as the dendritic cell (DC) depicted here. The DC carries antigens which bind to immunosuppressive CTLA-4 and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). As these receptors are used, the cell increases their presentation and the cell becomes exhausted—too burdened with inhibition to launch a cytotoxic response to the tumor. At the tumor site (right), the tumor presents programmed death-ligand 1 (PD-L1), which binds to programmed cell death protein 1 (PD-1) and further inhibits immune activity by the T cell, which in turn further expresses PD-1. Meanwhile, active STAT3 assists in promoting metastasis and sending inhibitory signals to the T cells. Lower half: Treatment scenarios. In contrast to tumor activity in the absence of intervention, treatment with monoclonal antibodies (shown here: anti-CTLA-4, anti-TIGIT, anti-PD-1) block key receptors and ligands from binding, thereby preventing immunosuppression. Oncolytic virotherapy such as talimogene laherparepvec (T-VEC) targets the tumor cells and lyses them; granulocyte-macrophage colony-stimulating factor (GM-CSF) recruits T cells. The burst tumor cells release large numbers of tumor antigens, which then DCs take to the lymph node to present to T cells. T cells (not exhausted) recognize the antigens and release cytokines to begin the inflammatory response. Toll-like receptor 7 (TLR7) and TLR9 agonists bind in the endosome, and these receptors activate additional immune activity. Bacillus Calmette–Guérin (BCG)-polarized macrophages recruit T cells to the tumor site and further increase immune activity while decreasing STAT3 activity, blocking off tumor escape mechanisms

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Viral vectors have been paired with several other tumor-targeting agents and tested in clinical trials for skin cancers to date (Table 1), and the next generation of clinical trials of oncolytic immunotherapies will no doubt revolve around optimizing the combinations, defining the limits of abscopal effects, exploring systemic possibilities, and optimizing the process of triaging the correct virus and agent for the individual patient [14]. For example, Hofbauer et al. compared the use of GM-CSF to interleukin-2 (IL-2) with a recombinant canarypox viral vector system called ALVAC in 2008 in a phase I trial [15]. Tested in both melanoma and cutaneous metastases of leiomyosarcoma, the IL-2 system seemed more effective in this study, exhibiting partial regression in three of eight tumors, whereas GM-CSF led only to stable disease. Coxsackievirus A21 (CVA21), an oncolytic using a live common cold virus, has displayed efficacy in a late-stage melanoma trial, with a best ORR of 24% [16]. Newcastle disease virus (NDV) studies have waned in cutaneous malignancies, but in the adjuvant setting, an NDV oncolysate demonstrated improved survival outcomes for patients with stage II melanoma [17]. A preclinical study of NDV demonstrated its potential as an intratumoral agent with systemic effects [18], but these results are yet to be replicated in a clinical context. In contrast, testing modified HSV remains popular. In a phase II study of T-VEC in stages IIIc and IV melanoma, 26% of 50 participants achieved a complete response [19]. A phase III study comparing T-VEC to GM-CSF alone then demonstrated an improved durable response rate (DRR) and ORR with T-VEC in cutaneous head and neck melanoma and in advanced melanoma in general [20, 21]. And while many combination studies have not yet reached completion (Table 2), results of a randomized phase II trial of T-VEC + ipilimumab versus ipilimumab alone show a 38.8% ORR with a combination of T-VEC and ipilimumab, compared to 18.0% for ipilimumab alone [22]. A trial of CVA21 with ipilimumab also has preliminary results in melanoma patients, and the ORR of 50% exceeds the sum of the average rate of either agent alone [23].

Table 1 Selected completed clinical trials on clinicaltrials.gov of adaptive intratumoral therapy
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Table 2 Ongoing clinical trials of novel adaptive intratumoral therapy and combination therapy
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At present, the novelty of oncolytic virotherapy precludes adequate long-term survival rates, though current studies are collecting this data and long-term efficacy should become apparent in the next few years. As seen in Table 1, oncolytic virotherapies make up almost half of the key clinical trials to date studying adaptive treatment approaches. Generally considered to be well-tolerated, the most common side effects are similar to those of systemic immunotherapy (fatigue, flu-like symptoms, and arthralgias) [15,16,17,18,19,20, 22, 23]. Viral shedding, as mentioned above, has been shown to be rare in phase I and II trials, further demonstrating the relative safety of the approach especially compared to chemotherapy [19, 24]. The highest enrolling completed T-VEC trial in melanoma thus far and its extension trial demonstrated common side effects of fatigue, fever, and chills, and a total (including unrelated to treatment) serious adverse event rate of 25.68% [21, 25, 26]. These trials also demonstrated superior efficacy when compared to GM-CSF alone in melanoma, though they do not approach the efficacy of checkpoint inhibitor therapy.

