Targeting therapeutics - immune checkpoint and small molecule inhibitors
Following migration to blood vessels and intravasation, metastatic melanoma cells must escape immune surveillance to survive, extravasate and enter the brain [
101]. This evasive capacity is generally mediated by the activation of immune suppressive regulatory T-cells (Tregs) by tumor cells through, for example, the secretion of TGFβ, reviewed in [
102]. Tregs accumulate in melanoma, which also exhibit an enrichment of activated CD8
+ T-cells [
103] and the ratio of CD8-positive T cells versus Treg has been found to be a predictor for melanoma patient survival [
104]. CD8
+ T-cells secrete pro-inflammatory cytokines to induce yet another means by which tumor cells evade immune surveillance, involving the up-regulation of programmed cell death-ligand 1 (PD-L1), [
103]. PD-L1 binds to the receptor PD-1, expressed on T cells, leading to a reduction in target cell activation [
105]. Soluble PD-L1 has been identified as a biomarker for melanoma [
106], which hints that a low expression of PD-L1 and PD-L2 might be correlated with favorable patient outcomes. But the expression of PD-1 ligands is restricted to certain melanoma cell subtypes [
107]. In the clinic, neutralizing antibodies blocking the interaction of PD-L1 (Pembrolizumab [
108], Nivolumab [
109]) with PD1, albeit their efficacy remains controversial [
110]. A recent study by Goldberg et al. (two-cohort, phase II, clinical trial NCT02085070) revealed that BM from melanoma exhibited partial or complete responses (22%) to pembrolizumab [
111]. But the high number of patients whose metastases failed to respond suggests that in many cases, melanoma cells take advantage of additional defense mechanisms that have yet to be identified.
The immune suppression of Tregs is further mediated by their expression of the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and its binding to CD80/CD86, reviewed in [
112]). The therapeutic application of the neutralizing antibody ipilimumab yet represents another mechanism of activation of the immune system to fight against tumor cells. The overall survival of patients with visceral metastases (M1c stage) who received ipilimumab was improved (10.1 months) compared to a control group administered a gp100 peptide vaccine (6.4 months) (NCT00094653, Additional file
1: Table S1) [
113].
Peritumoral edema is frequently observed alongside with brain metastases formation, leading to increased intracranial pressure and neurological disturbances; symptoms are controlled by administration of corticosteroids [
114]. Margolin et al. assessed the efficacy of ipilimumab in patients with either asymptomatic or symptomatic brain metastases who had not or had received corticosteroid treatment at study entry. The study revealed that ipilimumab elicited a disease control in 24% of patients with small and asymptomatic brain metastases, whereas 10% of patients with symptomatic brain metastases, received a disease control within 12 months (
NCT00623766, Additional file
1: Table S1, Metadata 1 and 2) [
115]. Hence, patients with larger and symptomatic brain metastases who require oral steroids to control peritumoral edema have an ongoing poor response to systemic therapy and are mostly excluded from clinical trials. Therefore, other treatment strategies improving the outcome of these patients are needed e.g. the combination of ipilimumab and the VEGF-neutralizing antibody bevacizumab, a combination which was successfully tested in glioblastoma [
116].
Overall, 26% of patients exhibited a refractory response to ipilimumab, and hence might benefit from PD1 inhibitor therapies [
108,
117]. A case which has been more comprehensively discussed elsewhere [
118,
119].
Strikingly, a dual strategy that used a combination of ipilimumab and nivolumab to block PD1 and CTLA4-mediated immune suppression evoked an intracranial objective response rate of 56%, as demonstrated by Tawbi et al. [
120]. And a multicenter US trial (CheckMate204 study, NCT02320058) of melanoma patients with one or more brain metastases achieved a complete response (CR) in 19% of patients; in these cases, the intracranial and extracranial responses largely overlapped. In addition, the survival of patients who received ipilimumab either prior to SRS or thereafter was significantly improved over that of patients who received SRS alone (19.9 months vs. 4.0 months;
P = 0.009), [
121]. Recently, a multicentre open-label randomized phase 2 trial (ABC, NCT02374242, Additional file
1: Table S1) by Long et al. [
122] performed in three cohorts of immunotherapy-naive patients revealed an intracranial response to the combination of nivolumab+ipilimumab or nivolumab alone in 46% or 20% of patients with asymptomatic brain metastases with no previous local brain therapy, respectively. The intracranial complete response to nivolumab+ipilimumab or nivolumab alone was 17 and 12%, respectively. However, the intracranial response was markedly reduced (6%) in patients brain metastases in whom local therapy had failed, or who had neurological symptoms, or leptomeningeal disease.
