Uveal melanoma (UM) with an inflammatory phenotype, characterized by infiltrating leukocytes and increased human leukocyte antigen (HLA) expression, carry an increased risk of death due to ...metastases. These tumors should be ideal for T-cell based therapies, yet it is not clear why prognostically-infaust tumors have a high HLA expression. We set out to determine whether the level of HLA molecules in UM is associated with other genetic factors, HLA transcriptional regulators, or microenvironmental factors.
28 enucleated UM were used to study HLA class I and II expression, and several regulators of HLA by immunohistochemistry, PCR microarray, qPCR and chromosome SNP-array. Fresh tumor samples of eight primary UM and four metastases were compared to their corresponding xenograft in SCID mice, using a PCR microarray and SNP array.
Increased expression levels of HLA class I and II showed no dosage effect of chromosome 6p, but, as expected, were associated with monosomy of chromosome 3. Increased HLA class I and II protein levels were positively associated with their gene expression and with raised levels of the peptide-loading gene TAP1, and HLA transcriptional regulators IRF1, IRF8, CIITA, and NLRC5, revealing a higher transcriptional activity in prognostically-bad tumors. Implantation of fresh human tumor samples into SCID mice led to a loss of infiltrating leukocytes, and to a decreased expression of HLA class I and II genes, and their regulators.
Our data provides evidence for a proper functioning HLA regulatory system in UM, offering a target for T-cell based therapies.
The aim of this paper is to inform on the surge of cases of acute macular neuroretinopathy (AMN) – a rare disease characterized by the sudden onset of acute scotomas caused by ischemia of the retinal ...capillary plexus – during the COVID-19 pandemic. In 2021, during the COVID-19 pandemic, a sudden rise in patients with AMN was observed in our clinic. In this paper, 4 cases from a hospital in the south of the Netherlands are reported, all of which could directly be linked to a COVID-19 infection or vaccination against the corona virus. A search for similar cases in the PubMed database produced thirteen relevant reports, which revealed that a similar increase in cases of AMN, all linked to COVID-19, has been observed worldwide. Analysis of the literature revealed that AMN is seen more often during the pandemic and that AMN after COVID-19 happens at a significantly older age than typically reported. This is the largest case series of patients with AMN after COVID-19 infection or vaccination. With the ongoing pandemic and extensive vaccination programs, it is expected that cases of AMN will surge. It is important for ophthalmologists to be aware of this disease, especially since typical patient characteristics may differ.
Aim To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk ...for metastases, following enucleation of the primary tumour. Methods Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining. Results The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010). Conclusions Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.
Uveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of ...infiltrating macrophages and T cells is associated with a poor prognosis rather than a good one. The clear link between inflammation and cancer in this malignancy provides a paradigm for macrophage plasticity and function. Macrophages in uveal melanoma have an M2-like phenotype and are associated with the loss of one specific chromosome – monosomy 3. The central players involved in this process and discussed in this review include macrophages, T lymphocytes, chemokines and cytokines, including the macrophage-attraction molecules. When a tumor acquires the ability to release significant amounts of macrophage-attraction molecules it causes the expansion of a population of myeloid immature cells that may not only help the tumor to suppress immune reactions but also aid in the construction of new blood vessels for tumor growth. A better understanding of the molecular basis of a local myelomonocytic cell population will bring a better understanding of the immunopathology of this disease and will lead to therapeutic interventions in uveal melanoma. This review focuses on the roles of the local inflammatory microenvironment in the development and progression of uveal melanoma.
