Genetic Profiling of Primary Orbital Melanoma: An Analysis of 6 Cases with Clinicopathologic Correlation.

PURPOSE: To analyze the genetic profile of 6 cases of primary orbital melanoma with clinicopathologic correlation. DESIGN: Retrospective noninterventional study to analyze the genetic profile of 6 cases of primary orbital melanoma and to correlate the genetic findings with prognosis and clinicopathologic features. Inclusion criteria were patients with primary orbital melanoma with no evidence of primary eyelid skin, conjunctival, uveal, or remote melanoma at extraocular sites. PARTICIPANTS: The study involved 6 primary orbital melanomas from 6 patients. Four patients were exenterated and 2 had incisional biopsies performed. METHODS: Clinical notes and radiologic records were assessed to ascertain clinical tumor behavior. Sections were stained with hematoxylin-eosin and exposed to immunohistochemistry for S100, MelA, HMB45, Sox10, and BAP1. Melanoma DNA was exposed to array comparative genomic hybridization to assess gross chromosomal copy number changes. Point mutation assessment and Sanger sequencing were performed for GNAQ, GNA11, BRAF, NRAS, pTERT, SF3B1, and EIF1AX. MAIN OUTCOME MEASURES: These were the presence of gross chromosomal copy number changes and the presence of mutations in GNAQ, GNA11, BRAF, NRAS, pTERT, SF3B1, and EIF1AX; the presence of metastases and time period between diagnosis and death from melanoma; and correlation between the tumor genetic profile and the clinical behavior of the tumor. RESULTS: One of the 6 cases was clinically associated with oculodermal melanocytosis. Of the 6 patients, 3 died of melanoma metastases and 1 of unrelated causes; 2 remain alive at last review. Three of the 6 cases were histologically associated with a benign precursor lesion. All melanomas expressed S100, MelA, HMB45, and Sox10. One patient showed loss of BAP1 nuclear staining. The most frequent chromosomal gains across the 6 cases, in order of frequency, were 6p, 8q, 17q, 6q, and 20p. The most frequently lost regions were 1p, 9p, 16q, and 17p. One patient showed monsomy 3 and gain of 8q (and showed the BAP1 loss). Mutations were found in GNAQ (1 case), GNA11 (1 case), SF3B1 (2 cases), NRAS (2 cases), and pTERT (2 cases). CONCLUSIONS: The data point to 2 genetic groups for primary orbital conjunctiva melanoma-like and a uveal melanoma-like group. A larger study would help confirm this suggestion.

Genetic Background of Iris Melanomas and Iris Melanocytic Tumors of Uncertain Malignant Potential.

PURPOSE: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Iris melanoma comprises 4% to 10% of all UMs and has a lower mortality rate. The genetic changes in iris melanoma are not as well characterized as ciliary body or choroidal melanoma. The aim of this study was to gain more insight into the genetic background of iris melanoma and iris nevi. DESIGN: Multicenter, retrospective case series. PARTICIPANTS: Patients diagnosed with iris melanoma or iris nevi who underwent surgical intervention as primary or secondary treatment. METHODS: Next-generation sequencing of GNAQ, GNA11, EIF1AX, SF3B1, BAP1, NRAS, BRAF, PTEN, c-Kit, TP53, and TERT was performed on 30 iris melanomas and 7 iris nevi. Copy number status was detected using single nucleotide polymorphisms (SNPs) included in the next-generation sequencing (NGS) panel, SNP array, or fluorescent in situ hybridization. BAP1 immunohistochemistry was performed on all samples. MAIN OUTCOME MEASURES: Mutation and copy number status were analyzed. Results of BAP1 immunohistochemistry were used for survival analysis. RESULTS: In 26 of the 30 iris melanoma and all iris nevi, at least 1 mutation was identified. Multiple mutations were detected in 23 iris melanoma and 5 nevi, as well as mutations in GNAQ and GNA11. Furthermore, 13 of 30 BAP1, 5 of 30 EIF1AX, and 2 of 30 SF3B1 mutations were identified in iris melanoma. No correlation between BAP1 status and disease-free survival was found. The iris nevi showed 1 EIF1AX and 3 BAP1 mutations. Two of the nevi, with a BAP1 mutation, were histologically borderline malignant. Mutations in NRAS, BRAF, PTEN, c-KIT, and TP53 were detected in 6 iris melanomas and 4 iris nevi. CONCLUSIONS: Mutations that are often found in uveal and cutaneous melanoma were identified in this cohort of iris melanomas and iris nevi. Therefore, iris melanomas harbor a molecular profile comparable to both choroidal melanoma and cutaneous melanoma. These findings may offer adjuvant targeted therapies for iris melanoma. There was no prognostic significance of BAP1 expression as seen in choroidal melanoma. Consequently, iris melanoma is a distinct molecular subgroup of UM. Histologic borderline malignant iris nevi can harbor BAP1 mutations and may be designated iris melanocytic tumors of uncertain malignant potential.

