GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi.

BACKGROUND: Mutations in GNAQ/11 genes are considered an early event in the development of uveal melanoma that may derive from a pre-existing nevus. The Hippo pathway, by way of YAP activation, rather than MAP kinase, has a role in the oncogenic capacity of GNAQ/11 mutations. METHODS: We investigated 16 nevi from 13 human eyes for driver GNAQ/11 mutations using droplet digital PCR and determined whether nevi are clonal by quantifying mutant nevus cell fractions. Immunohistochemistry was performed on 15 nevi to analyse YAP activation. RESULTS: For 15 out of 16 nevi, a GNAQ/11 mutation was detected in the nevus cells albeit at a low frequency with a median of 13%. Nuclear YAP, a transcriptional co-activator in the Hippo tumour-suppressor pathway, was detected in 14/15 nevi. CONCLUSIONS: Our analysis suggests that a mutation in GNAQ/11 occurs in a subset of choroidal nevus cells. We hypothesise that GNAQ/11 mutant-driven extracellular mitogenic signalling involving YAP activation leads to accumulation of wild-type nevus cells.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

PURPOSE: To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. METHODS: Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. RESULTS: The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and ß4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and ß1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. CONCLUSION: The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.

The genetic basis of uveal melanoma.

Uveal melanoma (UM) is the most common intraocular malignancy in adults with an incidence of about 1/100,000 new cases per year in the Western world. Risk factors are having a light skin, blond hair and blue eyes. As some UM patients have a young age at diagnosis or an affected family history for UM or other malignancies, there may be an underlying genetic basis. This review discusses known or suspected risk factors for UM, the cancer risk in UM patients and their family members, and the genes that have been reported to predispose to UM (germline mutations) and tumor development (somatic mutations).

[Decellularized collagen matrix from tilapia fish scales for corneal reconstruction (BioCornea)]. / Dezellularisierte Kollagenmatrix aus der Schuppe des Tilapia-Fisches als Hornhautersatz ("BioCornea").

BACKGROUND: The worldwide need for donor corneal tissue clearly exceeds the availability of transplantable human tissue; therefore, recent efforts aim to identify and characterize alternative tissues, such as decellularized collagen scaffolds. OBJECTIVES: The transparent fish scales of tilapia (Oreochromis mossambicus) were analyzed as a potential alternative for corneal reconstruction ("BioCornea"). MATERIAL AND METHODS: The article gives a review of the literature and own preliminary results. After decellularization the tissue characteristics of the fish scales, the repopulation with corneal epithelium and stromal cells, immunogenicity, the feasibility of corneal transplantation and the angiogenic properties were analyzed in vitro and in various animal models. RESULTS: The fish scales mainly consist of collagen type I and show an architecture that is similar to the human cornea. Corneal epithelium and stromal cells are able to grow over and into the scaffold. It is possible to transplant fish scales in various animal models without severe inflammatory responses. Furthermore, in mice, less blood and lymphatic vessels grow into the xenograft when compared to conventional allogenic transplants. CONCLUSION: Preliminary results with decellularized tilapia fish scales as an alternative for corneal reconstruction ("BioCornea") are promising.

Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.

Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.

Inflammation in uveal melanoma.

Leukocytic infiltration is a common feature of human cancers, including those that develop in immunoprivileged sites, such as the eye. The infiltration of myeloid and T cells into tumours is part of the host response against cancer. In uveal melanoma, high densities of immune cells seem to be involved in tumour progression, as they are associated with the loss of one chromosome 3. The nature of this tumour microenvironment might offer therapeutic opportunities.

New therapeutic agents in uveal melanoma.

Uveal melanoma is the most common primary intraocular malignant tumour in adults. Five, ten and fifteen years after primary tumour treatment, up to 25%, 34% and 50% of patients may develop metastases, respectively. There are only a few systemic therapies that have been approved for uveal melanoma, all with doubtful efficacy. As the molecular knowledge over cancer has improved, new therapies are being developed. Several drugs, such as bortezomib, celecoxib, dacarbazine, anti-angiogenic agents (such as bevacizumab, sorafenib and sunitinib), temsirolimus, mitogen-activated protein kinase kinase (MEK) inhibitors, ipilimumab and AEB071 are candidate drugs, and studies are underway to determine the therapeutic effects of these drugs in uveal melanoma.

Synergistic growth inhibition based on small-molecule p53 activation as treatment for intraocular melanoma.

The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1α induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1α. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1α protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1α. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.

