Increased Histological Tumor Pigmentation in Uveal Melanoma Is Related to Eye Color and Loss of Chromosome 3/BAP1.

Purpose: Heavy 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. Design: Retrospective comparison of clinical, histopathological, and genetic features and survival in UM with different pigmentation. Participants: A total of 1058 patients with UM from a White European population with diverse eye colors enucleated between 1972 and 2021. Methods: Cox 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 Measures: Uveal melanoma-related survival based on tumor pigmentation and chromosome status, correlation of tumor pigmentation with prognostic factors. Results: The 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). Conclusions: Patients 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. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.

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.

Chromosome 3 and 8q Aberrations in Uveal Melanoma Show Greater Impact on Survival in Patients with Light Iris versus Dark Iris Color.

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 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.

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.

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.

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing.

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.

Absent B cells, agammaglobulinemia, and hypertrophic cardiomyopathy in folliculin-interacting protein 1 deficiency.

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1-/- animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.

HDAC Inhibition Increases HLA Class I Expression in Uveal Melanoma.

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.

Prognostic Factors Five Years After Enucleation for Uveal Melanoma.

Purpose: A subgroup of uveal melanoma (UM) gives rise to metastases at a late stage. Our objective was to identify patient and tumor characteristics that are associated with UM-related death in patients who survived 5 years following enucleation. Methods: A retrospective analysis was performed in 583 primary UM cases, enucleated at the Leiden University Medical Center between 1983 and 2013. Univariable and multivariable Cox regression analyses were performed in the total cohort and separately in those surviving more than 5 years (n = 297). Results: In the total cohort, the median age was 62.6 years, and the median tumor diameter was 12.0 mm. Monosomy 3 was detected in 53% of cases and gain of 8q in 47%. In the cohort surviving 5 years, the median age was 59.5 years, and the median tumor diameter was 11.0 mm. Monosomy 3 and gain of 8q were detected in 33% and 31% of cases, respectively. In the total cohort, male gender (P = 0.03), tumor diameter (P < 0.001), mitotic count (P < 0.001), extravascular matrix loops (P = 0.03), extraocular growth (P < 0.001), and gain of 8q (P < 0.001) were independently associated with UM-related death. In patients surviving 5 years after enucleation, univariable analysis revealed that age (P = 0.03), tumor diameter (P < 0.001), monosomy 3 (P = 0.04), and 8q gain (P = 0.003) were associated with subsequent UM-related death. Using a multivariable analysis, only male gender (P = 0.03) and gain of 8q (P = 0.01) remained significant. Conclusions: Predictors of UM-related death change over time. Among UM patients who survived the initial 5 years following enucleation, male gender and chromosome 8q status were the remaining factors related to UM-related death later on.

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Aqueous Humor Biomarkers Identify Three Prognostic Groups in Uveal Melanoma.

Purpose: To investigate whether we can identify different patterns of inflammation in the aqueous humor of a uveal melanoma (UM)-containing eye, and whether these are related to prognosis. Methods: Ninety samples of aqueous humor from UM-containing eyes were analyzed using a high-throughput multiplex immunoassay that enables simultaneous analysis of 92 predefined protein biomarkers. Cytokine expression was compared to clinical and histopathological characteristics. Cluster analysis was performed, after which the clusters were compared with clinical and histopathological tumor characteristics. Results: Cluster analysis revealed three distinct clusters, with one cluster showing hardly any inflammatory cytokines, one showing intermediate levels, and one showing a high expression of inflammation-related biomarkers. Significant differences between the clusters were seen with regard to patient age (P = 0.008), tumor prominence (P = 0.001), ciliary body involvement (P < 0.001), American Joint Committee on Cancer (AJCC) stage (P < 0.001), monosomy of chromosome 3 (P = 0.03), and gain of chromosome 8q (P = 0.04), with the cluster with a highest cytokine expression having the worst prognostic markers. Especially apoptosis-related cytokines were differentially expressed. Conclusions: Analysis of cytokines in the aqueous humor shows distinct differences between aqueous humor samples and allocates these samples into three different prognostic tumor clusters. Especially large tumors with ciliary body involvement and monosomy 3 were associated with many cytokines, especially apoptosis-related cytokines. The presence of these cytokines in the aqueous humor may play a role in the lack of effective antitumor immune responses.

Do GNAQ and GNA11 Differentially Affect Inflammation and HLA Expression in Uveal Melanoma?

