A simple blood test could soon become the latest monitoring tool for the early detection of melanoma in the eye.
University of Queensland scientists have discovered markers in the blood that can differentiate between a benign mole and a melanoma, while also identifying if the cancer has spread to other areas of the body.
University of Queensland Diamantina Institute’s Dr. Mitchell Stark said the blood test could monitor very early signs of the disease.
“This blood test was able to detect the difference between a benign mole located at the back of the eye and a melanoma in the eye,” Dr. Stark said.
“The test also has the potential to show if the melanoma has metastasised and spread to other areas of the body.
“Moles or naevi in the eye are common, but can be difficult to monitor because changes to their shape or colouring can’t always be seen as easily as on the skin.
“Outcomes are poor for people with melanoma in their eye if their cancer spreads to the liver.
“Given that having naevi in the eye is fairly common, this test may allow us to better screen these patients for early signs of melanoma formation.”
The study is a progression of research conducted by Dr. Stark at QIMR Berghofer, where the panel of biomarkers was first developed and used to detect melanoma on the skin.
In this research, blood samples were collected from people with either benign naevi or melanoma in the back of their eye, in addition to a small number of metastasised cases.
The samples were then tested against the panel of microRNA biomarkers to distinguish the stage of disease.
Dr. Stark said after further development, the blood test had the potential to be used as a monitoring tool in conjunction with optometrists, GPs, and specialists.
“If someone went to their optometrist for a regular check-up and a mole was found, you could have this blood test at each routine visit to help monitor mole changes,” he said.
“If the biomarker in the blood had increased, it might be an early warning sign of melanoma.
“Knowing this patient was high-risk means they could be monitored more closely for the potential spread of cancer and be progressed more rapidly through the healthcare system.”
Queensland Ocular Oncology Service Director and ophthalmologist Dr. Bill Glasson AO said the test would be extremely helpful in clinical practice.
“These research findings are exciting for our patients with ocular tumours,” Dr. Glasson said.
“It will allow for earlier diagnosis as well as giving doctors an earlier indication of the development of metastatic disease and importantly, a better outcome for our patients.”
No standard treatment has been established for metastatic uveal melanoma (mUM). Immunotherapy is commonly used for this disease even though UM has not been included in phase III clinical trials with checkpoint inhibitors. Unfortunately, only a minority of patients obtain a clinical benefit with immunotherapy. The immunological features of mUM were reviewed in order to understand if immunotherapy could still play a role for this disease.
Standard treatments for metastatic uveal melanoma (mUM) have not been defined yet. Several treatments have been employed with poor results [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]. UM was not included in phase III clinical trials with immunotherapy for melanoma, due to the specific biological behavior and the different clinical outcome [3,4,5,6,7,8,30,31,32,33]. Nevertheless, immune checkpoint inhibitors (ICIs) are commonly used for the treatment of metastatic UM [3,4,5,6,7,8].
The available reports with ICIs in UM showed limited results in terms of efficacy, whereas they demonstrated a favorable tolerability of these agents (Table 1).
Clinical studies with immune checkpoint inhibitors (ICIs) in uveal melanoma.
|Authors||Treatment||Type of Study||No. of Enrolled Patients||Year|
|Zimmer et al. ||Ipilimumab||Phase II trial. Pre-treated and naïve patients.||53||2015|
|Maio et al. ||Ipilimumab||Retrospective analysis. Pre-treated patients.||82||2013|
|Kelderman et al. ||Ipilimumab||Retrospective analysis. Pre-treated patients.||22||2013|
|Luke et al. ||Ipilimumab||Retrospective, multi-center analysis. Pre-treated and naïve patients.||39||2013|
|Piulats Rodriguez et al. ||Ipilimumab||Phase II trial. Naïve patients||32||2014|
|Danielli et al. ||Ipilimumab||Retrospective analysis. Pre-treated patients.||13||2012|
|Khattak et al. ||Ipilimumab||Retrospective analysis, single center analysis. Pre-treated patients.||5||2016|
|Deo ||Ipilimumab||Retrospective, single center analysis. Pre-treated patients.||24||2014|
|Shaw et al. ||Ipilimumab||EAP.||18||2012|
|Joshua et al. ||Tremelimumab||Phase II trial. Naïve patients.||11||2015|
|Algazi et al. ||Pembrolizumab, Nivolumab, Atezolizumab||Retrospective, multi-center analysis. Pre-treated and naïve patients.||56||2016|
|Mignard et al ||Pembrolizumab, Nivolumab, Ipilimumab||Retrospective, multi-center analysis.||100||2018|
|Bender et al. ||Pembrolizumab, Nivolumab||Retrospective, multi-center analysis. Pre-treated patients.||15||2017|
|Heppt et al. ||Pembrolizumab, Nivolumab, Ipilimumab||Retrospective, multi-center analysis. Pre-treated and naïve patients.||96||2017|
|Piperno-Neumann et al. ||Pembrolizumab, Nivolumab||Retrospective, single center analysis. Naïve patients.||21||2016|
|Karydis et al. ||Pembrolizumab||Retrospective analysis. Pre-treated patients.||25||2016|
|Rossi et al. ||Pembrolizumab||Prospective. Naïve patients.||17||2019|
|Kottschade et al. ||Pembrolizumab||Retrospective, single-center analysis. Pre-treated patients.||8||2016|
|Van der Kooij et al. ||Pembrolizumab||Prospective. Pre-treated and naïve patients.||17||2017|
|Schadendorf et al. ||Nivolumab||Phase II. Pre-treated patients.||75||2017|
|Jung et al. ||Ipilimumab||Named patient use. Pre-treated patients.||10||2017|
|Shoushtari et al. ||Nivolumab, Ipilimumab||Expanded access program.||6||2016|
EAP: Expand Access Program.