Establishment and molecular characterisation of seven novel soft-tissue sarcoma cell lines.

BACKGROUND: Soft-tissue sarcomas (STS) are a diverse group of malignancies that remain a diagnostic and therapeutic challenge. Relatively few reliable cell lines currently exist. Rapidly developing technology for genomic profiling with emerging insights into candidate functional (driver) aberrations raises the need for more models for in vitro functional validation of molecular targets. METHODS: Primary cell culture was performed on STS tumours utilising a differential attachment approach. Cell lines were characterised by morphology, immunocytochemistry, proliferation assays, short tandem repeat (STR) and microarray-based genomic copy number profiling. RESULTS: Of 47 STS cases of various subtypes, half formed adherent monolayers. Seven formed self-immortalised cell lines, including three undifferentiated pleomorphic sarcomas, two dedifferentiated liposarcomas (one of which had received radiotherapy), a leiomyosarcoma and a myxofibrosarcoma. Two morphologically distinct yet genetically identical variants were established in separate cultures for the latter two tumours. All cell lines demonstrated genomic and phenotypic features that not only confirm their malignant characteristics but also confirm retention of DNA copy number aberrations present in their parent tumours that likely include drivers. CONCLUSIONS: These primary cell lines are much-needed additions to the number of reliable cell lines of STS with complex genomics available for initial functional validation of candidate molecular targets.

Clinical Implications and Molecular Features of Extracellular Matrix Networks in Soft Tissue Sarcomas.

PURPOSE: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterized. We aimed to investigate the tumor ECM and adhesion signaling networks present in STS and their clinical implications. EXPERIMENTAL DESIGN: Proteomic and clinical data from 321 patients across 11 histological subtypes were analyzed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS), and undifferentiated pleomorphic sarcomas (UPS). RESULTS: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct coregulated ECM networks which are associated with tumor malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the lymphocyte cytosolic protein 1 cytoskeletal protein as a prognostic factor in LMS. Characterization of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signaling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodeling proteins as candidate antistromal therapeutic targets. Finally, we define a proteoglycan signature that is an independent prognostic factor for overall survival in DDLPS and UPS. CONCLUSIONS: STS comprise heterogeneous ECM signaling networks and matrix-specific features that have utility for risk stratification and therapy selection, which could in future guide precision medicine in these rare cancers.

Atypically low spontaneous sister chromatid exchange formation in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular cancer of adults and is characterized by several well-established chromosomal changes. More recently, a specific mutation of guanine nucleotide binding protein Gq alpha subunit (GNAQ) has also been identified in a proportion of UM. Although some of these alterations have been suggested to be early changes, the genetic alterations responsible for the development of UM have yet to be clearly determined. Cancers are characterized by increased genetic instability, and analysis of established cancer cell lines and blood from cancer patients has universally been associated with an increased level of sister chromatid exchange (SCE). We have observed that the spontaneous frequency of SCE in primary cultures of UM and UM-derived cell lines is decreased below normal baseline levels, a phenomenon unique to UM when compared with multiple other cancers. This finding was specific to the tumor and not found in lymphocytes from the patients. Although we cannot exclude the possibility that low SCE (LSCE) is peculiar to the uveal melanocytes lineage, as it was consistently observed in all UM studied, regardless of other genetic defects, we propose that this phenomenon contributes to the molecular pathogenesis of UM.

Multi-Modal Mass Spectrometric Imaging of Uveal Melanoma.

Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI), was used to obtain images of lipids and metabolite distribution in formalin fixed and embedded in paraffin (FFPE) whole eye sections containing primary uveal melanomas (UM). Using this technique, it was possible to obtain images of lysophosphatidylcholine (LPC) type lipid distribution that highlighted the tumour regions. Laser ablation inductively coupled plasma mass spectrometry images (LA-ICP-MS) performed on UM sections showed increases in copper within the tumour periphery and intratumoural zinc in tissue from patients with poor prognosis. These preliminary data indicate that multi-modal MSI has the potential to provide insights into the role of trace metals and cancer metastasis.

