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Purpose: To analyze the clinical characteristics of a serous retinopathy associated with mitogen-activated protein kinase kinase (MEK) inhibition with binimetinib treatment for metastatic cutaneous melanoma (CM) and uveal melanoma (UM), and to determine possible pathogenetic mechanisms that may lead to this retinopathy., Design: Prospective observational, cohort-based, cross-sectional study., Participants: Thirty CM patients and 5 UM patients treated with the MEK inhibitor binimetinib (CM) or a combination of binimetinib and the protein kinase C inhibitor sotrastaurin (UM)., Methods: Extensive ophthalmic examination was performed, including Early Treatment of Diabetic Retinopathy Study best-corrected visual acuity, applanation tonometry, slit-lamp examination, indirect ophthalmoscopy, digital color fundus photography, and optical coherence tomography (OCT). In selected cases, additional examinations were performed, including visual field testing and electro-oculography (EOG). Blood samples were obtained from 3 CM patients and 3 UM patients to analyze the presence of autoantibodies against retinal and retinal pigment epithelium (RPE) proteins., Main Outcome Measures: Visual symptoms, visual acuity, fundus appearance, characteristics on OCT, fundus autofluorescence (FAF), and EOG., Results: Six CM patients (20%) and 2 UM patients (40%) reported visual symptoms during the study. The median time to the onset of symptoms, which were all mild and transient, was 3.5 days (range, <1 hour to 3 weeks). On OCT, subretinal fluid (SRF) was detected in 77% of CM patients and 60% of UM patients. In the 26 patients with SRF, the fovea was affected in 85%. After the start of the medication, an EOG was performed in 19 eyes of 11 patients; 16 of these eyes (84%) developed SRF on OCT. Fifteen of these eyes (94%) showed an abnormal Arden ratio (<1.65). A broad pattern of anti-retinal antibodies was found in 3 CM patients and 2 UM patients tested, whereas anti-RPE antibodies were detected in all 6 tested patients., Conclusions: A time-dependent and reversible serous retinopathy can develop both in patients with metastatic CM and UM treated with binimetinib. A minority of patients develop visual symptoms, which are generally mild and transient. A cause of binimetinib-associated serous retinopathy may be toxicity of medication, but autoantibodies also may be involved., (Copyright © 2015 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

To further elucidate the genetic underpinnings of uveal melanoma (UM) and identify new markers that correlate with disease outcome, archival formalin-fixed, paraffin-embedded enucleation specimens from 25 patients with UM and a mean follow-up of 14 years were analyzed for whole-genome copy-number alterations using OncoScan analysis. Copy-number alterations of chromosomes 1, 3, 6, and 8 were also analyzed in these tumors using multiplex ligation-dependent probe-amplification, and mutations in GNAQ, GNA11, and BAP1 were searched for by Sanger sequencing. Our study confirms the previously reported GNAQ and GNA11 mutation frequencies in UMs as well as the presence of monosomy 3 as a factor strongly indicating poor prognosis. Two cases with metastatic disease, but without monosomy of chromosome 3, showed loss of a small region in the distal part of chromosome 2p. Also, UMs leading to metastatic disease had more chromosomal aberrations than those without metastases. Three UMs lacking a GNAQ or a GNA11 mutation showed a gain of chromosome 8q; one of these cases showed extensive chromothripsis. Another case (with suspect lung metastasis) showed focal chromothripsis. Our whole-genome copy-number analysis shows that focal loss of chromosome 2p may be involved in the metastatic spread of UMs without monosomy 3; metastatic UMs carry more chromosomal aberrations than those without metastases; and chromothripsis may play a role in the oncogenesis of UMs, but does not necessarily indicate a poor prognosis. The clinical and particularly diagnostic utility of these findings needs to be corroborated in a larger set of patients with UM.