Clinical presentation and outcome of perforating ocular injuries due to BB guns: a case series.

PURPOSE: To describe the clinical presentations and treatment modalities of a series of BB gun-related perforating ocular injuries. METHODS: Clinical records of all consecutive cases of perforating BB gun injuries to the globe seen between September 2004 and September 2008 were reviewed retrospectively. At the time of the trauma and after final treatment, all patients underwent a complete ocular examination, including visual acuity,applanation tonometry for intraocular pressure, slit lamp biomicroscopy, indirect ophthalmoscopy and fundus photography, if possible. In all cases, primary globe repair was performed in the first session, and then appropriate surgery took place based on the individual situation. RESULTS: In this study, 13 patients (11 males and 2 females) with a mean age of 20.8 years (range 9­50 years) were enrolled. The mean follow-up period was 7.2 4.3 months (range 1­25 months). Initial visual acuity (VA) ranged from no-light perception (NLP) to finger counting (CF). Vitreous haemorrhage and retinal detachment were present in all involved eyes. Hyphema (30.76%), uveal and retinal prolapse (30.8%), retinalin carceration (30.8%) and retinal haemorrhage (53.8%) were other ocular findings. VA remained stable in 46.2% of the patients (6 cases). The best achieved final VA was CF at 2 min one case after 6 months follow-up.After several surgical procedures, enucleation was necessary in only 2/13 (15.4%) cases. CONCLUSION: Despite several surgical procedures which decreased the number of enucleations, BB gunperforating ocular injuries still lead to a grim visual outcome. This implies the importance of political strategies targeting on education of parents and restriction for children to access to these guns.

Episodic Src activation in uveal melanoma revealed by kinase activity profiling.

BACKGROUND: The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM. METHODS: We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays. RESULTS: We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced. CONCLUSION: We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.

Expression and distribution of MMPs and TIMPs in human uveal melanoma.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Mechanisms of local immunosuppression in cutaneous melanoma.

Cutaneous melanoma is highly immunogenic, yet primary melanomas and metastases develop successfully in otherwise immunocompetent patients. To investigate the local immunosuppressive microenvironment, we examined the presence of suppressor T lymphocytes and tolerising dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFbeta1 and TGFbeta2) and the enzyme indoleamine 2,3-dioxygenase (IDO) using qRT-PCR and immunohistochemistry in primary skin melanomas, negative and positive sentinel lymph nodes (SLN), and lymph nodes with advanced metastases. Our results indicate that tolerogenic DCs and suppressor T lymphocytes are present in melanoma at all stages of disease progression. They express transforming growth factor beta receptor 1 (TGFbetaR1), and are therefore susceptible to TGFbeta1 and TGFbeta2 specifically expressed by primary melanoma. We found that expression of IDO and interleukin 10 (IL-10) increased with melanoma progression, with the highest concentration in positive SLN. We suggest that negative SLN contain immunosuppressive cells and cytokines, due to preconditioning by tolerogenic DCs migrating from the primary melanoma site to the SLN. In primary melanoma, TGFbeta2 is likely to render peripheral DCs tolerogenic, while in lymph nodes IDO and TGFbeta1 may have a major effect. This mechanism of tumour-associated immunosuppression may inhibit the immune response to the tumour and may explain the discrepancy between the induction of systemic immunity by anti-melanoma vaccines and their poor performance in the clinic.

S-100B protein and melanoma inhibitory activity protein in uveal melanoma screening. A comparison with liver function tests.

AIM: Our purpose was to determine whether S-100B or melanoma inhibitory activity (MIA) concentrations in the serum of patients with large uveal melanomas were better markers for the presentation of metastases than liver function tests. We also investigated whether increased marker levels were related to known clinical and histopathological prognostic parameters. METHODS: Total S-100B (A1B + BB) and MIA concentrations were measured in the sera from 104 patients with uveal melanoma prior to enucleation and in the sera from 50 healthy controls. Concentrations were also determined in the sera from 30 patients with known uveal melanoma metastases. Liaison Sangtec 100, an automated immunoluminometric assay measuring the total S-100B, and Roche MIA ELISA were used to quantify these proteins in serum. Results were compared with liver function tests [alkaline phosphatase, lactate dehydrogenase (LD), aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transpeptidase]. RESULTS: The mean S-100B and MIA concentrations were significantly higher in patients with metastases compared to melanoma patients without metastases. At the time of enucleation, S-100B and MIA were not prognostic for metastases in uveal melanoma, but S-100B and LD were the best tests to predict the occurrence of metastatic disease during the follow-up period. CONCLUSIONS: In our study, the S-100B and MIA serum concentrations were not correlated with any tested established prognostic parameter. S-100B and LD showed better performance in identifying melanoma metastases than gamma-glutamyl transpeptidase and MIA. A prospective follow-up study is needed to evaluate S-100B and MIA in identifying early micrometastasis in uveal melanoma.