Common genetic changes in leiomyosarcoma and gastrointestinal stromal tumour: implication for ataxia telangiectasia mutated involvement.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract. Formerly GISTs were commonly classified histologically as leiomyosarcomas; however, they are now known to arise from the interstitial cells of Cajal. Majority of GISTs overexpress KIT and have characteristic mutations within the gene, which are the targets of drug treatment with tyrosine kinase inhibitors. Leiomyosarcoma is a malignant tumour of smooth muscle differentiation and falls into a group of sarcomas that show complex karyotypic changes with no consistent recurrent genetic abnormality. We have used comparative genomic hybridization in combination with fluorescence in situ hybridization to determine genetic differences between the tumour types. We found leiomyosarcomas and GISTs share common regions of chromosomal 13q and 11q imbalance, in addition to more specific 1p and 8p losses in leiomyosarcoma and 15q and 22q losses in GISTs. More importantly, we have shown for the first time a deletion in the ataxia telangiectasia mutated (ATM) gene locus with decreased/absent expression of ATM protein, and amplification in the region 13q21-q32 in both tumour types, suggesting both regions may play a role in leiomyosarcoma and GIST biology.

Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Aggressive Ciliary Body Adenocarcinoma with Bilateral Lung Metastases: Histological, Molecular, Genetic and Clinical Aspects.

PURPOSE OF THE STUDY: To describe the clinical and histopathological features of an aggressive ciliary body adenocarcinoma with pulmonary metastases and skull base spread. PROCEDURES AND RESULTS: A 45-year-old female patient presented with a post-traumatic phthisical eye that was eviscerated. This showed an unexpected carcinoma (positive for cytokeratins and melanocytic markers), the histological differential diagnosis for which included a primary ciliary body adenocarcinoma or a metastasis. The patient developed rapid post-surgical localized recurrence that required an orbital exenteration. This showed identical tumour to the evisceration specimen, with vascular invasion in orbital blood vessels and a contaminated orbital soft tissue margin. Staging imaging revealed multiple lung metastases, which were biopsied and shown to be a disseminated ciliary body adenocarcinoma rather than a disseminated primary lung carcinoma. The tumour spread locally to the skull base for which radiotherapy was given. Unfortunately, the patient passed away a few weeks later. CONCLUSIONS: To our knowledge, this is the first case of ciliary body adenocarcinoma with bilateral lung metastases. The malignant potential of these tumours should be considered as a possibility, and appropriate screening and staging tests should therefore be considered to guide appropriate management.

Sister Chromatid Exchange and Genomic Instability in Soft Tissue Sarcomas: Potential Implications for Response to DNA-Damaging Treatments.

Sarcomas are rare heterogeneous malignancies of mesenchymal origin characterised by complex karyotypes but no specific abnormalities. Recurrence is common, and metastatic disease carries poor survival despite standard DNA-damaging radiotherapy or chemotherapy. DNA double-strand breaks (DSBs) are either repaired by mechanisms such as homologous recombination (HR) or result in cell death by apoptosis. Endogenous γH2AX formation and SCE formation are early and late events, respectively, and their levels are considered surrogate measures of genomic instability. Combined γH2AX and SCE analysis was used to evaluate endogenous DNA DSB levels (and their subsequent repair) in 9 primary sarcoma cell lines and compared with well-established commercial lines. All the sarcoma cell lines had elevated γH2AX and SCE levels, but there was no correlation between the DNA DSB frequency and subsequent SCE. Typically, radioresistant osteosarcoma cells had relatively low γH2AX frequency but high SCE counts suggestive of efficient DNA repair. Conversely, liposarcoma cells derived from a radiosensitive tumour had high H2AX but relatively lower SCE levels that may imply inefficient DNA DSB repair. To our knowledge, this is the first report that correlates H2AX and SCE levels in primary sarcoma cell lines and may provide insight into potential response to DNA-damaging treatments.

Reduced expression of autotaxin predicts survival in uveal melanoma.

AIM: In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour-related mortality. METHODS: The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence-verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques--hierarchical cluster analysis and multidimensional scaling--were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10,000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded. RESULTS: Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real-time quantitative PCR. CONCLUSIONS: Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.

Effects of prolonged exposure to low dose metformin in thyroid cancer cell lines.