Purpose: To assess the safety and efficacy of dendritic cell vaccination in metastatic uveal melanoma., Design: Interventional case series., Methods: We analyzed 14 patients with metastatic uveal melanoma treated with dendritic cell vaccination. Patients with metastatic uveal melanoma received at least 3 vaccinations with autologous dendritic cells, professional antigen-presenting cells loaded with melanoma antigens gp100 and tyrosinase. The main outcome measures were safety, immunologic response, and overall survival., Results: Tumor-specific immune responses were induced with dendritic cell vaccination in 4 (29%) of 14 patients. Dendritic cell-vaccinated patients showed a median overall survival with metastatic disease of 19.2 months, relatively long compared with that reported in the literature. No severe treatment-related toxicities (common toxicity criteria grade 3 or 4) were observed., Conclusions: Dendritic cell vaccination is feasible and safe in metastatic uveal melanoma. Dendritic cell-based immunotherapy is potent to enhance the host's antitumor immunity against uveal melanoma in approximately one third of patients. Compared with other prospective studies with similar inclusion criteria, dendritic cell vaccination may be associated with longer than average overall survival in patients with metastatic uveal melanoma., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the long-term effects of ruthenium-106 plaque radiation therapy for iris and iridociliary melanomas in terms of tumour regression and complications., Methods: Between 1 November 1997 and 31 December 2007, 36 patients with an iris or iridociliary melanoma were treated with Ruthenium-106 (Ru-106) ophthalmic plaque radiation therapy. The median follow-up was 6.5 years with a range of 2-11 years. The mean tumour apex dose was 151.5 Gy. Main outcome measures were local tumour control and radiation-related ocular complications., Results: The mean age of the patients at the time of treatment was 54 years (range 14-82). The tumours had a median largest basal dimension of 4.8 mm (range 3-11) and a prominence of 2.3 mm (range 0.8-5), with a median involvement of three clock hours (range 1-6). The tumours were confined to the iris in 14 patients (39%), extended into the anterior ciliary body in 12 (33%), while the anterior ciliary body tumour extended into the iris in ten patients (28%). Tumours regressed in all patients (100%) with a mean regression of 80% of the original tumour size at 7 years of follow-up. Radiation-related complications included corneal erosions on the first postoperative day in nine patients (25%), cataract in four of 11 patients free of cataract before treatment (36%) and postradiation glaucoma in one patient (3%). Visual acuity of 20/200 or worse was present in one of 20 patients (5%) at 6 years of follow-up. Local recurrences occurred in two patients (5%), 2 and 5 years after the radiation therapy respectively, and both underwent enucleation. Distant metastases occurred in one of these enucleated patients (3%) 2 years after treatment., Conclusion: Ru-106 plaque radiation therapy for iris and iridociliary melanoma resulted in good local tumour control and preservation of visual acuity with few and treatable side-effects., (© 2010 The Authors. Journal compilation © 2010 Acta Ophthalmol.)

