PurposeHeavy pigmentation is known to be a prognostic risk factor in uveal melanoma (UM). We analyzed whether genetic tumor parameters were associated with tumor pigmentation and whether... Show morePurposeHeavy pigmentation is known to be a prognostic risk factor in uveal melanoma (UM). We analyzed whether genetic tumor parameters were associated with tumor pigmentation and whether pigmentation should be included in prognostic tests.DesignRetrospective comparison of clinical, histopathological, and genetic features and survival in UM with different pigmentation.ParticipantsA total of 1058 patients with UM from a White European population with diverse eye colors enucleated between 1972 and 2021.MethodsCox regression and log-rank tests were used for survival analysis; the chi-square test and Mann–Whitney U test were used for correlation analysis.Main Outcome MeasuresUveal melanoma–related survival based on tumor pigmentation and chromosome status, correlation of tumor pigmentation with prognostic factors.ResultsThe 5-year UM-related mortality was 8% in patients with nonpigmented tumors (n = 54), 25% with lightly pigmented tumors (n = 489), 41% with moderately pigmented tumors (n = 333), and 33% with dark tumors (n = 178) (P < 0.001). The percentage of tumors with monosomy 3 (M3) or 8q gain increased with increasing pigmentation (31%, 46%, 62%, and 70% having M3 [P < 0.001], and 19%, 43%, 61%, and 63% having 8q gain [P < 0.001] in the 4 increasing pigment groups, respectively). BRCA-associated protein 1(BAP1) loss (known for 204 cases) was associated with increased tumor pigmentation (P = 0.001). Cox regression analysis on survival showed that when chromosome status and pigmentation were both included, pigmentation was not an independent prognostic indicator. Preferentially expressed antigen in melanoma (PRAME) expression was a significant prognostic marker in light tumors (P = 0.02) but not in dark tumors (P = 0.85).ConclusionsPatients with moderately and heavily pigmented tumors showed a significantly higher UM-related mortality than patients with unpigmented and light tumors (P < 0.001), supporting prior reports on the relation between increased tumor pigmentation and a worse prognosis. Although we previously showed that a dark eye color was associated with tumor pigmentation, we now show that the tumor’s genetic status (chromosome 3 and 8q/BAP1 status) is also related to tumor pigmentation. When pigmentation and chromosome 3 status are both included in a Cox regression analysis, pigmentation is not an independent prognostic factor. However, evidence from this and previous studies shows that chromosome changes and PRAME expression have a stronger association with survival when they occur in light tumors than in dark ones. Show less
Uveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there... Show moreUveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there is a great need to harness the limited material available from primary tumors and metastases for advanced research and preclinical drug screening. We established a platform to isolate, preserve, and transiently recover viable tissues, followed by the generation of spheroid cultures derived from primary UM. All assessed tumor-derived samples formed spheroids in culture within 24 h and stained positive for melanocyte-specific markers, indicating the retention of their melanocytic origin. These short-lived spheroids were only maintained for the duration of the experiment (7 days) or re-established from frozen tumor tissue acquired from the same patient. Intravenous injection of fluorescently labeled UM cells derived from these spheroids into zebrafish yielded a reproducible metastatic phenotype and recapitulated molecular features of the disseminating UM. This approach allowed for the experimental replications required for reliable drug screening (at least 2 individual biological experiments, with n > 20). Drug treatments with navitoclax and everolimus validated the zebrafish patient-derived model as a versatile preclinical tool for screening anti-UM drugs and as a preclinical platform to predict personalized drug responses. Show less