Inflammation, characterized by high numbers of infiltrating leukocytes and a high HLA Class I expression, is associated with a bad prognosis in uveal melanoma (UM). We wondered whether mutations in GNA11 or GNAQ differentially affect inflammation and HLA expression, and thereby progression of the disease. We analyzed data of 59 primarily enucleated UM eyes. The type of GNAQ/11 mutation was analyzed using dPCR; chromosome aberrations were determined by Fluorescence in Situ Hybridization (FISH), karyotyping, and single nucleotide polymorphism (SNP) analysis, and mRNA expression by Illumina PCR. Comparing tumors with a GNAQ mutation with those with a GNA11 mutation yielded no significant differences in histopathological characteristics, infiltrate, or HLA expression. When comparing the Q209L mutations with Q209P mutations in tumors with monosomy of chromosome 3, a higher mitotic count was found in the Q209P/M3 tumors (p = 0.007). The Kaplan-Meier (KM) curves between the patients of the different groups were not significantly different. We conclude that the type (Q209P/Q209L) or location of the mutation (GNA11/GNAQ) do not have a significant effect on the immunological characteristics of the tumors, such as infiltrate and HLA Class I expression. Chromosome 3 status was the main determinant in explaining the difference in infiltrate and HLA expression.

Loss of BAP1 Is Associated with Upregulation of the NFkB Pathway and Increased HLA Class I Expression in Uveal Melanoma.

One of the characteristics of prognostically infaust uveal melanoma (UM) is an inflammatory phenotype, which is characterized by high numbers of infiltrating T cells and macrophages, and a high HLA Class I expression. We wondered how this inflammation is regulated, and considered that one of the most important regulators of inflammation, the NFkB pathway, might play a role. We analyzed 64 UM samples for expression of HLA Class I, its regulators, and of members of the NFkB transcription family, using an Illumina HT12V4 array. HLA Class I expression and infiltrating immune cells were also determined by immunohistochemical staining. Information was obtained regarding chromosome status by Affymetrix Nsp array. Our analysis shows that expression of NFkB1, NFkB2 and RELB positively correlates with the level of HLA Class I expression and the number of infiltrating T cells and macrophages, while SPP1 and PPARγ are negatively correlated. Increased levels of NFkB1 and NFkB2 and decreased levels of SPP1 and PPARγ are seen in Monosomy 3/BAP1-negative tumors. This is also the case in non-inflammatory UM, indicating that our observation not only involves infiltrating leukocytes but the tumor cells themselves. We report that the NFkB pathway is associated with inflammation and HLA Class I expression in UM, and is upregulated when BAP1 expression is lost.

Differential Expression of DNA Repair Genes in Prognostically-Favorable versus Unfavorable Uveal Melanoma.

Expression of DNA repair genes was studied in uveal melanoma (UM) in order to identify genes that may play a role in metastases formation. We searched for genes that are differentially expressed between tumors with a favorable and unfavorable prognosis. Gene-expression profiling was performed on 64 primary UM from the Leiden University Medical Center (LUMC), Leiden, The Netherlands. The expression of 121 genes encoding proteins involved in DNA repair pathways was analyzed: a total of 44 genes differed between disomy 3 and monosomy 3 tumors. Results were validated in a cohort from Genoa and Paris and the The Cancer Genome Atlas (TCGA) cohort. Expression of the PRKDC, WDR48, XPC, and BAP1 genes was significantly associated with clinical outcome after validation. PRKDC was highly expressed in metastasizing UM (p < 0.001), whereas WDR48, XPC, and BAP1 were lowly expressed (p < 0.001, p = 0.006, p = 0.003, respectively). Low expression of WDR48 and XPC was related to a large tumor diameter (p = 0.01 and p = 0.004, respectively), and a mixed/epithelioid cell type (p = 0.007 and p = 0.03, respectively). We conclude that the expression of WDR48, XPC, and BAP1 is significantly lower in UM with an unfavorable prognosis, while these tumors have a significantly higher expression of PRKDC. Pharmacological inhibition of DNA-PKcs resulted in decreased survival of UM cells. PRKDC may be involved in proliferation, invasion and metastasis of UM cells. Unraveling the role of DNA repair genes may enhance our understanding of UM biology and result in the identification of new therapeutic targets.

Tumour Angiogenesis in Uveal Melanoma Is Related to Genetic Evolution.

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 (p = 0.008 and p = 0.004, respectively). Within BAP1-positive lesions (n = 20), 8q-gain did not relate to MVD (p = 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (p = 0.041), Von Willebrand Factor (VWF) (p = 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (p = 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+, p = 0.017; CD68+CD163+, p = 0.031) and lymphocytes (CD4+, p = 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.