Currently, T-VEC is only FDA approved for recurrent melanoma; which means it cannot be used as a first-line therapy or in combination with any first-line therapy. There is also no evidence to suggest that the use of oncolytic virotherapy can improve long-term survival. Several ongoing studies (Table 2) have begun testing whether oncolytic virotherapies can be paired with other treatment agents to improve responses and survival rates, including in non-melanoma skin cancers. Most of these studies are in phase I as the risk profiles for these combinations have not yet been determined. In theory, local therapy could enhance the efficacy of a systemic treatment. This combination concept can be seen generally in Fig. 1. The next several years should provide some insight into whether oncolytic virotherapies have further applications outside of recurrent local melanoma treatment.

2.3 Intralesional Immunotherapy

2.3.1 Cytokines

The combination of electroporation and gene therapy led to the development of in vivo electroporation, which has since been established as a safe and effective method of transfecting immune-regulating DNA via a plasmid vector [27, 28]. Harnessing this technology with the right tumor-specific targets presents exciting possibilities in the field. One of the strongest predictors of immune checkpoint inhibitor success so far is the presence of tumor-infiltrating lymphocytes (TILs) [2]. TILs have the capacity to secrete cytokines to recruit effector T cells and initiate an immune response, and this may be a mechanism through which patients can achieve an abscopal response (Fig. 2b). TILs have become a focus for studies in both melanoma and Merkel cell carcinoma (MCC) for their role in cancer immunoediting [29]. High levels of TILs in both melanoma and MCC tumors correlate with improved responses to treatment and longer progression-free survival (PFS) with intravenous programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitors [2, 30]. These results have led to studies testing whether TIL numbers in tumor microenvironments can be increased via intralesional injection to improve outcomes in patients with naturally TIL-poor tumors. An important application of this concept is seen in the approach of intralesionally upregulating cytokines to both kill tumor cells and stimulate immune system activity, demonstrated in Fig. 1. An early phase I, dose-escalation study in melanoma utilized a plasmid vector to produce IL-12 paired with the electroporation technique and demonstrated its safety and tolerability [31]. The subsequent phase II trial enrolled 51 subjects and saw efficacy both locally and systemically with an ORR of 33% and an abscopal effect in 62% of patients [32]. The same plasmid was tested in 15 MCC patients in a phase II trial [33]. Of 12 evaluable subjects, ten had at least double the local expression of IL-12 following treatment, and two saw a response in uninjected lesions. The promising results of pIL-12 studies raise the question of whether anti-PD-1 could synergize with pIL-12. Combination therapy clinical studies utilizing this method have not yet released results (Table 2), though preclinical studies suggest that patients may see a marked improvement over monotherapy [34].

Fig. 2
figure 2

Immunotherapy: Intravenous vs. Intratumoral vs. combination therapy. a (1) Patient receives systemic immunotherapy. (2) Immune response occurs systemically; activity present throughout the body, but  targeted at tumor sites. (3) Over time, T cells become laden with inhibitory signals and stop killing tumor cells. The tumor escapes the immune response and migrates. (4) Disease progression. b (1) Intralesional drug injected into tumor. (2) Drug kills local tumor cells and/or fosters an immune response; response seen in injected tumor. (3) Result of the intralesional therapy creates memory B and T cells which circulate and recognize the metastatic site. (4) Non-injected tumor site experiences immune response followed by regression. c (1) Systemic drug injected into vein; intralesional therapy injected into tumor. (2) Immune response arises locally as well as throughout the body; abscopal effect occurs as with monotherapy. (3) Systemic immune activity further promotes abscopal effect, and systemic and local treatments work in tandem to renew T cells and prevent their exhaustion

