Immunomodulatory Drugs in Melanoma Brain Metastases

Brain metastases are about ten times more frequent than a brain primary tumor, being present in 20-40% of adults with systemic cancer. Together with lung cancer and breast cancer, skin cancers such as melanoma are top primary tumors which metastasizes to the brain. Advanced melanoma is well known for its propensity to metastasize to the brain, with 80% of patients presenting brain metastasis at the autopsy. However, current therapies are not very efficient and brain metastases are in most of the cases lethal. Treatment of melanoma brain metastases with surgery and/or radiation therapy results in a median overall survival of only about four months after diagnosis. New immunotherapies such as targeted or immunomodulatory drugs, many in clinical trials, have shown promise, with some immunomodulatory drugs being able to at least double the overall survival rates for patients with melanoma brain metastases. This review focuses on the recent advances and future potential of using immunotherapy, such as the newly developed immunomodulatory drugs, for melanoma brain metastases therapy. Immunomodulatory drugs bring a great promise as new tools for melanoma treatment in particular and for the treatment of other types of malignancies in general.


Introduction
Brain metastasis, the spread of a tumor from a primary neoplasm to the brain, is about 10 times more frequent than a primary brain tumor 1 . Noteworthy, 20-40% of cancer patients with systemic pathology have or will develop brain metastases 2,3 . Most common brain metastases have their primary tumor in the lung (~45%), breast (20%) and skin (e.g. melanoma, 10%) 4 . Brain metastases have a very poor prognosis, being characterized by a progressive Central Nervous System (CNS) damage and functional decline, significantly affected quality of life and shortened patient survival. Advanced melanoma is well known for its potential to metastasize to the brain. Approximately 80% of the melanoma patients present brain metastases at autopsy 5,6 .

KEY POINTS
◊ Some immunomodulatory drugs are able to at least double the overall survival rates for patients with melanoma brain metastases ◊ Immunotherapy, including the immunomodulatory drugs, bring a great promise as new tools for melanoma treatment in particular, and for the treatment of other types of malignancies in general.
However, current therapies are not very efficient and brain metastases are in most of the cases lethal. Treatment of melanoma brain metastases with surgery and/or radiation therapy results in a median overall survival of only about 4-6 months after diagnosis and they cause notable complications and morbidity (stroke, radiationinduced necrosis and cognitive defects) 7 . New immunotherapies such as targeted or immunomodulatory drugs, many in clinical trials, have shown promise, with some immunomodulatory drugs being able to at least double the overall survival rates for patients with melanoma brain metastases 8 . Immunotherapy uses components of the body's own immune system to fight against cancer. It works in several ways, for example by enhancing the capacity of the immune system to attack cancer cells or giving the immune system specific components artificially produced 9 . In particular, immunomodulators, antibodies stimulating T-cell function either by blocking or activating regulatory receptors, have shown to cause regression of several types of tumors and an exponential number of clinical trials are underway. Several immunomodulatory drugs/ checkpoint inhibitors are already approved by the US Food and Drug Administration (FDA) for the treatment of melanoma, non-small cell lung cancer, breast cancer, bladder cancer, kidney cancer, Hodgkin lymphoma 10,11 . Noteworthy, pembrolizumab was recently approved by the FDA for solid tumors with microsatellite instability-high (MSI-H) or mismatch repair-deficient 11 .
Here, we aim to review the most important advances and future potential of using immunotherapy, such as the newly developed immunomodulatory drugs, for melanoma brain metastases therapy.

Melanoma Brain Metastasis
Melanoma brain metastases have been detected in about 45-60% of the patients, with 75%-80% presenting brain metastases at autopsy examinations. MRI is the gold standard for both diagnosis and monitoring of brain metastases 12 . Patients diagnosed with melanoma brain metastasis have an overall survival of only 4 to 6 months with standard available treatments, such as surgery and/or radiation therapy 13 . This is definitely not the desired outcome and sustain efforts are currently underway to develop better therapies.
The tumor microenvironment is an important factor influencing all steps of metastasis development, from metastasis formation to its progression and response to different therapies. In addition to the tumor cells, tumor microenvironment also contains other types of cells, such as fibroblasts, immune cells, pericytes and endothelial cells. The main features distinguishing the brain tissue from any other tissues are the presence of blood-brain barrier (BB) and unique resident cells (microglia, astrocytes and neurons) 14 . Recent results suggest that tumor cells from brain metastases can communicate with local astrocytes through gap junctions and program them to produce and secrete tumorstimulating cytokines. These cytokines will then promote NF-kB-mediated survival and/or proliferation of cancer cells. Gap junctions can be successfully targeted 15 .
Interestingly, the report of a functional lymphatic vasculature along dural sinuses in mice caused a revision of the previous view of CNS as an immune privileged site 16,17 . Moreover, CNS-derived antigens can induce an immune response in cervical lymph nodes 18 , while some reports show that the BB can be affected in brain tumors resulting in signifi-cant accumulation of immune cells from outside CNS 19 . Noteworthy, previous studies reported that the brain metastases, unlike normal brain parenchyma and primary CNS tumors, have an immunoregulatory environment significantly infiltrated by lymphocytes. For example, over 99.1% of the analyzed brain metastases in a study shown the presence of the CD3+ lymphocytes, with over 55% having high density of tumor-infiltrating lymphocytes 20 . This is in contrast with the earlystage brain tumors, which, at least in part, were previously shown to have an immunosuppressive environment, with no presence of peripheral immune cells 21,22 . Taken into consideration these results, it makes sense to consider immunotherapy as a potentially promising tumor-targeting strategy in melanoma brain metastases. Recent clinical trials have confirmed that his hypothesis is correct.