Purpose: In trans-scleral thermotherapy (TSTT), heat is applied through the sclera in order to target an intraocular uveal melanoma. Previously, it had been shown that in uveal melanoma, hyperthermia and transpupillary thermotherapy influenced expression of immunologically relevant proteins, such as S100, HLA and heat-shock proteins (HSPs). We investigated whether TSTT induced similar changes., Methods: Experimental TSTT was applied on eleven uveal melanomas prior to enucleation. Each tumour sample was processed for histopathological examination; immunohistochemical analysis was performed to determine expression of S100, HLA, HSPs and macrophage markers., Results: In TSTT-treated areas, expression of S100 and different HSPs was lost, while an upregulated expression of HSP GP96 was observed at the border of these areas. Expression levels of HLA-A and HLA-B varied between tumours and were not influenced by TSTT. The borders of the TSTT-treated areas showed high numbers of infiltrating macrophages, which were predominantly of the M2 phenotype., Conclusion: TSTT has an effect on immunological parameters with local loss of expression of HSPs and S100. The influx of M2 macrophages around the TSTT-treated areas indicates the presence of an innate immune reaction against the induced necrosis, suggesting that TSTT-treated tumour cells are removed by a macrophage-mediated tissue repair mechanism., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To evaluate long-term outcomes of eye-conserving treatment using Ruthenium-106 plaque brachytherapy with or without transpupillary thermotherapy (TTT) for small to intermediate size choroidal melanomas., Methods: Outcomes of 425 consecutive patients were analysed. The median basal tumour diameter was 10.9 mm (range 4.8-15.9 mm), and the median apical height 4.2 mm (range 1.2-9.3 mm). Brachytherapy doses ranged from 400 to 600Gy with TTT (86%), or from 600 to 800Gy without TTT (14%), specified at the scleral surface. Kaplan-Meier survival curves, log-rank tests and Cox regression analysis were used for analysis., Results: Median follow-up was 50 months. Five-year actuarial local control was 96%. Five-year overall and metastases-free survival rates were 79.6% and 76.5%. Prognostic factors for metastasis-free survival were peripheral location (p=0.02) and smaller basal diameter (p<0.001). No dose effect relationships were found. Radiation side effects were frequent, with 2- and 5-year rates free of radiation complications of 60% and 35%. Five-year enucleation rate was 4.4% (10 for local recurrence, 7 for complications). Cosmetic and functional (visual acuity >0.10) eye preservation rates were 96% and 52% at 5 years., Conclusions: Ruthenium-106 brachytherapy for choroidal melanoma provides excellent rates of local control and eye preservation., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The differential diagnosis between primary uveal melanoma and cutaneous melanoma metastasis in the eye may be difficult, both clinically and histologically. We report successful application of combined mutational analysis of the NRAS and the CDKN2A gene to discriminate between these two entities. The patient had a history of a superficial spreading cutaneous melanoma of the left shoulder. Nine years later, she developed a lymph node metastasis in the left axilla, and 13 years later she presented with an atypical, pigmented tumor in the uvea. Histologically, the origin of the uveal melanoma could not be determined with certainty. We performed molecular analysis on the skin melanoma, the lymph node metastasis and the uveal melanoma. We detected an NRAS codon 61 mutation (c.182A>G, p.Gln61Arg) in all three tumor specimens. This mutation was absent in the normal control tissue of the patient, thereby excluding a germline mutation. To confirm a clonal relationship between the tumors, we also performed CDKN2A mutational analysis. We detected a CDKN2A mutation ((p16) c.238C>T, p.Arg80X, (p14) c.404C>T, p.Pro135Leu)) in the tumor samples, but not in the normal control tissue of the patient. We concluded that the uveal melanoma is a metastasis from the cutaneous melanoma removed 13 years before.

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., (Copyright (c) 2007 S. Karger AG, Basel.)

Objectives: To determine the presence of vascular endothelial growth factor A (VEGF-A) in the aqueous humor of eyes with uveal melanoma and to identify its source., Methods: The VEGF-A concentrations were determined in aqueous humor samples obtained after enucleation from 74 eyes with untreated uveal melanoma and from 8 eyes with treated uveal melanoma. Patient survival and clinical and histopathological tumor variables were compared. In situ hybridization, Western blot analysis, and enzyme-linked immunosorbent assay were used to determine expression of VEGF-A in tumor tissue and in overlying retina., Results: Aqueous VEGF-A concentrations ranged from 18 to 826 pg/mL in 74 untreated eyes, while concentrations in 30 control eyes were significantly lower (median, 50.1 pg/mL) (P<.001). Concentrations in 8 treated eyes were much higher (median, 364 pg/mL). In situ hybridization on tissue sections and Western blot analysis and enzyme-linked immunosorbent assay on tissue extracts revealed VEGF-A in uveal melanoma tissue and in retinal tissue., Conclusions: Uveal melanoma is associated with increased concentrations of VEGF-A in aqueous humor. Aqueous VEGF-A concentration correlates with largest basal tumor diameter and with the tumor height. In eyes with uveal melanoma, tumor and retinal tissues are sources of VEGF-A.