These results clearly demonstrate that therapeutic interventions that block immune suppressive mechanisms are effective in brain metastases, however the identification of strategies for further improving the response to and efficacy of checkpoint inhibitors is mandatory and will provide more insight in the interaction of melanoma and immune cells. A very recent study uncovered the role of the oral and gut microbiome, discriminating melanoma patients who respond and those who do not respond to anti–PD-1 immunotherapy [
123]. In addition, since the brain is immune privileged, extracranial and brain metastases very likely feature distinct immune evasion mechanisms. At least for brain metastases, microglia might play an important role. In breast cancer, microglia were associated forced brain metastasis [
124].
The identification of mutations of BRAF in human cancers [
125] is a milestone, opened new avenues in the therapy of melanoma and was a prerequisite for the development of small molecule BRAF inhibitors, most important vemurafenib and dabrafenib. Besides the high response rate of 53% of patients to vemurafenib accompanied by a median OS of 15.9 months [
126], vemurafenib also induces clinical responses in melanoma brain metastases. Albeit, the access of vemurafenib to the brain is restricted by an ABCB1-mediated efflux [
127]. Dummer et al. demonstrated in an open-label pilot study that 42% of patients showed an overall partial response (PR) to vemurafenib at both intracranial and extracranial sites, 38% achieved a stable disease and 37% of patients showed a remarkable (> 30%) regression of brain metastases [
128]. In addition, a open-label, phase 2, multicentre study of 146 patients with or without previous vemurafenib treatment showed a intracranial BORR (best overall response rate) of 18% as assessed by an independent reviewer committe (IRC). However, 32% or 34% of patients progressed in cohorts without or with previous treatments, respectively [
129]. Furthermore, the BREAK-MB trial (
NCT01266967 and Additional file
1: Table S1) assessed the response of patients with confirmed BRAFV600E/K mutations to dabrafenib. The study revealed that patients who either had or had not received previous local treatment for brain metastases and progressive brain metastases after previous local treatments showed an intracranial response of 39.2% or 30.8%, respectively. Hence, dabrafenib was effective in brain metastases irrespective of whether they were untreated or have been previously treated and progressed [
130]. The intracranial response of patients who either had or had not received previous local treatment for brain metastases was further increased by the combination of dabrafenib and trametinib (56% vs. 58%) in the COMBI-MB study (NCT02039947, Additional file
1: Table S1) [
20]. However, the median duration of response was relatively short and might reflect the different modes of activation of signaling pathways and molecular profiles of melanoma brain and extracranial metastases [
46].
In summary, targeting therapies have significantly improved the outcome of patients both with extracranial and brain metastases. However, their impact and efficacy might depend on the spatial distribution of metastases.
Radiosurgery
Single metastases that occur in accessible areas of the brain can be resected or successfully treated with stereotactic radiosurgery (SRS) using gamma or cyber knifes, minimal invasive state-of-the-art techniques which have proven more effective than whole brain radiotherapy (WBRT) in extending OS, with the median reaching 13.9 months [
131‐
133]. Both, WBRT and SRS are associated with severe side effects, most important adverse neurocognitive effects for patients who received WBRT as well as radiation necrosis which is also common but more frequently observed in SRS patients (reviewed in [
134]). Radiation necrosis is characterized by fibrinoid necrosis of small arteries and arterioles, likely induced by extensive damage to the vascular endothelium [
135].
In a comprehensive clinical study of patients diagnosed with brain metastases from several cancer types except leptomeningeal disease, small-cell lung cancer and hematologic cancer surgical resection followed by SRS of the surgical cavity was proven more effective and significantly lowered local recurrence compared with patients with surgical resection alone (
NCT00950001 and Additional file
1: Table S1) [
136]. Initially, SRS or the combination of SRS + WBRT was associated with a higher local and distant control than observed for SRS alone, hence adopted to patients with a limited number (1–4) of brain metastases. Albeit this strategy did not improve OS (reviewed in [
137]). A multicohort prospective study of 1194 patients with brain metastases of mainly breast and lung cancer, revealed that even in patients with multiple brain metastases (5–10) who had not received previous WBRT [
132], SRS was as effective as in patients with 2–4 brain metastases.
To further increase the time to recurrence and improve melanoma patients survival, radiotherapy is combined with targeting drugs or immune checkpoint inhibitors, capable of passing the blood brain barrier. Currently, clinical trials combining ipilimumab and nivolumab with SRS (< 5 progressing BM) or WBRT (≥6 progressing BM) in melanoma patients are in progress (NCT03340129, NCT02097732 and Additional file
1: Table S1). In addition, a recent retrospective study revealed a high (70%) complete or partial response (CR/PR) to concurrent pembrolizumab and SRS [
138]. However, the efficacy of these combinatorial trials needs to be proven more comprehensively. Since both extracranial metastases and BM also exhibit a broad response to vemurafenib, dabrafenib and other drugs targeting oncogenic BRAF [
20,
129,
139,
140]. Dabrafenib has particularly potent effects on melanoma BM irrespective whether patients had received previous local treatment including surgical resection, WBRT, or SRT and progressed (clinical study
NCT01266967) [
130].