Current Treatments in Melanoma Brain Metastasis
Current therapies employed for brain metastases are generally inefficient, with very low median overall survival. They include whole brain radiation therapy (WBRT), surgery and stereotactic radiosurgery (SRS) 23 . WBRT is the standard treatment for metastatic brain tumors, with WBRT and surgical removal being used for multiple and/or large tumors and MRI-assisted SRI for smaller tumors. Tumor Treating Fields method is an additional option used in treating brain metastases [24][25][26][27] . Although successful, it may result in seizures and other CNS symptoms, such as insomnia or anxiety 27 .
Treatment of melanoma brain metastases with surgery and/or radiation therapy results in a median overall survival of only about 4-6 months after diagnosis and they cause notable complications and morbidity 7 ( Table 1). In general, SRS is preferred to WBRT in the treatment of melanoma brain metastasis 28 . Melanoma cells usually have a powerful DNA damage repair machinery, resulting in the need of delivery of larger fractions/doses of radiotherapy 29 . In contrast, chemotherapy has produced disappointing results in melanoma patients with brain metastases, and the results are similar to those obtained in melanoma treatment in general 30 .
More promising results were obtained with targeted treatments in patients presenting BRAF activating mutations. For example, vemurafenib, dabrafenib or dabrafenib in combination with trametinib are FDA approved for metastatic melanoma patients that show the BRAFV600 mutation 31,32 (see Table 2, Targeted Therapies). However, although these targeted inhibitors are effective when used, resistance emerges in most of the treated cases. Resistance occurrence in melanoma brain metastases is not well studies and the specific CNS environment may contribute to different resistance mechanisms compared to the mechanisms already described in melanoma located outside the brain 35,36 .
In recent years, already FDA approved or in clinical trials immunotherapies have shown significant promise, with several immunomodulatory drugs being able to at least double the overall survival rates for patients with melanoma brain metastases 8 . These therapies may potentially be improved by radiation 37 and they may have decreased adverse effects (e.g. decreased neurotoxicity) 38 .

Immunotherapy in Metastatic Melanoma Tumors
Current FDA approved treatment for melanoma are summarized in Table 2. Three of the most important targeted therapies used in the clinic are vemurafenib, trametinib, dabrafenib and some of their combinations, which are FDA approved regimens for melanoma treatment acting by blocking BRAF with activatory mutations, such as V600E or V600K. Notable, the chemotherapy treatment is ineffective, with only 5-20% of patients having their tumor shrink, but with no improvement in overall survival, although it was the only approved drug to treat metastatic melanoma ( Table 2).
The first immunotherapeutic to show effect against melanoma brain metastasis was high dose interleukin 2 (hdIL-2). Melanoma patients with CNS involvement require higher doses of IL-2, which is challenging due to adverse events such as neurotoxicities and the need for hydration 39 . Recently, several immunomodulatory drugs were approved for melanoma treatment, with a recent study showing that the checkpoint blockade immunotherapy can double survival rates for patients with melanoma brain metastases 8 . Patients receiving these immunomodulatory drugs showed a mean survival of ~12.5 months compared to ~5.2 months for those not receiving immunotherapy, with a 4-year survival of ~28% versus only ~11% 8,40 .
Research studies have demonstrated the CD4 and CD8 are required for limitation or prevention of brain metastases, with an important role assigned to the regulatory T cells (Treg) 41 . The most important molecules as immune checkpoints are the programmed cell death protein 1 (PD-1) and its ligand (PD-L1) and the cytotoxic T lymphocyteassociated protein 4 (CTLA-4). PD-1 is found on the T cells and its interaction with PD-L1 expressed on the cancer cells surface, causes apoptosis of cytotoxic T lymphocytes, while preventing apoptosis of Treg cells 42 . In addition, CTLA-4 is a costimulatory protein which interacts with receptors on T lymphocytes, inhibiting effector T cells. Both pathways are significant modulators of immunetumor interaction (Figure 1) and targeting them focused significant energy in the past several years, with notable successes 38 .
Stimulation of T cells in the periphery with immunomodulators have also benefic effects against CNS tumors. A recent study has shown that pembrolizumab-induced PD-1 inhibition results in 20-30% responses in CNS, in patients with melanoma of non-small lung cancer CNS metastases. Moreover, combined regimen of nivolumab and ipilimumab, which acts by both inhibiting PD-1 and CTLA-4 has notable 55% CNS response in melanoma brain metastases patients 38 . Additionally, radiation therapy (e.g. SRS) is known to sensitize melanoma brain metastases to the action of checkpoint inhibitors, such as ipilimumab 43 .
It is important to point out that, currently, there are several clinical trials underway for melanoma brain metastasis. A summary of the most important clinical trials is presented in Table 3.
Immunomodulatory drugs, such as PD-1/PD-L1 or CTLA-4 inhibitors, have a great therapeutic potential in metastatic melanoma, including melanoma brain metastases. Noteworthy, only a small percentage of the patients are actually