Background: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment. Some sub-types however have a poor prognosis. Metformin, an oral anti-diabetic drug is shown to have anti-cancer effects in several types of cancer (breast, lung and ovarian cancer). The proposed mechanisms include activation of the Adenosine Mono-phosphate-activated Protein Kinase (AMPK) pathway and inhibition of the mTOR pathway (which promotes growth and proliferation). By inhibiting hepatic gluconeogenesis and increasing glucose uptake by muscles, metformin decreases blood glucose and circulating Insulin levels. Aims: Explore the effect of metformin on the growth and proliferation of thyroid cancer cell lines. Methods: The effects of metformin on thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1, 8305C and TT) and normal thyroid follicular cells (Nthy-ori 3-1) were investigated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for cell proliferation; clonogenic assays; FACS analysis for apoptosis and cell cycle, H2A.X phosphorylation (γH2AX) assay for DNA repair and scratch assay for cell migration. Results: Metformin inhibited cell proliferation and colony formation at 0.03 mM and above and inhibited cell migration at 0.3 mM. At concentrations of 0.1 mM and above metformin increased the percentage of apoptotic cells and induced cell cycle arrest in G0/G1 phase at minimum concentration of 0.3 mM. Unlike previous reports, no effect on DNA repair response was demonstrated. Conclusion: Metformin suppressed growth of all thyroid cancer cell lines, at concentrations considered to be within in the therapeutic range for diabetic patients on metformin (<0.3 mM).

Phenotypic Plasticity in Uveal Melanoma Is Not Restricted to a Tumor Subpopulation and Is Unrelated to Cancer Stem Cell Characteristics.

Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and approximately half of those diagnosed will die of metastasis. This study investigates whether UM progression is driven by a subpopulation of stem-like cells, termed "cancer stem cells" (CSCs). Methods: Expression of postulated stem cell markers aldehyde dehydrogenase (ALDH), CD44, and CD133 was analyzed in UM cell lines and primary UM short-term cultures (STCs) established from tumor samples. Additionally, the notion of a "cellular hierarchy" within UM was investigated. Finally, the phenomenon of phenotypic plasticity in response to environmental factors was explored. Results: We demonstrate that expression of ALDH, CD44, and CD133 does not select for a subpopulation of stem-like cells in either UM cell lines or UM STCs. Furthermore, there is an absence of a cellular hierarchy in cell lines and all cells in culture are able to drive tumor progression. Last, we show that established UM cell lines and UM STCs are plastic in nature and switch their phenotype in response to environmental stimuli. Conclusions: We hypothesize that this capacity to undergo phenotypic plasticity may be a consequence of neural crest lineage and renders the exploration of the CSC hypothesis extremely challenging in UM.

Late Solitary Extraocular Recurrence From Previously Resected Iris Melanoma.

PURPOSE: To report on cases of late extraocular relapse of previously resected iris melanoma, without concurrent intraocular recurrence. DESIGN: Retrospective case series. METHODS: A retrospective chart review of 4 patients diagnosed with late subconjunctival relapse of previously resected iris melanoma. RESULTS: Three female patients and 1 male patient underwent iris tumor resection and presented to our service with suspicious conjunctival lesions at a median of 22 years later (mean: 21 years). None showed intraocular relapse. Treatment of the conjunctival tumors included excisional biopsy (n = 4), followed by cryotherapy (n = 3) and/or brachytherapy (n = 3). In all cases, histopathology confirmed malignant melanoma, with no intraepithelial component or associated melanosis. Genetic sequencing (n = 3) showed wild-type BRAF and NRAS in all. GNA11 mutation was found in 1 case. On array-based comparative genomic hybridization (n = 3), gain of 6p was found in 2 cases and gain of 8 in 2. Overall, findings were strongly suggestive of a diagnosis of late extraocular relapse from previously resected iris melanoma. In a median of 2.5 years (mean: 7.7 years) from the subconjunctival relapse, no further episodes of intraocular/extraocular recurrence were recorded, and all patients were free from distant metastasis. CONCLUSIONS: Patients undergoing iris melanoma resection are at risk of developing late solitary extraocular relapse even more than 30 years after surgery. In the absence of an intraocular component, diagnosis may be challenging, as tumors mimic a primary conjunctival lesion. Management by excisional biopsy followed by adjuvant therapy was successful, and histopathology and genetic analysis supported a diagnosis of extraocular uveal tumor spread rather than a primary conjunctival tumor.

The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro.

Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.

A potential role for TGFbeta in the regulation of uveal melanoma adhesive interactions with the hepatic endothelium.