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IL-2, which can promote naïve T-cell differentiation into effector T cells, has been dosed systemically with an overall response rate of 10–20% [35], albeit with high toxicity [36]. More recently, IL-2 has also been tested intralesionally. Combining results of six phase II trials of intralesional IL-2 results in a 50% complete response rate, though stage IV patients did not experience responses and regressions were short lived [30]. These results, while preliminary, are promising enough to warrant further study. Some data already exist regarding intralesional IL-2 use in combination with other agents. For example, a retrospective case series of 11 metastatic melanoma patients combined IL-2 injections with topical imiquimod, a toll-like receptor 7 (TLR7) agonist, and retinoic acid [37]. All patients in the series experienced complete local response, and nine lived for at least 2 years post-treatment, seven without recurrence. Another study of the combined action of imiquimod and IL-2 injection also demonstrated that the IL-2 injections offer an increased response rate compared to topical imiquimod alone [38]. Further, a phase I trial in advanced melanoma tested the combination of both intratumoral IL-2 and intratumoral ipilimumab [39]. Even though the 12 participants were split into three ipilimumab dose levels, nine of the 12 patients experienced an abscopal effect and four achieved a partial response by immune-related response criteria. These results beg further testing in both monotherapy and combination therapy with IL-2, especially in the context of supplementing checkpoint inhibition.

Similar to IL-2, interferons (IFNs) are immune stimulating cytokines that can be highly toxic when applied systemically. However, significant success has been shown intralesionally in MCC, basal cell carcinoma (BCC), and melanoma. In a single-patient MCC case study, for example, IFN-α2a followed by IFN-α2b was administered intralesionally combined with ongoing topical imiquimod [40]. The patient first experienced some reduction in tumor burden, though not all injected lesions responded. Subsequently, several instances of treatment cessation for the patient's safety correlated with new metastases, most of which were injected with the IFN regimen and then regressed. To further study the potential for IFNs in intralesional treatment, Paulson et al. performed cellular analyses on MCC cell lines and MCC patient samples [41]. This study demonstrated upregulation of major histocompatibility complex (MHC)-I following IFN introduction in vitro and in vivo. MHC-1 presents antigens to effector T cells to stimulate an immune response, and this upregulation suggests that IFNs may work well in tandem with other immunotherapy approaches which utilize the MHC-I pathway. In BCC, Chimenti et al. treated 140 patients with intralesional IFN-α2b and produced results strongly favoring efficacy, with a complete response rate of 67.1% and an ORR of 90.7% [42]. Since then, another study has demonstrated long-term efficacy, with low recurrence rates [43]. Melanoma patients have also seen benefit from intralesional IFNs, as in Fierlbeck et al.’s study in which IFN-β exhibited a dose-dependent relationship, with regression in 19 metastases [44]. IFNs have the most combination data in melanoma thus far, with examples utilizing radiotherapy, tumor necrosis factor, and IL-2, to name a few [45,46,47]. However, combinations with intralesional IFNs have thus far been broad in terms of secondary agent choice and thus relatively unreplicated.

2.3.2 Monoclonal Antibodies

Monoclonal antibodies (mAbs) have long been utilized in the systemic setting, with a relatively low toxicity profile compared to chemotherapy when applied intravenously (see Fig. 1). Given its efficacy and availability, the reduced adverse event rates seen with intratumoral therapies would make the immune checkpoint inhibitor nivolumab a prime target for further intralesional study. If effective, this application could potentially result in reduced side effects in settings outside widely metastatic melanoma. L19-IL2 and L19-TNF are mAb-cytokine fusion proteins, which, for example, provide a tumor-targeting mechanism. Weide et al. demonstrated L19-IL2 efficacy at lower intratumoral doses when compared to the cytokine alone in melanoma [48]. Danielli et al. expanded on this finding with a 20-subject melanoma clinical trial using a combination of both fusion proteins; 55% of treated lesions exhibited a response, and one subject experienced a complete response [49]. Outside of melanoma, the switch from intravenous to intralesional anti-PD-1 alone would particularly benefit immunocompromised patients, such as HIV + patients with cutaneous malignancies. Currently, skin cancer patients with infections like HIV and hepatitis struggle to find effective treatments that do not present an unacceptably high risk of death or disability. There is currently one ongoing clinical trial (see Table 2) intending to test the safety of intralesional nivolumab in patients with Kaposi’s sarcoma (KS) [14]. This study is estimated to enroll 12 participants (HIV + and HIV −) with KS, and these patients will receive four doses of 10 mg nivolumab in a 1-mL injection intratumorally over 8 weeks. If safe enough, this study will shed light on T-cell responses in KS patients, which could ultimately lead to testing in other skin cancers for patients with comorbid HIV or hepatitis. Intralesional delivery of anti-PD-1 has been similarly lacking in trials across all cutaneous malignancies, though Samoylenko et al. have released an abstract of their pilot study on intralesional nivolumab and pembrolizumab in metastatic melanoma, with dosing proportional to lesion size (maximum 50 mg in 5 mL) [50]. As of the interim analysis, seven patients were dosed with one of the anti-PD-1 drugs intratumorally, and four of the seven patients experienced an overall response to therapy, and two of those four had response in non-injected lesions as well. TIL values were measured at baseline and following intervention, with responders displaying higher TIL values than non-responders. This further supports the necessity of characterizing the microenvironment of an individuals’ disease with respect to T cells prior to choosing a treatment method [2, 51]. And while the current default mechanism for mAbs is checkpoint inhibition, some attention is now being paid to agonistic antibodies. For example, ADC-1013, a CD40 agonist, has now been demonstrated to be both active pharmacodynamically and safe enough for further testing following NCT02379741, a first-in-human trial in 23 patients with advanced solid tumors [52]. In the same study, Irenaeus et al. also studied the drug in mice in combination with a PD-1 inhibitor, and the efficacy of these experiments combined with the low treatment-related adverse event severity (especially for intratumoral patients) of the clinical trial suggests that testing a combination of PD-1 and ADC-1013 may be on the horizon. Further testing is certainly warranted to establish an application and indications for intralesional immune checkpoint inhibitors, especially with regard to immunosuppressed patients.