Challenges and Limitations
Treatment of melanoma brain metastases with surgery and/or radiation therapy results in a very low median overall survival and there are important complications and morbidity associated with these treatments, with a prominent cognitive decline (see Table 1). For example, WBRT can result in radiation toxicity, headaches, nausea, vomiting, bone marrow suppression, skin reactions, fatigue, while SRS is usually associated with neurocognitive decline, brain swelling, fatigue, skin problems, local hair loss, nausea, vomiting, headaches ( Table 1). Focal treatments such as SRS and surgery are limited to the treatment of the area of interest, which may result if tumor relapse from other not treated sites which were under the limit of detection of our imaging methods 25 .
Resistance to radiation, chemotherapy, targeted treatments and recently developed immunotherapies is one of the major challenges in treating melanoma, melanoma brain metastases and other types of malignancies. For example, in the case of immunotherapy, a significant number of patients do not respond to existing immunotherapy treatments, and the exact causes are under investigation 38 . Brain metastases are generally resistant to cancer immunotherapy. An extensive understanding of these mechanisms and causes of resistance for brain metastases is required in order to overcome this resistance. One limitation to these investigations are the current methods used to investigate the tumor and in situ tumor microenvironment of the brain, which provide limited information of a heterogeneous tissue, spatially and dynamically, in time 27 . Another limitation is the lack of preclinical models which can mimic with high accuracy human brain metastases and that can recapitulate all the steps of brain metastases development 12 . As some research group suggest, the development of intravital microscopy technologies for high resolution imaging of brain metastases can be an important step forward 27 .
The majority of patients with melanoma brain metastases will receive some form of radiation therapy. Thus, it is important to investigate how  radiotherapy interferes with targeted and immunotherapy. Although initially the radiation treatment was believed to be immunosuppressive, recent studies showed that it can actually serve a booster of the anti-tumor immune response, by increasing the availability of antigens available after radiation-induced necrosis and other mechanisms. Noteworthy, the combination of radiation with immunomodulatory drugs is more efficient than the use of each drug alone 38 .
It is now imperative to detect better biomarkers within the CNS which can guide the therapeutic strategy and can predict the response to therapy, in particular immunotherapy. For example, some studies show that higher density of CD3 and CD8 tumor-associated lymphocytes is a good prognostic factors correlated with increased survival 38 . Additionally, some treated patients with brain metastases may need control of their symptoms with steroids, which can make immunotherapy ineffective 38 . In conclusion, there are many challenges and limitations to overcome in order to better investigate, understand, develop effective therapies and significantly treat melanoma brain metastases.

Conclusion
Melanoma patients with metastatic brain tumors have very poor prognosis. However, recent therapeutic strategies, such as the use of immunomodulatory drugs, are now emerging, with several compounds already approved by the FDA and other ones in clinical trials. Immunotherapy has already revolutionized the treatment of melanoma and other malignancies, with very effective results and low adverse events for some of the treated patients. However, many of the patients are resistant to immunotherapy and it is imperative to find out the exact mechanisms and how the tumor can be rendered sensitive. The preliminary studies in melanoma brain metastasis show significant promise and require additional investigation. In conclusion, immunotherapy and immunomodulatory drugs bring a great promise as new tools for melanoma treatment in particular and for the treatment of other types of malignancies in general.