Increasing evidence demonstrates that uveal melanoma cells employ escape mechanisms to elude the immune system . The environment of the eye is an important immune-privileged site, with many immunosuppressive mechanisms that primary UM cells retain to gain immune-protection even when they leave the eye.
The immune-modulatory microenvironment of the liver, the typical site of metastasis for UM, could further protect escaped UM cells from immune surveillance . Furthermore, the low mutational burden is considered another relevant characteristic of uveal melanoma, which could justify the limited activity of immunotherapy .
Nevertheless, a minority of patients treated with immunotherapy obtain a clinical benefit .
In this review, we aim to describe the immunological features of UM in order to understand if it could still be a rationale for using immunotherapy in this disease.
Microenvironment of the Eye
The eye is considered a “privileged immunological site”  due to different mechanisms of immune protection.
The first is represented by the blood–ocular barrier: the tight junctions and the lack of lymphatic vessels in the cornea and uvea limit the circulation of immune cells .
The other mechanism involved is represented by soluble immunosuppressive factors in the aqueous humor . They are: transforming growth factor (TGF)-β, α-melanocyte-stimulating hormone (α-MSH), calcitonin gene-related peptide (CGRP), vasoactive intestinal protein (VIP), and indoleamine 2,3 dioxygenase (IDO). TGF-β inhibits the activation of macrophages, T lymphocytes, and natural killer (NK) cells and enhances the tolerance of antigen-presenting cells (APCs) . TGF-β is also required for CTLA-4 up-regulation on CD8+ T cells. CTLA-4 stimulation leads to T cell inactivation and generation of T regulatory (Treg) cells . α-MSH reduces neutrophil activities and stimulates Tregs. α-MSH and CGRP downregulate the production of pro-inflammatory factors. VIP inhibits NK-cells mediated cytolysis. VIP and IDO inhibit T cell activation .
Moreover, the cornea, iris, and retina cells express immunosuppressive ligands on their surface, such as PD-L1 and FasL. PD-L1 suppresses proliferation and induces T-lymphocyte and neutrophil apoptosis when it recognizes its ligand, PD-1, on these cells. First apoptosis signal ligand (FasL, a member of the TNF family) promotes apoptosis of activated T cells . Furthermore, complement regulatory proteins (CRPs) are capable of interrupting the complement cascade with the inhibition of complement-mediated cytolysis in the eye .
Corneal and retinal cells express MHC-Ib molecules (such as HLA-G and HLA-E); in this way, they are able to inhibit natural killer cell cytotoxic activity, binding the inhibitory receptors, such as CD94-NKG2. The interaction between MHC-I molecules and inhibitory receptors delivers ‘‘off signals’’ to NK cells, blocking their ability to kill target cells. On the other hand, the MHC class Ia molecules are down-regulated on corneal and retinal cells, reducing the susceptibility to T-cell mediated cytolysis .
When antigens enter the eye, a unique mechanism of immune privilege is involved: it is termed “anterior chamber-associated immune deviation” (ACAID), an immunomodulatory phenomenon involving the eye, but also the thymus, spleen, and sympathetic nervous system . In mouse models an antigen injected into the anterior chamber of the eye is captured by ocular APCs. Then, these APCs migrate to the spleen and thymus, inducing immunomodulatory cells, such as Tregs, regulatory B cells, and NK T cells, which cause immune deviation. The sympathetic nervous system promotes the maintenance of a functional ACAID .
Immune Infiltrate in Uveal Melanoma
The crosstalk between tumor and microenvironment influences the inflammatory response: cancer cells interact with both the innate and the adaptive immune system and use immune cells for tumor survival and protection from immunological attacks. The main immune cells in uveal melanoma are the M2-type macrophages, which foster tumor growth through angiogenesis and immunosuppression [48,49].