Uveal melanoma develops in one of the most capillary-rich tissues of the body and is disseminated hematogenously. Knowledge of the nature and the spatiotemporal expression of angiogenic factors in uveal melanoma is essential to the development of new treatment strategies, especially with regard to improving survival. In this study, we measured the angiogenic potential of several angiogenic factors in different uveal melanoma cell lines, in an in vivo model, and in primary tumor material from patients with melanoma. Most uveal melanoma cell lines expressed vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF-B, VEGF-C, VEGF-D, and basic fibroblastic growth factor (b-FGF) to various extents. The expression of VEGF-A 121 was always higher than that of the other VEGF-A isoforms, suggesting that VEGF-A 121 is the most abundant VEGF-A isoform. All experimentally induced tumors expressed VEGF-A, VEGF-B, VEGF-C, VEGF-D, and basic fibroblastic growth factor (b-FGF). Similarly, significant amounts of mRNA for VEGF-B, VEGF-C, VEGF-D, and b-FGF were detected in uveal melanoma material from patients. In contrast, VEGF-A mRNA (121, 165, 189) was low (9/28) or not detectable in the tumor samples. The synthesis of VEGF-A 165 and b-FGF protein by various cell lines was measured by enzyme-linked immunosorbent assay (ELISA). Most uveal melanoma cell lines, but not normal melanocytes, strongly synthesized and secreted VEGF-A 165 and b-FGF during cell culture. Our data suggest that the expression of (lymph) angiogenic factors may play a causal role in the angiogenesis and progression of uveal melanoma and distant metastasis.

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.

Purpose: Human uveal melanoma develops in one of the most capillary-rich tissues of the body and has a pure hematogenous dissemination. Radiodiagnostic examinations, such as ultrasonic diagnostic resonance imaging and chest radiographs plus liver enzyme studies in blood, are methods used to detect liver and other distant metastases in patients. Nevertheless, the mortality rate is high, because of the frequent occurrence of metastases and the lack of systemic therapy. Therefore, the development of novel anticancer strategies is urgent, and more sensitive and less invasive methods of detecting and monitoring in vivo tumor growth and metastatic disease in cancer models are needed., Methods: A luciferase (Luc)-positive human uveal melanoma cell line (OCM-1 FRT/luc) was established. Tumor cells were inoculated into the anterior chamber of murine eyes for induction of orthotopic growth or into the left heart ventricle to mimic hematogenous micrometastatic spread. Development of metastases and tumor growth was monitored weekly by whole-body bioluminescent reporter imaging (BLI)., Results: Injection of cancer cells into the anterior chamber of the eye of mice closely mimicked orthotopic tumor growth of uveal melanoma. Tumor progression could be quantitatively monitored 3 weeks after inoculation of 10(5) OCM-1 FRT/luc cells. Of the mice injected, 83% exhibited a detectable tumor within 5 weeks. Intracardiac injection of tumor cells resulted in metastatic growth, especially in bone. Mice had bone (maxillofacial region and femora) and visceral (lung and mediastinum) metastases after 4 to 6 weeks. OCM-1 FRT/luc cells may also have a propensity to colonize the eye after intracardiac inoculation., Conclusions: BLI enables continuous quantitative monitoring in the same animal of growth kinetics for each tumor and its metastases. This model will accelerate the understanding of the pathogenesis and treatment of uveal melanoma and metastasis.

The purpose of this study was to investigate the cytotoxic effect of heat as induced by transscleral thermotherapy (TSTT), which may be of interest in the treatment of patients with choroidal melanoma. The aim of TSTT is to heat both the sclera and the tumor up to a cytotoxic temperature of about 60 degrees C. TSTT was performed in hamsters with subcutaneously implanted Greene melanoma covered by a specimen of human donor sclera of thickness 0.5, 0.7 or 0.9 mm. A newly developed applicator, which combines conductive episcleral heating at 60 degrees C with laser-induced heating, was used at laser powers ranging from 500 to 1500 mW delivered by an 810 nm diode laser, beam diameter 3 mm, and exposure time 1 min. Temperatures were measured at the scleral surface and at the sclera-tumor interface. The extent of tumor necrosis was examined by light microscopy and the sclera was examined by polarized light microscopy. Maximal depth of tumor necrosis without scleral damage was 4.4 (SD 1.5) mm. The temperature at the scleral surface after TSTT was 58.8 (SD 2.4) degrees C. The temperature at the sclera-tumor interface ranged from 56.4 (SD 3.7) degrees C at 500 mW to 65.3 (SD 4.4) degrees C at 1250 mW laser power. Structural changes to the scleral collagen started to develop at 1250 mW. TSTT with combined laser-induced and conductive heating caused cytotoxic temperatures in the tumor and the sclera, which were well tolerated by the scleral collagen.