Increased angiogenesis is associated with a higher metastasis- and mortality rate in uveal melanoma (UM). Recently, it was demonstrated that genetic events, such as 8q-gain and BAP1-loss, influence ...the level of immune infiltrate. We aimed to determine whether genetic events, and specific cytokines, relate to angiogenesis in UM. Data from UM patients who underwent enucleation between 1999 and 2008 were analysed. Microvascular density (MVD) and the presence of infiltrating immune cells were determined with immunohistochemistry (IHC) and immunofluorescence in 43 cases. Chromosome status, BAP1 IHC and mRNA expression of angiogenesis-related genes were known in 54 cases. Tumours with monosomy 3/BAP1-loss showed a higher MVD compared to tumours with disomy 3/normal BAP1 expression (
= 0.008 and
= 0.004, respectively). Within BAP1-positive lesions (
= 20), 8q-gain did not relate to MVD (
= 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (
= 0.041), Von Willebrand Factor (VWF) (
= 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (
= 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+,
= 0.017; CD68+CD163+,
= 0.031) and lymphocytes (CD4+,
= 0.027). Concluding, vascular density of UM relates to its genetic profile: Monosomy 3 and BAP1-loss are associated with an increased MVD, while an early event (gain of 8q) is not independently related to MVD, but may initiate a preparation phase towards development of vessels. Interestingly, VEGF-B expression is decreased in UM with a high MVD.
In contrast to many other malignancies, in uveal melanoma (UM) the presence of an immune infiltrate is associated with a bad prognosis. An analysis of the different functional phenotypes of ...tumor-infiltrating leukocytes (TIL) and a comparison with the genetic background of the tumors may help to explain this apparent anomaly.
We performed a comprehensive immunohistochemical study by evaluating the density of CD8(+) and CD4(+) T lymphocytes, forkhead box p3 (Foxp3(+)) regulatory T cells (Tregs), and CD68(+) and CD68(+)CD163(+) macrophages in 43 cases of UM in relation to tumor characteristics. Expression of the chemokines CCL2, CCL17, and CCL22 in cultured human UM cells and peripheral blood monocytes was analyzed by quantitative PCR (qPCR).
The presence of TILs was highly variable between tumors and was dominated by CD8(+) T cells with fewer CD4(+) T cells and Tregs. When tumors were infiltrated by immune cells, the infiltrate generally comprised all different subsets of lymphocytes (P < 0.001) and M2 macrophages (P < 0.001). Different T-cell ratios did not influence clinical outcome. In addition, the presence of TIL correlated with the loss of one chromosome 3 (P < 0.04). UM cells express CCL2 and CCL22, two chemokines known to mediate trafficking of immune cells to the tumor.
All studied subtypes of tumor-infiltrating immune cells were collectively increased and showed an association with monosomy of chromosome 3 suggesting that tumor intrinsic factors control the leukocyte influx, possibly through local chemokine secretion.
The presence of a high number of infiltrating macrophages in uveal melanoma is associated with a bad prognosis. However, there are several known types of macrophages, of which the M2 is considered to ...be proangiogenic and tumor-promoting. This study was conducted to determine whether the tumor-infiltrating macrophages in uveal melanoma are of this M2 subtype.
Macrophages were identified in sections from 43 uveal melanomas by immunofluorescence histochemistry, using monoclonal antibodies directed against CD68 and CD163. The immunopositive cell density was measured visually and with a confocal microscope and calculated per square millimeter. Results were compared with clinical and tumor characteristics.
Infiltrating macrophages in uveal melanoma were predominantly CD68(+)CD163(+), thus of the M2 phenotype. The density of CD68(+), CD163(+), and CD68(+)CD163(+) cells was significantly increased in uveal melanomas with monosomy 3 compared with cases with disomy of chromosome 3 and was associated with ciliary body involvement. High CD68(+)CD163(+) staining was associated with an increased microvascular density. Survival was significantly better among patients with low CD68(+) and CD68(+)CD163(+) staining.
The main type of macrophage present in uveal melanoma was the M2 type. Tumors with monosomy of chromosome 3 contained a higher number of M2-macrophages than tumors with disomy of chromosome 3. Infiltration of M2-type macrophages gives a worse prognosis for survival. As M2-type macrophages are proangiogenic, a high density of these cells may contribute to the previously noticed positive association between the density of CD68(+) macrophages and blood vessels.