PURPOSE: TGFbeta has been shown to have a regulatory effect on uveal melanoma invasion, but it is not known which processes are specifically influenced. The purpose of this study was to analyze the effect of TGFbeta stimulation on the adhesive interactions of uveal melanomas with the extracellular matrix (ECM) and endothelium and, in addition, its effect on the secretion of collagenases. METHODS: Invasive and a noninvasive uveal melanoma cell lines, supported by short-term primary uveal melanoma cultures, were used to assess the effect of TGFbeta on ECM and endothelial adhesion and degradation of the ECM. Changes in cell adhesion molecule expression were assessed by flow cytometry, and conditioned media were analyzed by gelatin zymography. Assays of adhesion to ECM substrates and endothelial cells were also performed. RESULTS: Treatment with TGFbeta increased low basal levels of adhesion molecule and latent MMP-2 expression, as well as adhesion to hepatic endothelial cells by the noninvasive cell line. Conversely, TGFbeta reduced adhesion to laminin and a laminin-binding integrin by invasive cells but had no effect on their adhesion to the endothelium. CONCLUSIONS: In this preliminary study, TGFbeta was found to upregulate levels of MMP-2, reduce adhesion to laminin, and downregulate expression of laminin-binding integrins. Specifically, TGFbeta was found to increase adhesion of noninvasive uveal melanoma cells to the hepatic, but not the dermal, endothelium and may therefore contribute to the preferential targeting of the liver by uveal melanomas.

High-Resolution Array CGH Analysis Identifies Regional Deletions and Amplifications of Chromosome 8 in Uveal Melanoma.

PURPOSE: Monosomy 3 (M3) and abnormalities of chromosome 8 associate with poor prognosis in uveal melanomas (UM). Although M3 has been the subject of more in-depth studies, none have intensively focused on chromosome 8. To elucidate the potential role of chromosome 8 abnormalities, array comparative genomic hybridization (aCGH) was performed on primary UM. METHODS: A specifically-designed custom high-resolution array was developed focusing on changes most implicated in UM. Probes for chromosome 8 had a mean spacing of 2.3 kb while chromosomes infrequently affected had a mean spacing of 36.6 kb. A series of 75 UM, including one formalin-fixed paraffin sample were analyzed, and where possible control DNA extracted from the patient's own peripheral blood was used. RESULTS: The most common copy number abnormalities were chromosome 8 (75%) and M3 (51%), with M3 and gain of the long arm of chromosome 8 (8q+) associated in 41% of cases. Also identified were partial deletions of chromosome 3 (3%) and regional 8q+ (23%), and the intensive coverage of chromosome 8 revealed small focal deletions and amplifications affecting both arms. The most significant predictor of prognosis was M3/8q+ having a hazard ratio of 10.1 (P < 0.0001). CONCLUSIONS: Neither 8p deletion nor focal changes affecting chromosome 8 were linked to outcome. The most significant indicator was M3/8q, and multiple 8q+ associated with shorter survival. Studying UM with this technology provides a powerful robust tool for predicting prognosis while considering other genetic changes, allowing the future incorporation of such data as it becomes clinically significant.

Tumor necrosis factor alpha increases and alpha-melanocyte-stimulating hormone reduces uveal melanoma invasion through fibronectin.

Iris melanomas are less likely to metastasize than posterior compartment melanomas. The anterior chamber of the eye is an immunosuppressed microenvironment where a wide range of immunosuppressive factors in aqueous humor contribute to the immune privilege. One such factor is alpha-melanocyte-stimulating hormone, a potent anti-inflammatory neuropeptide that exhibits efficacy in many studies of acute and chronic inflammation. The aim of this study was to investigate whether the different metastatic behavior of iris melanomas versus posterior compartment melanomas might be explained by the differing immunosuppressive/anti-inflammatory environments of these tumors in vivo. To investigate this hypothesis, we studied the effect of human aqueous and vitreous fluids, of the proinflammatory cytokine tumor necrosis factor alpha, and of the anti-inflammatory peptides alpha-melanocyte-stimulating hormone and melanocyte-stimulating hormone 11-13 (KP-D-V) on the invasion of three human uveal melanoma cell lines through human fibronectin. Fresh aqueous humor samples significantly decreased the invasion in two out of three uveal melanoma cell lines. In contrast, vitreous humor did not reduce invasion. Tumor necrosis factor alpha significantly increased the invasiveness of uveal melanoma cell lines by approximately 50%-80% over 20 h. Full-length alpha-melanocyte-stimulating hormone, at concentrations present in the aqueous humor (10-9 M), as well as melanocyte-stimulating hormone 11-13 (KP-D-V) reduced the invasion of cells through human fibronectin by 45%-50% and also protected uveal melanoma cells from the pro-invasive actions of tumor necrosis factor alpha. These data are consistent with inflammation playing a major role in affecting the metastatic ability of uveal melanomas. Thus, ocular microenvironments that differ in their immunosuppressive/anti-inflammatory properties may influence the invasiveness of developing tumors.