Uveal melanoma is the most common primary intraocular tumour in adults. After treatment of the primary tumour, up to 50% of patients will ultimately develop metastases. Treatment options for metastases are limited. When uveal melanoma metastases are confined to the liver, isolated hepatic perfusion (IHP) could be a treatment option. Herein, we report the results of a small group of patients with uveal melanoma metastases of the liver treated with IHP. Eight patients with uveal melanoma metastases confined to the liver underwent IHP with high-dose melphalan (200 mg) for 1 h. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria and tumour response was assessed according to World Health Organization criteria. The tumour response rate (complete or partial remission) was 50%. The median time to progression was 6.7 months (range, 1.7-16.9 months). The overall median survival was 9.9 months (range, 4.7-34.6 months), with a 1 year survival of 50% and a 2 year survival of 37.5%. Three patients experienced grade 3-4 hepatotoxicity which was transient within 3 months. Although only a small group of patients has been treated and evaluated so far, IHP is a treatment option for uveal melanoma metastases confined to the liver which can result in tumour responses and may lead to survival benefits in a selective group of patients.

Local tumour control in uveal melanoma has improved in the last decades. However, 5-year mortality due to metastases from large uveal melanomas remains high. Recently both isolated liver perfusion therapy and chemotherapy have reached encouraging results in improving metastasis survival. As such screening at an early stage, especially for liver metastases, becomes imperative.

A new tool in the search for tumour markers is proteomic technology. Proteomics (or protein profiling) is the study of the proteome, the protein complement of the genome. The advantage of this technique in comparison with genomics is that the actual protein production can be measured. Gene microarrays determine levels of mRNA but do not necessarily predict the level of the corresponding proteins in a cell. In this study, we evaluated the use of proteomics in the aqueous humour of uveal melanoma patients compared with control patients using the surface-enhanced laser desorption ionization time-of-flight technique. The protein mass spectra of aqueous humours from 24 uveal melanoma eyes were compared with 24 control eyes using a strong anion exchange surface protein chip array. On the basis of two proteins (4543.43 and 6853.30 kDa), the aqueous humour of melanoma eyes and control eyes could be distinguished in 89% of cases. Therefore, proteomic evaluation might be helpful in finding diagnostic markers for uveal melanoma patients.

Aim: To evaluate the results of combined plaque radiotherapy and transpupillary thermotherapy (TTT) in 50 consecutive patients 5 years after treatment., Methods: 50 adult patients with choroidal melanoma were treated with ruthenium-106 ((106)Ru) plaque radiotherapy combined with TTT. A flat scar was the preferred end point of treatment. The mean tumour thickness was 3.9 mm (range 1.5-8.0 mm), the mean tumour diameter was 11.3 mm (range 5.8-15.0 mm). TTT was performed with an infrared diode laser at 810 nm, a beam diameter of 2-3 mm, and 1 minute exposures. Tumours >5 mm thick received an episcleral contact dose of 800 Gy (106)Ru; tumours 3 mm thick (log rank test p = 0.01). Eight melanomas were amelanotic, seven of which required multiple TTT sessions. In one patient the tumour recurred at the central margin of the treated area; this eye was enucleated. In one patient the tumour failed to regress 6 months after treatment and enucleation was performed at the patient's request. Three eyes developed severe proliferative retinopathy. Radiation maculopathy caused a loss of the best corrected visual acuity: before treatment 31 patients had a best corrected visual acuity of 20/60 or better but in only 12 patients did it remain in this range 5 years after treatment. Three patients developed distant metastasis to the liver., Conclusion: The 5 year results for combined plaque radiotherapy and TTT as treatment for choroidal melanoma are favourable in terms of complete tumour regression and low rate of recurrences; however, there was considerable loss of visual acuity as a result of radiation maculopathy.