The consequences of drug resistance
Following a positive response towards BRAF-targeting drugs, tumor cells begin to exhibit resistance within 6–7 months [
141]. This is marked by an up-regulation of receptor tyrosine kinase receptors PDGFRB [
142] or EGFR [
143], signaling mediators such as CRAF or NRAS, and the acquisition of mutations in MEK1, MEK2 and NRAS that trigger a stimulation of the RAS/RAF/MAPK pathway (reviewed in [
144]). This suggests that melanomas resistant to BRAF inhibitors might be treated with the potent MEK inhibitor trametinib [
145]. However, the combination of dabrafenib and trametinib was only moderately effective in patients with BRAF inhibitor–resistant melanoma [
146]. Nevertheless, the combination provoked a better response of intracranial BRAFV600-mutant brain metastases from melanoma (clinical trial
NCT01266967) compared to monotherapies based on the BRAF inhibitor [
130]. At this point another hurdle commonly appeared: cells developed resistance to MEK inhibitors, and the MAPK pathway was reactivated through an acquisition of MEK activating mutations [
147] or BRAF gene amplification [
148]. Furthermore, two recent studies reported increases in BM or a spontaneous formation of new lesions in patients being treated with vemurafenib [
149,
150], most likely due to the mechanisms described above.
Therapy-induced changes in the expression of markers
Several lines of evidence suggest a therapy-induced enrichment for stem-like tumor cells a mechanism potentially responsible for therapeutic failures and tumor relapse. Kim et al. demonstrated that CD133 -a putative marker of melanoma-initiating cells [
151,
152]- acts in concert with the chemokine receptor CXCR4 to facilitate a metastatic phenotype [
151]. The expression of CD133 (PROM1), was modified by drug-treatment. Furthermore, in glioblastoma, Bao et al. demonstrated a radiation-induced mechanism responsible for the enrichment of radio-resistant CD133
+ glioblastoma stem-like cells by activation of the DNA-damage response [
153]. In addition, breast cancer-related BM [
154] have been associated with a high expression of DNA repair genes and the activation of the PI3K/AKT signaling [
155] pathway. These factors may also determine whether radiation therapy will be effective against BM arising from melanoma [
156]. Alongside an increased capacity to repair DNA damage, metastatic cells may undergo drug-induced changes in gene expression that help account for their low response to chemotherapies. These therapies in particular induce a DNA damage response in the cells.
The expression of CD271, which is induced by DNA-damaging drugs, may serve as a critical factor in the regulation of DNA repair genes (Fig.
4d) and modifies the migratory phenotype of melanoma cells. Cell migration of melanoma cells was indeed modified in response to the levels of CD271. Hence, increased migration was observed for drug-resistant (Fote, Vind) cells or cells with forced expression of CD271 (NGFR/CD271) [
88]. In contrast, the CD271 knock-down was accompanied by a marked reduction in migration [
21], (Fig.
4e). Moreover, Lehraiki et al. demonstrated that CD271 promotes vemurafenib-resistance of A375 cells through a NFκB-regulated mechanism. Furthermore, the expression of CD271 was strongly increased in relapsed tumors [
157].
Very recently Haueis and Imafuku et al. have reported that melanoma patients undergoing vemurafenib therapy develop more brain metastatic lesions/ tumors than those who do not receive the drug [
149,
150]. Along the same lines, Klein and Zubrilov et al. identified CCR4, JARID1B and CD271 among the top up-regulated genes in vemurafenib-resistant, brain metastatic melanoma cells [
70,
158]. This raises the provocative question of whether vemurafenib treatment enhances metastasis generally, or is an effect specific to BM in melanoma patients, potentially because the treatment induces the expression of metastasis-promoting factors. Seifert et al. investigated how melanoma metastases responded to vemurafenib in previously drug naive patients. They found that extracranial metastases were more prone to respond completely (CR) or partially (PR) than metastases of the brain or bone. Moreover, the brain was the most common site of progression for patients receiving vemurafenib; among patients who had previously developed brain metastases, 79% experienced a recurrence of BM [
159]. The mechanisms responsible for the low response have not been fully elucidated. One suggestion from Seifert et al. is that growth factors from the cerebrospinal fluid (CSF) trigger PI3K/AKT signaling pathways, making BM resistant to vemurafenib [
159]. This idea could explain the findings made by Haueis and Imafuku et al.