Purpose: Individuals with gray, blue, or green eyes have a higher chance of developing uveal melanoma (UM) than those with brown eyes. We wondered whether iris pigmentation might be related not... Show morePurpose: Individuals with gray, blue, or green eyes have a higher chance of developing uveal melanoma (UM) than those with brown eyes. We wondered whether iris pigmentation might be related not only to predisposition to UM but also to its behavior; therefore, we compared the clinical, histopathologic, and genetic characteristics of UM between eyes with different colors. Design: We determined iris color in a large cohort of patients enucleated for UM. Clinical and histopathologic tumor characteristics, chromosome status, and survival were compared among 3 groups on the basis of iris color. Participants: A total of 412 patients with choroidal/ciliary body UM, who had undergone primary enucleation at the Leiden University Medical Center, Leiden, The Netherlands, between 1993 and 2019, were divided into 3 groups based on iris color: gray/blue, green/hazel, and brown. The validation cohort included 934 patients with choroidal/ciliary body UM treated at Wills Eye Hospital (WEH). Methods: Comparison of clinical, histopathologic, and genetic characteristics of UM in patients with different iris colors. Main Outcome Measures: Melanoma-related survival in UM patients, divided over 3 iris color groups, in relation to the tumor's chromosome 3 and 8q status. Results: Moderate and heavy tumor pigmentations were especially seen in eyes with a brown iris (P < 0.001). Survival did not differ between patients with different iris colors (P = 0.27); however, in patients with a light iris, copy number changes in chromosome 3 and 8q had a greater influence on survival than in patients with a dark iris. Likewise, chromosome 3 and chromosome 8q status affected survival more among patients with lightly pigmented tumors than in patients with heavily pigmented tumors. The WEH cohort similarly showed a greater influence of chromosome aberrations in light-eyed individuals. Conclusions: Although iris color by itself did not relate to UM-related survival, chromosome 3 and 8q aberrations had a larger influence on survival in patients with a light iris than those with a brown iris. This suggests a synergistic effect of iris pigmentation and chromosome status in the regulation of oncogenic behavior of UM. Iris color should be taken into consideration when calculating a patient's risk for developing metastases. (C) 2021 by the American Academy of Ophthalmology. Show less
Souri, Z.; Wierenga, A.P.A.; Kroes, W.G.M.; Velden, P.A. van der; Verdijk, R.M.; Eikmans, M.; ... ; Jager, M.J. 2021
Simple Summary Uveal melanoma (UM) is a rare type of intraocular malignancy, which often gives rise to metastases. While treatment with immune checkpoint inhibitors is often effective in the... Show moreSimple Summary Uveal melanoma (UM) is a rare type of intraocular malignancy, which often gives rise to metastases. While treatment with immune checkpoint inhibitors is often effective in the treatment of cutaneous melanoma metastases, it is hardly effective in the case of UM metastases. Lymphocyte-activation gene 3 (LAG3) is a recently recognized immune checkpoint; we determined the distribution of LAG3 expression and its ligands in three sets of primary UM. High-risk UM (epithelioid cell type, loss of chromosome 3/BAP1 staining) had a higher expression of LAG3 and its ligands, which correlated with the presence of infiltrating immune cells. We concluded that not only LAG3 but also its ligands Galectin-3 and HLA class II are especially expressed in high-risk UM and may be a target for adjuvant immunotherapy in UM. Uveal melanoma (UM) is a rare ocular malignancy which originates in the uveal tract, and often gives rise to metastases. Potential targets for immune checkpoint inhibition are lymphocyte-activation gene 3 (LAG3) and its ligands. We set out to analyse the distribution of these molecules in UM. The expression of mRNA was determined using an Illumina array in 64 primary UM from Leiden. The T lymphocyte fraction was determined by digital droplet PCR. In a second cohort of 15 cases from Leiden, mRNA expression was studied by Fluidigm qPCR, while a third cohort consisted of 80 UM from TCGA. In the first Leiden cohort, LAG3 expression was associated with the presence of epithelioid cells (p = 0.002), monosomy of chromosome 3 (p = 0.004), and loss of BAP1 staining (p = 0.001). In this Leiden cohort as well as in the TCGA cohort, LAG3 expression correlated positively with the expression of its ligands: LSECtin, Galectin-3, and the HLA class II molecules HLA-DR, HLA-DQ, and HLA-DP (all p < 0.001). Furthermore, ligands Galectin-3 and HLA class II were increased in monosomy 3 tumours and the expression of LAG3 correlated with the presence of an inflammatory phenotype (T cell fraction, macrophages, HLA-A and HLA-B expression: all p < 0.001). High expression levels of LAG3 (p = 0.01), Galectin-3 (p = 0.001), HLA-DRA1 (p = 0.002), HLA-DQA1 (p = 0.04), HLA-DQB2 (p = 0.03), and HLA-DPA1 (p = 0.007) were associated with bad survival. We conclude that expression of the LAG ligands Galectin-3 and HLA class II strongly correlates with LAG3 expression and all are increased in UM with Monosomy 3/BAP1 loss. The distribution suggests a potential benefit of monoclonal antibodies against LAG3 or Galectin-3 as adjuvant treatment in patients with high-risk UM. Show less
Simple Summary We hypothesize that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, may play a role in tumor development in uveal melanoma.... Show moreSimple Summary We hypothesize that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, may play a role in tumor development in uveal melanoma. We determined the expression of these cytokines in both uveal melanoma tissue as well as in aqueous humor, which provides a unique combination of data. We observed that ANG-2, in contrast to ANG-1, showed more expression in high-risk tumors and was associated with the development of metastases. Its presence in aqueous humor correlated with expression in tumor tissue. Knowledge about the expression of these cytokines may help to identify targets for personalized treatment. Uveal melanoma (UM) metastasize haematogeneously, and tumor blood vessel density is an important prognostic factor. We hypothesized that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, might play a role in tumor development and metastatic behavior. mRNA levels of ANG-1 and ANG-2 were determined in 64 tumors using an Illumina HT-12 v4 mRNA chip and compared to clinical, pathologic, and genetic tumor parameters. Tissue expression was also determined by immunohistochemistry (IHC). Samples of aqueous humor were collected from 83 UM-containing enucleated eyes and protein levels that were determined in a multiplex proximity extension assay. High tissue gene expression of ANG-2, but not of ANG-1, was associated with high tumor thickness, high largest basal diameter, involvement of the ciliary body, and with UM-related death (ANG-2 mRNA p < 0.001; ANG-2 aqueous protein p < 0.001). The presence of the ANG-2 protein in aqueous humor correlated with its mRNA expression in the tumor (r = 0.309, p = 0.03). IHC showed that ANG-2 was expressed in macrophages as well as tumor cells. The presence of ANG-2 in the tumor and in aqueous humor, especially in high-risk tumors, make ANG-2 a potential targetable cytokine in uveal melanoma. Show less
Simple Summary Uveal melanoma (UM) is an ocular malignancy which is derived from melanocytes in the uveal tract. Epigenetic regulators such as Histone Deacetylase (HDACs) inhibitors are being... Show moreSimple Summary Uveal melanoma (UM) is an ocular malignancy which is derived from melanocytes in the uveal tract. Epigenetic regulators such as Histone Deacetylase (HDACs) inhibitors are being tested as treatment of UM metastases. Expression of different HDACs is variable, and some are increased in high-risk tumors with loss of one chromosome 3. As this genetic abnormality is also associated with an inflammatory phenotype, we analyzed whether HDAC expression was influenced by inflammation. In two cohorts of UM cases, expression of several HDACs showed a positive correlation with tumor-infiltrating T cells, while HDACs 2 and 11 showed a negative association with macrophages. Interferon-gamma stimulated expression of some HDACs on UM cell lines. These data suggest that cytokines produced by T cells may be responsible for the increased expression of some HDACs in UM with monosomy 3. In Uveal Melanoma (UM), an inflammatory phenotype is strongly associated with the