Monocytes/macrophages of M2 lineage exceed T cells in tumor-infiltrating immune cells. They secrete anti-inflammatory cytokines (IL-10 and TGF-β), which inhibit dendritic cell activation, as well as T- and NK-cell functions [35,48] (Figure 1). Treg and UM cells trigger inflammation and M2-polarization through the production of factors, such as CCL22, CCL2, VEGF, M-CSF, TGF-β, IL-6, IL-10, CCL17, CCL22, PGE2, and endothelial monocyte-activating polypeptide (EMAP)-II [35,48,50]. Moreover, M2-macrophages can secrete soluble factors, which enhance the invasive capabilities of neoplastic cells, such as the melanoma inhibitory activity (MIA), a growth-regulatory protein, which inhibits the cellular adhesion to the extracellular matrix .
The analysis of uveal melanoma suspensions has revealed that the majority of tumor infiltrating lymphocytes (TILs) are CD8+ T cells with fewer CD4+ T cells . Lagouros reported a limited number of Tregs in primary UM . Nevertheless, the count of Tregs within primary tumor correlates with the development of systemic metastases. In addition, Tregs and cyclooxygenase-2 (COX-2) expression in primary tumors are associated with a poor prognosis . Tregs are recruited by IL-10, chemokine (C–C motif) ligand (CCL) 17, and CCL22 produced by M2 macrophages .
Tumor-associated macrophages (TAMs) are a negative prognostic factor for UM . Indeed, TAMs are associated with highly malignant tumors characterized by negative prognostic features, such as epithelioid cells, high microvascular density, intense pigmentation, and larger size . TAMs seem to promote tumor growth through angiogenesis and metastatic spread [55,57].
In primary UM, the expression of HLA class I and II exerts a prognostic role . It has been reported that, in primary tumors, down-regulation of HLA class I is a mechanism for evading CD8+ cell cytotoxicity. Therefore, tumors should be more sensitive to NK cells [59,60,61,62]. Nevertheless, only 50% of primary UM expresses MHC class I related chain (MIC) A and B, which are the ligands for NK cell receptor (NKG2D). In UM metastases, MIC A and B are absent. Consequently, the activity of NK cells in metastatic UM is limited. Vetter reported a single case of MIC expression induced after chemotherapy, suggesting a possible role for immunotherapy following cytotoxic therapy . It has also been proved that UM cells are capable of producing the macrophage migration inhibitory factor (MIF), a cytokine that inhibits cytolytic activity of NK cells, contributing to tumor growth and metastatic spread [63,64,65]. The expression of FasL is a further mechanism in UM explaining the escape from NK cells . Additionally, TILs and TAMs of UM produce IL-2 and IL-15, which bind receptors on tumor cells, promoting UM cell growth and reducing sensitivity to NK cell activity .
Moreover, the loss of the maturation marker CD-40 on APCs has been observed in primary UM. This lack does not allow correct lymphocyte T-mediated anti-tumor activity because of an inadequate functioning of APCs to induce T cell activation .
Overall, “inflammatory phenotype” has been proposed to define UM with infiltrating macrophages and lymphocytes in addition to a high expression of HLA class I and II molecules. It identifies tumors with a worse prognosis [48,49].
It is doubtless that the immune cells in the UM inflammatory phenotype do not stimulate an antitumor response. They contribute to angiogenesis, immunosuppression, tumor growth, and metastatic spread . Moreover, comparing the circulating immune cells between primary and metastatic UM, a weaker immune surveillance has been spotted in case of metastases. Lower circulating CD3−CD56dim NK cells, CD8+, and NK T cells, as well as an increase in Tregs and MSDCs have been detected during the metastatic phase. Furthermore, metastases are associated with increasing plasma levels of several miRNAs involved in immune regulation .
In the metastatic sites, CD4+ cells are present in perivascular aggregates, while CD8+ cells are scarce and mainly surround the liver metastases [71,72,73,74]. Tumor cells and resident hepatic cells recruit in metastatic sites two different types of myeloid-derived suppressor cells (MDSCs): monocytic MDSCs and polymorphonuclear MDSCs. Monocytic MDSCs, more frequent than polymorphonuclear MDSCs in metastases, promote neoplastic growth after their differentiation towards TAMs. MDSCs are able to induce Tregs releasing IL-10 and TGF-β [35,71] (Figure 1).
Nevertheless, Rothermel found that a subset of TILs associated with a lack of melanin pigmentation is more effective in antitumor response, similarly to TILs in cutaneous melanoma .
More information: Mitchell S. Stark et al, A Panel of Circulating MicroRNAs Detects Uveal Melanoma With High Precision, Translational Vision Science & Technology (2019). DOI: 10.1167/tvst.8.6.12
Provided by University of Queensland