A new model for limbal transplantation using E-GFP for follow-up of transplant survival.

Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.

Immunophenotypic markers to differentiate between benign and malignant melanocytic lesions.

BACKGROUND/AIMS: The authors investigated the expression of S100A1, S100A6, S100B, MelanA, and CEA in conjunctival naevi, primary acquired melanosis (PAM), conjunctival melanoma, and uveal melanoma in order to assess their potential usefulness in the pathological differential diagnosis of these entities. METHODS: Paraffin embedded sections of 18 conjunctival naevi, 14 PAM, 16 conjunctival melanomas, and 20 uveal melanomas were immunostained for S100A1, S100A6, S100B, MelanA, and CEA, and expression was scored semiquantitatively. RESULTS: Expression of S100A1 differed significantly between conjunctival naevi and conjunctival melanoma, with percentages of positive cells of 30.6% and 71.4%, respectively. Conjunctival melanomas had high average scores for S100A1 and S100B (71.4%, 62.9%, respectively), while uveal melanomas also had high S100A1 but low S100B scores (88.5%, 18.5%, respectively). MelanA was highly variable; naevi and uveal melanoma had higher average scores than conjunctival melanoma. CEA was hardly detectable in all four groups. CONCLUSION: S100A1 seems to be a possible candidate to differentiate conjunctival naevi from conjunctival melanoma. S100B seems to differentiate between uveal melanoma and conjunctival melanoma. However, the study size was small and therefore the data have to be confirmed by others.

Ciliary tissue transplantation in the rabbit.

Irreversible damage of the ciliary body can be responsible for prolonged ocular hypotony and phthisis bulbi, which, currently, cannot be treated. The aim of this study was to achieve survival of morphologically normal ciliary tissue (CT) transplants in the anterior chamber of a rabbit's eye. Outbred female New Zealand albino rabbits received CT allografts, which were placed on to the surface of the host iris. We evaluated the influence of ciclosporin (CsA), VEGF and donor perfusion on graft survival. Operated eyes were assessed clinically and histologically, and revascularization of the grafts was determined by fluorescein angiography. All grafts became dark and ischemic during the first five to seven days after transplantation. Reperfusion of the grafted tissue was complete at approximately ten days after transplantation. In untreated animals, transplants became infiltrated by inflammatory cells, which led to destruction of the tissue. This was prevented by systemic use of CsA. Transplants treated with VEGF prior to transplantation had fewer ischemic areas but epithelial cell survival was not improved. Whole body donor perfusion prior to preparation of the grafts resulted in less inflammation and, histologically, in a better quantity and quality of the epithelial cells in the CT transplants. Ciliary tissue can be successfully transplanted but the ciliary epithelium suffers from ischemia and in untreated animals the whole transplant is rejected in the classical fashion. If the donor is perfused and the host immunosuppressed, histologically normal ciliary epithelium can be preserved together with rapid revascularization, minimal inflammation and good survival of the transplant, although fibrosis continued to occur during the two months after transplantation.

Normal HLA class I, II, and MICA gene distribution in uveal melanoma.

PURPOSE: The molecules of the HLA class I and II molecules as well as the MHC class I chain-related gene A (MICA), a polymorphic and stress-induced cell surface molecule, are involved in T-cell and natural killer-cell (NK-cell) mediated immune responses. In this study we looked for any genetic susceptibility contributed by HLA class I, class II, or MICA genes with regard to the development of uveal melanoma. METHODS: Between 1998 and 2001, 159 uveal melanoma patients were typed for HLA class I and II, and 168 uveal melanoma patients were evaluated for MICA by microsatellite typing. The HLA antigen and MICA allele frequencies were compared with control groups of, respectively, 2,440 and 247 healthy Dutch individuals. RESULTS: HLA class I, HLA class II, and MICA gene frequencies in uveal melanoma patients and healthy Dutch controls showed no significant deviations after correction for the number of comparisons. CONCLUSIONS: We conclude that there is no genetic susceptibility or increased risk attributed to any HLA class I, class II, and MICA polymorphism with regard to the development of uveal melanoma.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.