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.

Upregulation of HLA Expression in Primary Uveal Melanoma by Infiltrating Leukocytes.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our data provides evidence for a proper functioning HLA regulatory system in UM, offering a target for T-cell based therapies.

Skewed expression of the genes encoding epigenetic modifiers in high-risk uveal melanoma.

PURPOSE: Monosomy 3 (M3) or the presence of a specific RNA expression profile, known as class 2, is strongly associated with death from uveal melanoma (UM). Given the important role of epigenetic processes in cancer development and progression, we compared the transcriptional profiles of a selection of epigenetic regulators between primary UM with a good and a bad prognosis. METHODS: Transcriptional levels of 59 epigenetic regulator genes were measured by quantitative PCR (qPCR) in 20 UM, 12 with monosomy of chromosome 3 (M3) and 8 with disomy of chromosome 3 (D3). Validation was performed in an independent cohort. Expression levels were compared to clinicopathological characteristics, including class type. Bisulfite sequencing was used to evaluate the role of DNA methylation in gene silencing. RESULTS: In the first set of tumors, general downregulation of transcription of the genes encoding epigenetic regulatory enzymes was seen in association with M3. The 10 genes with the highest differential expression between M3 and D3 were selected and were analyzed in a second set of tumors. In the validation set, significantly lower levels of KAT2B (P = 0.008), HDAC11 (P = 0.009), KMT1C (P = 0.05), KDM4B (P = 0.003), KDM6B (P = 0.04), and BMI-1 (P = 0.001) transcripts were found in tumors with M3/class 2. Methylation of C-phosphate-G (CpG) residues was not observed on the putative regulatory regions of KAT2B, KDM4B, or KDM6B. CONCLUSIONS: Expression levels of a number of histone-modifying genes and polycomb family members are significantly lower in uveal melanoma with monosomy 3/class 2, supporting a general dysregulation of epigenetic modifiers in UM with a bad prognosis.

Prognostic parameters in uveal melanoma and their association with BAP1 expression.

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.

A fish scale-derived collagen matrix as artificial cornea in rats: properties and potential.

PURPOSE: A fish scale-derived collagen matrix (FSCM) is proposed as an alternative for human donor corneal tissue. Light scatter and light transmission of the FSCM were measured and compared with human cornea, and its short-term biocompatibility was tested in a rat model. METHODS: light scatter was determined with a straylight measuring device, whereas light transmission was measured using a broadband absorption spectrometer. for evaluation of the biocompatibiliy, three approaches were used: the FSCM was implanted as an anterior lamellar keratoplasty (ALK), placed in an interlamellar corneal pocket (IL), and placed subconjunctivally (SC). Transparency, neovascularization, and epithelial damage were followed for 21 days. Morphology and cellular infiltration were assessed histologically. RESULTS: The amount of scattered light was comparable to that seen in early cataract and the percentage of light transmission was similar to the transmission through the human cornea. Implantation of the FSCM as an ALK led to mild haziness only, not obscuring the pupil, despite the development of neovascularization around the sutures; IL placement led to a moderate haze, partly obscuring the pupil, and to (partial) melting of the anterior corneal lamella. The SC group exhibited local swelling and induration, which decreased over time. Histology showed a chronic inflammation varying from mild and moderate in the ALK and IL group, to severe in the SC group. CONCLUSIONS: In spite of technical difficulties, it was feasible to use the FSCM for ALK, whereas IL placement led to melting of the anterior lamella. Further studies are necessary for better understanding of its immunogenicity. The light scatter and transmission data show that the first version of this FSCM is comparable to human cornea tissue in this respect.

A comparison of HLA genotype with inflammation in uveal melanoma.

PURPOSE: Human leukocyte antigen (HLA) polymorphisms have been associated with the development of autoimmune diseases. In uveal melanoma, a high expression of HLA classes I and II, and infiltration with lymphocytes and macrophages are associated with a bad prognosis. Inflammation has an important role in this malignancy. The goal of our study was to determine whether specific HLA alleles are associated with increased inflammation. METHODS: Records were analyzed of 45 patients who underwent enucleation for uveal melanoma. HLA typing, tumor HLA expression and tumor macrophage infiltration were determined in each case. RESULTS: Before correction for multiple testing, macrophage infiltration was less in HLA-A2 positive patients. Patients with HLA-DR6 had a higher tumor cell expression of HLA-DR. After correction for the number of analyses, no associations remained statistically significant. CONCLUSION: The results before correction suggest that the HLA genotype may influence inflammation as indicated by HLA expression and macrophage infiltration in uveal melanoma. However, after correction this association did not prove significant.