Ischemia Is Related to Tumour Genetics in Uveal Melanoma.

Hypoxia-inducible factor 1-alpha (HIF1a) and its regulator von Hippel-Lindau protein (VHL) play an important role in tumour ischemia. Currently, drugs that target HIF1a are being developed to treat malignancies. Although HIF1a is known to be expressed in uveal melanoma (UM), it is as yet unknown which factors, such as tumour size or genetics, determine its expression. Therefore, we aimed to determine which tumour characteristics relate to HIF1a expression in UM. Data from 64 patients who were enucleated for UM were analysed. Messenger RNA (mRNA) expression was determined with the Illumina HT-12 v4 chip. In 54 cases, the status of chromosomes 3 and 8q, and BRCA1-associated protein 1 (BAP1) protein expression (immunohistochemistry) were determined. Findings were corroborated using data of 80 patients from the Cancer Genome Atlas (TCGA) study. A significantly increased expression of HIF1a, and a decreased expression of VHL were associated with monosomy 3/loss of BAP1 expression. The relationship between BAP1 loss and HIF1a expression was independent of chromosome 3. The largest basal diameter and tumour thickness showed no relationship with HIF1a. HIF1a expression related to an increased presence of infiltrating T cells and macrophages. From this study, we conclude that HIF1a is strongly related to tumour genetics in UM, especially to loss of BAP1 expression, and less to tumour size. Tumour ischemia is furthermore related to the presence of an inflammatory phenotype.

Heterogeneity revealed by integrated genomic analysis uncovers a molecular switch in malignant uveal melanoma.

Gene expression profiles as well as genomic imbalances are correlated with disease progression in uveal melanoma (UM). We integrated expression and genomic profiles to obtain insight into the oncogenic mechanisms in development and progression of UM. We used tumor tissue from 64 enucleated eyes of UM patients for profiling. Mutations and genomic imbalances were quantified with digital PCR to study tumor heterogeneity and molecular pathogenesis. Gene expression analysis divided the UM panel into three classes. Class I presented tumors with a good prognosis and a distinct genomic make up that is characterized by 6p gain. The UM with a bad prognosis were subdivided into class IIa and class IIb. These classes presented similar survival risks but could be distinguished by tumor heterogeneity. Class IIa presented homogeneous tumors while class IIb tumors, on average, contained 30% of non-mutant cells. Tumor heterogeneity coincided with expression of a set of immune genes revealing an extensive immune infiltrate in class IIb tumors. Molecularly, class IIa and IIb presented the same genomic configuration and could only be distinguished by 8q copy number. Moreover, UM establish in the void of the immune privileged eye indicating that in IIb tumors the infiltrate is attracted by the UM. Combined our data show that chromosome 8q contains the locus that causes the immune phentotype of UM. UM thereby provides an unique opportunity to study immune attraction by tumors.

Radiation Treatment Affects Chromosome Testing in Uveal Melanoma.

PURPOSE: The purpose of this study was to determine whether radiation treatment induces chromosomal aberrations in uveal melanoma (UM) and to evaluate which tumor features determine success of karyotyping and FISH. METHODS: Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH for chromosome 3 was performed in 248 samples. Thirty-six UMs had previously undergone irradiation. Karyotypes were analyzed, and the success rate of karyotyping/FISH was evaluated and compared with clinicopathologic tumor characteristics and prior irradiation. RESULTS: Aberrations were observed in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16, and Y being altered in at least 15% of the tumors. Aberrations were more common and more complex in previously irradiated tumors (significant for chromosomes 5 [P = 0.004] and 13 [P = 0.04]). Karyotyping and FISH failed significantly more often in irradiated tumors (both P < 0.001). In nonirradiated cases, successful karyotyping was related to a large tumor prominence (P = 0.004) and a high mitotic count (P = 0.007). The success of FISH in these tumors was not associated with any of the studied parameters. In irradiated tumors, karyotyping succeeded more frequently in cases with a high mitotic count (P = 0.03), whereas FISH was more often successful in tumors with a high mitotic count (P = 0.001), a large diameter (P = 0.009) and large prominence (P = 0.008). CONCLUSIONS: Karyotyping and FISH are more often successful in UMs with features characteristic of high tumor aggressiveness, whereas prior irradiation leads to multiple chromosome aberrations and to unsuccessful tests. It will be interesting to determine whether other techniques can provide reliable information on the chromosome status of previously irradiated UMs.

Digital PCR validates 8q dosage as prognostic tool in uveal melanoma.

BACKGROUND: Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM. METHODS: A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping. RESULTS: Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk. CONCLUSION: Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.