Background: In cutaneous melanoma, the S-100-beta serum level is recognized as a marker of metastatic disease., Objectives: To determine whether S-100-beta is present in the serum of patients with uveal melanoma and to test whether the serum concentration of S-100-beta is related to known clinical and histopathological prognostic factors in these patients., Methods: The S-100-beta concentration was measured in serum samples collected from 64 patients with uveal melanoma before enucleation and from 58 healthy control subjects. A 2-site immunoluminometric assay was used to quantify the S-100-beta concentration in serum. S-100-beta concentrations in the serum from patients were compared with clinicopathological tumor variables, sex, occurrence of metastasis, and survival., Results: Thirty-seven (57.8%) of 64 patients with uveal melanoma showed detectable levels of serum S-100-beta. There was, however, no significant difference between serum levels of patients and control subjects (P =.71). Statistical analysis showed no significant correlation between S-100-beta concentration and any of the clinicopathological tumor variables, occurrence of metastases, or survival. Only sex was correlated with S-100-beta serum levels, which was not observed in the control group., Conclusions: In our study on patients with uveal melanoma, the S-100-beta serum concentration was not correlated with any investigated prognostic factor and was not of prognostic value itself. Female patients appeared to have higher S-100-beta concentrations than male patients.

Purpose: Expression of heat shock proteins (HSPs) is of prognostic significance in several tumor types, whereas HSPs may also have clinical use as stimulators in tumor vaccination. HSP expression levels were determined in normal eyes and in uveal melanoma and tested whether HSPs expression was associated with prognostic parameters in the uveal melanoma., Methods: Expression of HSP27, HSP70, HSP90, and glycoprotein96 (GP96) were determined on paraffin-embedded and frozen sections from seven healthy eyes, 20 primary uveal melanomas without prior treatment, and 18 uveal melanomas after prior treatment. HSP expression was determined by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry, using appropriate monoclonal antibodies and scored semiquantitatively. Expression of HSPs was validated on retinal tissue of a normal eye and in two uveal melanoma cell lines by Western blot analysis., Results: Expression of HSPs was observed in epithelial and pigment cells of the normal eyes. In uveal melanoma, the level of expression of HSPs varied. Expression of HSP27 and GP96 was noted in more than 30 of 38 uveal melanomas (with, respectively, a mean of 66% and 53% positive cells). HSP70 and HSP90 were expressed in 6% of tumor cells. The amount of expression of any of the HSP types was not significantly associated with known prognostic factors. There was not a significant difference in expression of the HSPs between uveal melanomas with or without any type of prior treatment., Conclusions: In this study, expression of HSPs in uveal melanoma is not correlated with known histopathologic prognostic factors. The high expression of GP96 indicates that this protein is a potential vector in tumor vaccination in patients with large uveal melanomas.

During the past 20 years of enucleation, which was the standard treatment for choroidal melanoma over more than a century, has largely been replaced by eye salvaging therapies such as radiotherapy or local resection. In 1995 transpupillary thermotherapy (TTT) using an infrared diode laser was introduced as a new conservative therapy for patients with choroidal melanoma. TTT can be defined as a heat treatment modality, which is delivered through a dilated pupil to the tumour surface. The technique uses a wide diode laser beam diameter with a low irradiance and a long exposure time. TTT induces tumour necrosis at sub-photocoagulation levels by a direct cell destructive effect with only a few ocular complications. TTT can be performed as sole therapy or combined with plaque radiotherapy, thus permitting a lower radiation dose. For amelanotic tumours dye-enhanced TTT with indocyanine green can be used. In this paper we review the role of sole or combined TTT, related to the current other treatment modalities for choroidal melanoma.