3 Addressing the Tumor Microenvironment

The immediate environment of a tumor can determine both its responsiveness to therapy and its ability to escape that therapy’s attack. The skin is the largest organ of the human body and has the greatest potential for external manipulation, which could present difficulty in studying the potentially vast variety of microenvironments. However, lesion accessibility can certainly be favorable in this context, and it is therefore imperative to study local therapies for patients with particularly resilient, tumor-favoring microenvironments.

3.1 Utilizing Bacteria

The immediate environment of a tumor can determine its responsiveness to therapy, and ultimately, its ability to escape the attack of such therapy. Microenvironments are so called because they can differ so vastly across the body. It is therefore imperative to study local therapies for patients with particularly resilient, tumor-favoring microenvironments. The success of immunotherapy in skin cancers has led to the study of any approach which can foster a pro-inflammatory environment surrounding the tumor. One possible mechanism of tumor escape involves macrophage polarization into M2, and then becoming tumor-associated macrophages, or TAMs. These macrophages then add to the tumor’s immunosuppressive capacity, counteracting the work of pro-inflammatory cytokines. Bacteria can regulate macrophage activity. Macrophages polarized in vitro with bacillus Calmette–Guérin (BCG) vaccine have demonstrated recruitment of both CD4 + and CD8 + T cells (Fig. 1) upon encountering melanoma cell lines [53]. TAMs are also known to facilitate tumor growth and metastases, assisting in tumor escape by promoting regulatory T-cell (Treg) activity. For example, Chen et al. demonstrated that the reduction of TAMs led to protection from metastases [54]. Bacteria may be the answer to effectively reducing TAMs and recruiting macrophages to pro-inflammatory roles. Further, polarizing these macrophages so they would not differentiate into M2 (via enzyme CYP4A, which is overexpressed in melanoma) decreased migration and STAT3 activity, thought to foster metastasis. However, reducing Tregs alone may not result in a robust therapy in itself, especially for patients with advanced disease. These data, when combined with STAT3 findings above, imply that a combined effort of macrophage manipulation and gene-targeted therapy may be required to prevent tumor signal migration and foster an anti-tumor microenvironment. Much of the bacterial research thus far is preclinical, with BCG currently being used only as an immune activating agent. The effect of macrophage polarization will likely be subtle and require many years’ worth of data to prove efficacy, especially since it is a method designed for use alongside other agents, to prevent recurrence and escape while the other agent fights the disease. This research is still presently relevant, however, as it will teach researchers how best to manipulate skin microenvironments surrounding malignancies for the desired effect, such as fostering immune activity to boost drug efficacy.