development of metastases and with chromosome 3/BAP1 expression loss. As an increased expression of several Histone Deacetylases (HDACs) was associated with loss of chromosome 3, this suggested that HDAC expression might also be related to inflammation. We analyzed HDAC expression and the presence of leukocytes by mRNA expression in two sets of UM (Leiden and TCGA) and determined the T lymphocyte fraction through ddPCR. Four UM cell lines were treated with IFN gamma (50IU, 200IU). Quantitative PCR (qPCR) was used for mRNA measurement of HDACs in cultured cells. In both cohorts (Leiden and TCGA), a positive correlation occurred between expression of HDACs 1, 3 and 8 and the presence of a T-cell infiltrate, while expression of HDACs 2 and 11 was negatively correlated with the presence of tumor-infiltrating macrophages. Stimulation of UM cell lines with IFN gamma induced an increase in HDACs 1, 4, 5, 7 and 8 in two out of four UM cell lines. We conclude that the observed positive correlations between HDAC expression and chromosome 3/BAP1 loss may be related to the presence of infiltrating T cells. Show less
Simple Summary Uveal melanoma (UM) is a rare ocular malignancy that often gives rise to metastases. Tumours with an inflammatory phenotype have an especially bad prognosis. As an increased HLA... Show moreSimple Summary Uveal melanoma (UM) is a rare ocular malignancy that often gives rise to metastases. Tumours with an inflammatory phenotype have an especially bad prognosis. As an increased HLA expression and the presence of tumour-infiltrating lymphocytes and macrophages may be regulated by miRNAs, we set out to investigate whether any miRNAs are associated with inflammatory parameters in this malignancy. Some miRNAs were increased in UM with a high HLA expression and high T cell numbers, while others were decreased, showing two opposing patterns; however, both patterns were related to the tumour's chromosome 3/BAP1 status. We conclude that specific miRNAs are related to the inflammatory phenotype and that these are differentially expressed between disomy 3/BAP1-positive versus monosomy 3/BAP1-negative UM. MicroRNAs are known to play a role in the regulation of inflammation. As a high HLA Class I expression is associated with a bad prognosis in UM, we set out to determine whether any miRNAs were related to a high HLA Class I expression and inflammation. We also determined whether such miRNAs were related to the UM's genetic status. The expression of 125 miRNAs was determined in 64 primary UM from Leiden. Similarly, the mRNA expression of HLA-A, HLA-B, TAP1, BAP1, and immune cell markers was obtained. Expression levels of 24 of the 125 miRNAs correlated with expression of at least three out of four HLA Class I probes. Four miRNAs showed a positive correlation with HLA expression and infiltration with leukocytes, 20 a negative pattern. In the first group, high miRNA levels correlated with chromosome 3 loss/reduced BAP1 mRNA expression, in the second group low miRNA levels. The positive associations between miRNA-22 and miRNA-155 with HLA Class I were confirmed in the TCGA study and Rotterdam cohort, and with TAP1 in the Rotterdam data set; the negative associations between miRNA-125b2 and miRNA-211 and HLA-A, TAP1, and CD4 were confirmed in the Rotterdam set. We demonstrate two patterns: miRNAs can either be related to a high or a low HLA Class I/TAP1 expression and the presence of infiltrating lymphocytes and macrophages. However, both patterns were associated with chromosome 3/BAP1 status, which suggests a role for BAP1 loss in the regulation of HLA expression and inflammation in UM through miRNAs. Show less
Roelofsen, C.D.M.; Wierenga, A.P.A.; Duinen, S. van; Verdijk, R.M.; Bleeker, J.; Marinkovic, M.; ... ; Jager, M.J. 2020