In uveal melanoma, both tumor size and an inflammatory phenotype have been shown to correlate with a poor clinical prognosis. The purpose of this study was to identify whether inflammatory cytokines ...were present in the vitreous of eyes with uveal melanoma and to determine whether the vitreal concentration of cytokines correlated with prognostic variables such as tumor dimensions and immune cell infiltrate.
Vitreous was acquired from patients with uveal melanoma (n = 33) and from control eyes with no known ocular conditions (n = 9), and analyzed using a 27-plex cytokine bead array. Immunofluorescence testing was performed to determine the presence of macrophages, CD4(+), CD8(+), and Foxp3(+) regulatory T cells (T(regs)).
Compared with control eyes, eyes with uveal melanoma demonstrated higher vitreal concentrations of many cytokines and chemokines, including IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, and RANTES. IL-1ra was decreased in the vitreous of tumor-bearing eyes compared with controls. Tumor prominence positively correlated with several cytokines, including IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, RANTES, GCSF, IFN-γ, and VEGF. IL-6 and IP-10 were found to positively correlate with increasing regulatory T-cell infiltrate and IL-6 alone positively correlated with macrophage infiltration.
Eyes with uveal melanoma contain higher vitreal concentrations of several inflammatory cytokines and chemokines that correlate predominantly with increasing tumor size; elevations in certain cytokines and chemokines also correlate with the presence of immune cell infiltrate.
IMPORTANCE Among the characteristics of uveal melanoma that are associated with a poor prognosis are a large tumor size and the presence of increased numbers of lymphocytes and macrophages. In ...rapidly growing tumors, reduction in oxygen tension may occur with increased distance from blood vessels, which we hypothesize may lead to an inflammatory microenvironment, further stimulating tumor growth. OBJECTIVES To analyze whether hypoxia induces uveal melanoma cells to express proinflammatory cytokines and whether tumor supernatant (TSN) affects monocyte migration and differentiation. DESIGN AND SETTING The expression of proinflammatory genes in freshly cultured uveal melanoma samples was studied in an in vitro 24-hour hypoxic culture system using quantitative polymerase chain reaction. In addition, cell lines cultured under normoxic and hypoxic conditions were used. The effect of TSN on monocyte chemotaxis was tested using a transwell migration system and by analyzing monocyte differentiation. The levels of the cytokines CCL2, IL6, and PGE2 in TSN were determined by enzyme-linked immunosorbent assay. PARTICIPANTS Five cell lines (OCM8, 92.1, Mel270,Mel290 and OMM2.5) and 11 primary short-term cultures. RESULTS Exposure of freshly cultured uveal melanoma cells to hypoxia led to an increased expression of the proinflammatory cytokines PLGF (OMIM 601121), TGFβ (OMIM 190180), END1 (OMIM +131240), and ICAM1 (OMIM 147840) and a lower expression of AIMP1 (OMIM 603605) (EMAP2), CCL2 (MCP-1) (OMIM +158105), and IL1b (OMIM *147720). The TSN from cultured melanoma cell lines induced chemotaxis of monocytes, but this was independent of the normoxic or hypoxic state. The TSN of 1 cell line and 2 primary uveal melanoma cultures inhibited the dendritic cell maturation and did not induce M2 macrophage polarization in vitro. CONCLUSIONS AND RELEVANCE Our results indicate that under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. The TSN from uveal melanoma cell lines is capable of affecting the chemotactic response and maturation of monocytes in vitro, but this is irrespective of hypoxia.
Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes ...influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes.
We used an in vitro coculture system in which UM cell lines and T cells were cultured together, but separated by a membrane. Uveal melanoma gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a transwell plate was used.
Gene-expression analysis of UM cell lines showed that coculture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, VEGF, intracellular adhesion molecule 1 (ICAM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant toward monocytes.
Cytokines derived from activated T cells shifted the UM cell transcriptome toward a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor-promoting inflammatory microenvironment.
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