3.2 T-Cell Exhaustion

Fighting a chronic condition, like a malignancy, is known to train T cells to express certain receptors with increasing frequency (see Fig. 1). With age, naïve T-cell abundance wanes, while memory and effector T cells generally persist [55]. In a murine melanoma model, depleting Tregs in combination with anti-PD-L1 antibodies proved able to reverse disease recurrence, and blocking lymphocyte-activation gene (LAG)-3 removed the need to deplete Tregs [56]. This study highlights the differences between a primary disease and recurrence, suggesting that T-cell exhaustion is an important mechanism by which malignancies might develop immunity to PD-1 blockades. Further, a phase I/II trial of stereotactic radiotherapy followed by ipilimumab in melanoma patients without brain metastases has provided an example of a sequential combination treatment of high-dose, short-term radiotherapy followed by anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) [57]. The tolerability of this combination, let alone efficacy, is not yet determined. However, in theory, this combination would work on both sides to increase the CD8 +/Treg ratio. Ultimately, the results of this trial had patients high in PD-L1 exhibiting exhausted T cells and progression, while murine models including anti-PD-L1 experienced improved outcomes with reinvigorated T cells. The study suggests that PD-L1 plays a key role in tumor escape. Patients may therefore receive a distinct benefit from the local action of stereotactic body radiation therapy (SBRT) in combination with anti-CTLA-4 and anti-PD-L1 (or anti-PD-1).

3.3 Regulating T-Cell Function

Similar to CTLA-4 and PD-1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is another inhibitory receptor on CD8 + cells and represents another promising target for intralesional and combination therapies following the concept of drug resistance in melanoma (Fig. 1). The presence of TIGIT may be able to mark exhausted T cells, indicating the need for further interventions as a patient’s immune response decreases in efficacy [58]. Chauvin et al. demonstrated that TIGIT and PD-1 are often co-expressed, and that TIGIT is upregulated in an anti-PD-1 environment [59]. This contradicts the finding that TIGIT is a T-cell exhaustion marker, and further work is needed to conclusively determine the significance of its presence on CD8 + cells. However, the study did indicate a supplementary effect of TIGIT blockade alongside anti-PD-1 therapy. Meanwhile, a combination of anti-TIGIT and anti-PD-L1 demonstrated that utilizing TIGIT as a co-inhibitory target has the potential to reduce effector T-cell exhaustion by reducing the inhibitory burden on CD8 + T cells so they continue producing higher cytokine volumes [60, 61]. Thus far, there is not enough data to assess whether TIGIT is an effective target for attacking skin cancers and preventing their recurrence. An ongoing currently recruiting phase I study intends to assess an anti-TIGIT mAb for safety; the study is currently ongoing and expected to conclude in October of 2019 [62]. Should this agent prove safe, it may provide a necessary agent to refresh immune responses where TILs have become exhausted in the anti-CTLA-4 and anti-PD-1 environments. And for patients who fail on currently approved mAbs, anti-TIGIT agents could provide a new treatment option.

Toll-like receptors (TLRs), especially those embedded in endosomes, have also become a target of interest in the study of immune exhaustion in skin cancers. TLR7 agonists have been used topically in dermatology for various skin pathologies since FDA approval of imiquimod in 1997. Plasmacytoid dendritic cell (pDC) precursors express TLR7 and TLR9, which makes these receptors especially attractive as a target in melanoma [63]. In studying TLR7/8 and TLR9 ligands, their presentation led to a reduction in Treg cells and increase cytokine production [64]. Studies involving TLR7-deficient mice led to the conclusion that TLR7 is able to recognize certain small antiviral compounds and produce IFN-α in response [65]. The other pDC precursor-derived target, TLR9, has gained traction recently in melanoma clinical trials. TLR9 agonists SD-101, CMP-001, and IMO-2125 are all intralesionally administered and are currently being tested in clinical trials as monotherapy and in various systemic immunotherapy combinations [66,67,68]. Thus far, these studies are ongoing, though preliminary data suggest that combination therapies including TLR9 agonists have the ability to counteract immune exhaustion, preventing tumor escape by regulating the chronic inflammation caused by systemic immunotherapies [69]. These studies include combination therapy arms and should thus clarify the benefit they may add to current therapies and suggest further work in intralesional partnering with currently approved therapies.