Background: In order to improve medical care for uveal melanoma (UM) patients, we need to monitor disease and survival to guide our research efforts. We analyzed the data of UM patients who... Show moreBackground: In order to improve medical care for uveal melanoma (UM) patients, we need to monitor disease and survival to guide our research efforts. We analyzed the data of UM patients who underwent an enucleation at the Leiden University Medical Center over the last five decades and investigated trends in patient and tumor characteristics and survival. Methods: Data were collected from charts and pathology reports from all patients who underwent an enucleation for UM between 1973 and 2019 (n = 1,212), of which 1,066 were primary enucleations; data were analyzed according to five time periods: 1973-1979 (n = 209), 1980-1989 (n = 148), 1990-1999 (n = 174), 2000-2009 (n = 280), and 2010-2019 (n = 401). Results: Over time, mean patient age at the time of enucleation for UM increased from 54.9 to 64.7 years (p < 0.001), more tumors showed histopathological involvement of the ciliary body (p < 0.001), and were classified in a high TNM/AJCC class (p < 0.001). Overall, the 5- and 10-year UM-related survival rates were 0.68 and 0.59, respectively. Over time, survival showed no change in patients with tumors in AJCC stages I or III, with recently a slightly worse survival in stage II UM (p = 0.02). Conclusion: Between 1973 and 2019, we found similar rates of UM-related survival following enucleation, although we noticed a strong increase in more unfavorable patient and tumor characteristics over time, such as an older age and larger tumor size. The lack of improvement indicates that more research should take place to develop adjuvant treatments to prevent metastases and efficient treatments once metastases develop. Show less
Souri, Z.; Jochemsen, A.G.; Versluis, M.; Wierenga, A.P.A.; Nemati, F.; Velden, P.A. van der; ... ; Jager, M.J. 2020
Simple SummaryChemotherapy and immunotherapy are both used to treat malignancies. The immunotherapy of cancer often involves T cells, which recognise the antigens presented in HLA molecules. Uveal... Show moreSimple SummaryChemotherapy and immunotherapy are both used to treat malignancies. The immunotherapy of cancer often involves T cells, which recognise the antigens presented in HLA molecules. Uveal melanoma (UM) is an intraocular malignancy, which often gives rise to metastases. We determined whether high-risk tumours expressed the target of two drugs, histone deacetylase (HDAC) inhibitor Quisinostat and Tazemetostat, an inhibitor of Enhancer of zeste homologue 2 (EZH2). We observed that especially high-risk UM tumours (monosomy 3, gain of 8q, loss of BAP1) expressed several HDACs, and showed a high HLA Class I expression. We further tested whether these drugs influenced HLA Class I expression on three UM cell lines. The drug Quisinostat led to an upregulation of HLA protein and mRNA levels in three UM cell lines, while Tazemetostat had little effect. We concluded that the use of drugs that influence epigenetic regulators may impact immunotherapy approaches.The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy. Show less
Uveal melanoma (UM) is a global disease which especially occurs in elderly people. Its incidence varies widely between populations, with the highest incidence among Caucasians, and a South-to-North... Show moreUveal melanoma (UM) is a global disease which especially occurs in elderly people. Its incidence varies widely between populations, with the highest incidence among Caucasians, and a South-to-North increase in Europe. As northern Europeans often have blond hair and light eyes, we wondered whether iris colour may be a predisposing factor for UM and if so, why. We compared the distribution of iris colour between Dutch UM patients and healthy Dutch controls, using data from the Rotterdam Study (RS), and reviewed the literature regarding iris colour. We describe molecular mechanisms that might explain the observed associations. When comparing a group of Dutch UM patients with controls, we observed that individuals from Caucasian ancestry with a green/hazel iris colour (Odds Ratio (OR) = 3.64, 95% Confidence Interval (CI) 2.57-5.14) and individuals with a blue/grey iris colour (OR = 1.38, 95% CI 1.04-1.82) had a significantly higher crude risk of UM than those with brown eyes. According to the literature, this may be due to a difference in the function of pheomelanin (associated with a light iris colour) and eumelanin (associated with a brown iris colour). The combination of light-induced stress and aging may affect pheomelanin-carrying melanocytes in a different way than eumelanin-carrying melanocytes, increasing the risk of developing a malignancy. Show less
