Microphthalmia-Associated Transcription Factor: A Differentiation Marker in Uveal Melanoma.

Microphthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. In cutaneous melanoma, MITF loss has been linked to an increased expression of stem cell markers, a shift in epithelial-to-mesenchymal transition (EMT)-related factors, and increased inflammation. We explored the role of MITF in Uveal Melanoma (UM) using a cohort of 64 patients enucleated at the Leiden University Medical Center. We analysed the relation between MITF expression and clinical, histopathological and genetic features of UM, as well as survival. We performed differential gene expression and gene set enrichment analysis using mRNA microarray data, comparing MITF-low with MITF-high UM. MITF expression was lower in heavily pigmented UM than in lightly pigmented UM (p = 0.003), which we confirmed by immunohistochemistry. Furthermore, MITF was significantly lower in UM with monosomy 3/BAP1 loss than in those with disomy 3/no BAP1 loss (p < 0.001) and with 8q gain/amplification 8q (p = 0.02). Spearman correlation analysis showed that a low MITF expression was associated with an increase in inflammatory markers, hallmark pathways involved in inflammation, and epithelial-mesenchymal transition. Similar to the situation in cutaneous melanoma, we propose that MITF loss in UM is related to de-differentiation to a less favourable EMT profile and inflammation.

Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction.

Assessment of specific ß-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to ß-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of ß-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as a reflection of the relative amount of lysed ß-cells.

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma.

BACKGROUND: Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. METHODS: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. RESULTS: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. CONCLUSIONS: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma.

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment.

Uveal melanoma (UM) is characterized by a number of genetic aberrations that follow a certain chronology and are tightly linked to tumor recurrence and survival. Loss of chromosome 3, bi-allelic loss of BAP1 expression, and gain in chromosome 8q have been associated with metastasis formation and death, while loss of chromosome 3 has been associated with the influx of macrophages and T cells. We used a set of genetically-classified UM to study immune infiltration in the context of their genetic evolution. We show in two independent cohorts that lack of BAP1 expression is associated with an increased density of CD3+ T cells and CD8+ T cells. The presence of extra copies of chromosome 8q in disomy 3 tumors with a normal BAP1 expression is associated with an increased influx of macrophages (but not T cells). Therefore, we propose that the genetic evolution of UM is associated with changes in the inflammatory phenotype. Early changes resulting in gain of chromosome 8q may activate macrophage infiltration, while sequential loss of BAP1 expression seems to drive T cell infiltration in UM.

EMT-associated factors promote invasive properties of uveal melanoma cells.

PURPOSE: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors. METHODS: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed. RESULTS: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth. CONCLUSIONS: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

PRAME Expression: A Target for Cancer Immunotherapy and a Prognostic Factor in Uveal Melanoma.

Purpose: Uveal melanoma (UM) is a rare disease with a high mortality, and new therapeutic options are being investigated. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, expressed in the testis, but also in cancers, including uveal melanoma. PRAME is considered a target for immune therapy in several cancers, and PRAME-specific T cell clones have been shown to kill UM cells. Methods: We studied the literature on PRAME expression in hematological and solid malignancies, including UM, and its role as a target for immunotherapy. The distribution of tumor features was compared between PRAME-high and PRAME-low UM in a 64-patient cohort from the Leiden University Medical Center (LUMC) and in the Cancer Genome Atlas (TCGA) cohort of 80 cases and differential gene expression analysis was performed in the LUMC cohort. Results: PRAME is expressed in many malignancies, it is frequently associated with a negative prognosis, and can be the target of T cell receptor (TCR)-transduced T cells, a promising treatment option with high avidity and safety. In UM, PRAME is expressed in 26% to 45% of cases and is correlated with a worse prognosis. In the LUMC and the TCGA cohorts, high PRAME expression was associated with larger diameter, higher Tumor-Node-Metastasis (TNM) stage, more frequent gain of chromosome 8q, and an inflammatory phenotype. Conclusions: We confirm that PRAME is associated with poor prognosis in UM and has a strong connection with extra copies of 8q. We show that PRAME-specific immunotherapy in an adjuvant setting is promising in treatment of malignancies, including UM.

Bevacizumab and intraocular tumors: an intriguing paradox.

PURPOSE: Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro. METHODS: B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated. RESULTS: Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures. CONCLUSIONS: In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

In aged mice, outgrowth of intraocular melanoma depends on proangiogenic M2-type macrophages.

Macrophages are part of the tumor microenvironment and have been associated with poor prognosis in uveal melanoma. We determined the presence of macrophages and their differentiation status in a murine intraocular melanoma model. Inoculation of B16F10 cells into the anterior chamber of the eye resulted in rapid tumor outgrowth. Strikingly, in aged mice, tumor progression depended on the presence of macrophages, as local depletion of these cells prevented tumor outgrowth, indicating that macrophages in old mice had a strong tumor-promoting role. Immunohistochemistry and gene expression analysis revealed that macrophages carried M2-type characteristics, as shown by CD163 and peroxisome proliferator-activated receptor gamma expression, and that multiple angiogenic genes were heavily overrepresented in tumors of old mice. The M2-type macrophages were also shown to have immunosuppressive features. We conclude that tumor-associated macrophages are directly involved in tumor outgrowth of intraocular melanoma and that macrophages in aged mice have a predisposition for an M2-type profile.

Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples.

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples.

A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus.

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA "scars" (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies.

Tumor-Infiltrating T Cells Can Be Expanded Successfully from Primary Uveal Melanoma after Separation from Their Tumor Environment.

Purpose: To evaluate whether expanded tumor-infiltrating lymphocytes (TILs) can be obtained from primary uveal melanoma (UM) for potential use as adjuvant treatment in patients at risk of developing metastatic disease. Design: Experimental research study. Participants: Freshly obtained primary UM from 30 patients. Methods: Three different methods were used to expand TILs: (1) direct culture from small fragments of fresh tumor tissue, (2) single-cell tissue preparation by enzymatic digestion and subsequent enrichment of mononuclear cells, and (3) selection of CD3+ T cells using magnetic beads. Surface expression of costimulatory and inhibitory T-cell markers and T-cell reactivity against autologous tumor cells was assessed. Clinical, histopathologic, genetic, and immunologic characteristics of the tumors were compared with the capacity to expand TILs and with their reactivity against autologous tumor cells. Main Outcome Measures: The feasibility of expanding TILs from primary UM, testing their reactivity to autologous UM cells, and evaluating the impact of an immunomodulatory environment. Results: Direct culture of tumor parts led to successful TIL culture in 4 of 22 tumors (18%), enrichment of mononuclear cells gave rise to TILs in 5 of 12 tumors (42%), while preselection of CD3+ T cells with magnetic beads resulted in TIL expansion in 17 of 25 tumors (68%). In 8 of 17 tumors (47%), the TIL cultures comprised UM-reactive T cells. The presence of UM-reactive T cells among TILs was not related to clinical, histologic, genetic, or immunological tumor characteristics. Interestingly, RNA-Seq analysis showed that approximately half of the UM tumors displayed an increased expression of immunomodulatory molecules related to T-cell suppression, such as galectin 3, programmed death-ligand 1, cytotoxic T-lymphocyte-associated protein 4, indoleamine 2,3-dioxygenase 1, and lymphocyte activating 3, potentially explaining why T cells require optimal removal of tumor components for expansion. Conclusions: The need to separate TILs from their tumor microenvironment for their successful expansion and the presence of UM-reactive T cells among TILs suggests that these UM-reactive T cells are strongly suppressed in vivo and that UM is immunogenic. These findings indicate that adoptive TIL therapy could be an option as an adjuvant treatment in primary UM patients at high risk of developing metastatic disease.

Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR.

An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as genetic instability can disturb the underlying mathematical model. For example, approximately 24% of the tumors from The Cancer Genome Atlas pan-cancer data set carried a copy number alteration affecting the TRB gene T-cell marker, which would cause an underestimation or overestimation of the T-cell fraction. In this study, we introduce a multiplex digital PCR experimental setup to quantify T cells in copy number unstable DNA samples. By implementing a so-called regional corrector, genetic alterations involving the T-cell marker locus can be recognized and corrected for. This novel setup is evaluated mathematically in silico and validated in vitro by measuring T-cell presence in various samples with a known T-cell fraction. The utility of the approach is further demonstrated in copy number altered cutaneous melanomas. Our novel multiplex setup provides a simple, but accurate, DNA-based T-cell quantification in both copy number stable and unstable specimens. This approach has potential clinical and diagnostic applications, as it does not depend on availability of T-cell epitopes, has low requirements for sample quantity and quality, and can be performed in a relatively easy experiment.

In uveal melanoma Gα-protein GNA11 mutations convey a shorter disease-specific survival and are more strongly associated with loss of BAP1 and chromosomal alterations than Gα-protein GNAQ mutations.

BACKGROUND AND AIM OF THE STUDY: Mutations in the Gα-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. METHODS: We analyzed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. RESULTS: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1-associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High-risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. CONCLUSIONS: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation.

Small deletions but not methylation underlie CDKN2A/p16 loss of expression in conventional osteosarcoma.

Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14(ARF) function.

Scientific and clinical implications of genetic and cellular heterogeneity in uveal melanoma.

Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago. Since uveal melanoma grow in the corpus vitreous, they present a unique tumour model because every cell present in the tumour tissue is actually part of the tumour and possibly plays a role. For an effective treatment of uveal melanoma metastasis, it should be clear whether precursor clones and normal cells play an active role in progression and metastasis. We propagate analysis of bulk tissue that allows analysis of tumour heterogeneity in a clinical setting.

LAG3 and Its Ligands Show Increased Expression in High-Risk Uveal Melanoma.

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.

In Uveal Melanoma, Angiopoietin-2 but Not Angiopoietin-1 Is Increased in High-Risk Tumors, Providing a Potential Druggable Target.

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.

MiRNAs Correlate with HLA Expression in Uveal Melanoma: Both Up- and Downregulation Are Related to Monosomy 3.

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.

Expression of HDACs 1, 3 and 8 Is Upregulated in the Presence of Infiltrating Lymphocytes in Uveal Melanoma.

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 IFNγ (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 IFNγ 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.