3.4 Current Challenges

The most obvious limitation of intralesional therapies lies in advanced disease with visceral metastases. In terms of delivery, it is much more complicated and risky to inject visceral lesions. Intralesional therapies have typically been administered repeatedly, which would certainly be less feasible for some disease sites given the current technology available. Then, should the lesion be accessible for ultrasound-guided injection, the microenvironment of that tumor is likely to be vastly different from cutaneous disease. Dendritic cell (DC) presence is a prime example. DCs were originally discovered in the mouse spleen [70]. Lymphoid tissues displayed the crucial role of DCs, and different subsets were then categorized in the lymph nodes, skin, intestine, lung, liver, and kidney [71,72,73,74]. In addition to this variation across organs, relative density is also a factor. In a murine model, for example, the liver was found to have the lowest density of DCs [75]. In theory, this would limit the rate at which tumor-derived antigens could be detected and destroyed. Liver metastases of melanoma have further demonstrated lower TIL counts and poorer outcomes for patients on systemic immunotherapy [76]. Manipulating DC maturation and concentration could prime tumor microenvironments for further drug mobilization. Current therapies are lacking in terms of DC manipulation testing in humans, but current studies’ biospecimens may be utilized retrospectively to characterize DCs’ behavior in metastasis and tumor escape prevention.

4 Novel Approaches in First-Line Treatment

4.1 Diagnosis and Technology

Immense interest has been directed to technological advances in two primary areas of skin cancer therapy: diagnosis and drug delivery. The diagnostic front has grown considerably in the last several years, with improved imaging possibilities, biological modeling, and artificial intelligence [77]. When combined with nanoparticles and robotics, these technologies could lead to an intralesional therapy for early stage skin disease, preventing the need for surgery, with fewer side effects and lower risk of infection [78,79,80]. New and improved in vivo DNA delivery systems are in development as well. Jet injection, iontophoresis, skin abrasion, thermal ablation, and microneedle patches represent some of the burgeoning progress which may resolve many of the costs and difficulties surrounding intralesional drug administration, especially in the case of metastasized disease and non-skin cancers [79,80,81,82,83,84]. While all of these approaches need further work before they can be contenders alongside standard of care therapies, the overall direction of minimizing disease burden and procedure invasiveness is assuring.

4.2 Neoadjuvant Therapy

Treating tumors prior to resection is a relatively novel concept in skin cancer treatment, especially in resectable cases. The potential lies in the possibility of using drugs to encourage an anti-tumor T-cell response, prior to excision. This approach is relatively novel and as such has fewer studies (Table 3). In a case series, three patients with rapidly growing ocular surface tumors (conjunctival melanoma or ocular surface squamous neoplasia) were treated intralesionally with IFN-α2a prior to resection [85]. In the 3- to 4-week period between injection and surgery, all three patients’ lesions reduced in size and improved in appearance. All three were then successfully managed by surgery. This case series exemplifies the possible benefits of neoadjuvant immunotherapy. First, in the time patients have to wait for their surgical appointments, they may not have to experience rapid tumor growth. Second, the course of surgical interventions can be smoother, safer, and more successful.

Table 3 Neoadjuvant clinical trials utilizing adaptive intratumoral therapy
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The potential for TLR agonists and oncolytic virotherapy in neoadjuvant therapy is also an area of study, though trials are mostly in early stages without posted results to date. Preclinical work and case studies have led researchers to hypothesize that using imiquimod locally in a neoadjuvant setting will simplify the patient’s subsequent dermatological course [86]. An interim analysis was recently presented on a study studying T-VEC neoadjuvant therapy compared to immediate resection [87]. The experimental arm of this study applies six injections of T-VEC to subjects prior to resection. As this trial is studying stage III/IV melanomas, all patients receive standard of care adjuvant systemic therapy. Thus far 76 subjects in the experimental arm and 74 in the control arm have completed the interventional portion of the study. Eleven subjects progressed on T-VEC therapy, compared to 17 control patients in the same amount of time. The overall response to neoadjuvant treatment was 14.7%, but of the T-VEC responders, 21% had no melanoma cells detectable in their resection specimen. There is potential for this trial to elucidate effectiveness on sequential intralesional T-VEC and adjuvant immunotherapy, but the value of these data ultimately relies on time and the subjects’ choices. Neoadjuvant intralesionals, especially in combination or sequence with other robust treatments, may be moving the melanoma climate in the direction of prevention. Additional data are required to improve patients’ risk/benefit ratio to the point where they would consider this relatively extensive approach compared to a single surgery.

4.3 Vaccines

As with any disease, prevention is the ultimate goal. The rise of genomic sequencing of tumors has led to the possibility of a personalized neoantigen vaccine. Logistically, this is complicated, as individualized cancer vaccines can be expensive and time consuming to produce. However, as immune escape mechanisms are better understood and gene sequencing results become easier to obtain, cancer vaccines will likely become a much more important frontier in research. For example, Stravodimou et al. recently demonstrated that effector T cells and Tregs increase prior to malignancy in both actinic keratosis and squamous cell carcinoma (SCC) [88]. This study also displayed differences in T-cell populations between invasive and in situ SCC. Investigating T-cell populations and microenvironments may allow for predictive, preventative therapy as opposed to reactive treatment. TLR ligands have also been investigated in the context of vaccine adjuvants. When tested for microbial vaccination, ligands for TLR7/8 and TLR9 incorporated into a vaccine were shown to grow the splenocyte population and reduce Treg presence [64]. While still in very early stages with regard to human trials, cancer vaccines are currently being tested in animal models. STINGVAX is a vaccine which applies stimulator of interferon genes (STING). Fu et al. applied STINGVAX treatment in vivo to several murine melanoma models and witnessed increased TIL proliferation and effective anti-tumor activity [89]. Application of the STING vaccine combined with anti-PD-1 in this trial resulted in tumor regression where anti-PD-1 alone failed. These results further illustrate the need to explore T-cell and tumor microenvironment manipulation alongside available established, effective drugs.

Vaccines are typically thought of as antibody-producing substances, but can also be defined as drugs imparting an acquired immunity in general. Adoptive T-cell therapy potentially takes out the “middle man” of antibodies and provides T cells primed to fight the patient’s malignancy.

While there is currently a wealth of active research in intratumoral immunotherapy, our understanding of how such treatments interact with systemic immunotherapy is in its infancy. Adoptive T-cell therapy is one possible approach which appears promising and presents a possibility for a cancer vaccine mechanism. Khammari et al. demonstrated that adoptive T-cell therapy and an IFN-γ–expressing adenovirus (TG1042) in late-stage melanoma patients could perform well in combination [90]. Fifty-one percent of injected lesions regressed or resolved in response to the combination, and 75% of lesions remained stable or improved. The authors of this study pointed to the possibility of selecting TILs for adoptive T-cell therapy based on the antigen abundance of the tumor. In theory, this would increase the efficiency of the effector T-cell activity, broadening the abscopal effects of whatever other agent was applied. Depending on the strength of the abscopal effect, an argument can then be made for intralesional anti-PD-1 therapy, which has indeed seen some success in early clinical trials [50]. Similarly, other current studies are testing DC vaccines in order to manipulate which antigens at which rate are presented to the T cells already surrounding the tumor (Table 2). As has been seen above, combination therapy has worked well with anti-PD-1, and there is some evidence in melanoma that PD-1 and TIGIT may have tandem effects in blocking TIL activity [59]. Therefore, a combination intralesional approach without the need for a systemic agent exemplifies a reasonable direction for future research with an incredible impact on patients’ experience in the course of their treatment. Dual intralesional therapies should be considered, even if only to decrease toxicity. More studies are needed to verify that this approach is at least as effective as systemic anti-PD-1 plus any intralesional agent, but even a slightly less effective result would lower patients’ risk to benefit ratio given the toxicities seen in intravenous immunotherapy.

5 Conclusion

Several intratumoral agents have demonstrated efficacy as skin cancer treatment over the past several years. None of these, however, exceed the current standards of care. Many more studies are required to clarify the role these agents will play in the future treatment landscape. However, results of recent studies suggest that the primary goal of this endeavor should not be for a replacement therapy but rather a complementary one. Today, intralesional therapies are a much needed option for patients who have exhausted their systemic possibilities and especially for those who cannot tolerate systemic treatment to begin with. The latter group will benefit most from agents with strong efficacy, harnessed for local administration to reduce adverse reactions. Logically, this would mean intratumoral anti-PD-1 or anti-CTLA-4, though this has not yet been demonstrated clinically to the point of conclusively meeting the performance of these agents delivered intravenously. For those who fail all currently approved treatments, their tumors are either better equipped to escape intervention or the tumor microenvironments are too weak and permissive to prevent metastasis. These patients need a more robust, multifaceted approach. Rather than testing intralesional agents one by one due to the assumption of inefficacy in all previously failed approaches, a more measured offense should be attempted. More research is needed in the effort to optimize therapy combinations, and the intratumoral angle presents the possibility of efficacy without having to endure the severe reactions which accompany the same agents systemically. These combinations are not only the future of treatment; they are also the future of prevention.