The aim of this study was to evaluate the effectiveness of transpupillary thermotherapy (TTT) in generating tumour necrosis by light and electron microscopy, as well as to evaluate additional cell damage in the area directly adherent to the necrotic zone. Four eyes of four patients diagnosed with intraocular malignant melanoma of the uvea were treated experimentally with diode laser TTT. In all cases a standard technique was used. All eyes were enucleated: one eye the day after TTT, two eyes 2 days after TTT, and one eye 6 weeks after TTT. Immediately after enucleation the eyes were immersed in standard Karnovsky's fixative with cocodylate buffer and prepared for light and electron microscopy. In the treated area of all four melanomas we found a dense band of necrotic tissue (zone A) consisting of an amorphous mass of dead cells sharply demarcated from the rest of the neoplastic tissue. Next to this zone was a more eosinophilic and also sharply demarcated band (zone B) that consisted of similar but less intensive changes. In the next band (zone C), marked injury to the cellular membrane and subcellular structures were seen on electron microscopy. The next band (zone D) consisted of changes mainly observed only within the cytoplasm of neoplastic cells and significantly less intensive than those in zone C. Outside zone D tumour cells that were normal in appearance were seen. No scleral alterations induced by heat were found. We concluded that after TTT the cytotoxic effect gradually decreases in proportion to the distance from the central point of the diode laser spot, with additional cell damage in the area adjacent to the necrotic zone. The interval between TTT and enucleation had no influence on the histological results.

Purpose: Allelic variations of the melanocortin-1 receptor (MC1R) gene have been linked to red hair and sun-sensitive skin types and may play a role in the susceptibility to develop cutaneous malignant melanoma (CMM). To define the role of MC1R gene in uveal melanoma, a case control study was performed, in which the presence of MC1R gene variations in uveal melanoma patients was compared with that of healthy controls., Methods: MC1R gene variants were analyzed in 162 uveal melanoma patients and 255 healthy controls. After genomic DNA was isolated from venous blood, the MC1R gene was amplified by polymerase chain reaction (PCR) and examined for the presence of variants by single-strand conformation polymorphism (SSCP) analysis. Participants were asked to complete a questionnaire regarding skin type, eye color, and hair color., Results: No disparity was found between the distribution of the MC1R gene variants in both groups. Furthermore, no associations between MC1R genotype and pigment phenotype were found. In contrast to CMM, uveal melanoma patients did not show specific MC1R gene variants. Compared with controls, most uveal melanoma patients had blue eyes (65%, P = 0.060) and skin type III (56%); however, in the uveal melanoma group the presence of dark blond hair was significantly elevated (46%, P = 0.030). These findings are in contrast with studies on CMM, where most patients have skin type II and red/fair hair., Conclusions: These data suggest that MC1R variants do not play a role in the susceptibility to develop uveal melanoma. Furthermore, most uveal melanoma patients share phenotypic characteristics that differ from findings in CMM patients.

Aims: To investigate whether corneal autofluorescence is different in patients with choroidal melanoma or choroidal naevus., Methods: Corneal autofluorescence was determined by fluorophotometry in both eyes of 32 patients with a unilateral choroidal melanoma, 32 patients with a unilateral choroidal naevus, and 32 age matched healthy controls. The corneal autofluorescence ratio between affected and contralateral eyes of patients or between randomly selected eyes of healthy controls was calculated., Results: Mean corneal autofluorescence ratio of patients with a choroidal melanoma was significantly higher than that of healthy controls (mean ratio: 1.09 (SD 0.15) and 1.00 (0.09), respectively, ANOVA p=0.014), and than that of patients with choroidal naevus (mean ratio 0.96 (0.09), p<0.001). Mean ratios of patients with choroidal naevus and healthy controls were not significantly different (p=0.27)., Conclusions: Corneal autofluorescence ratio of patients with a unilateral choroidal melanoma is increased. This is probably due to an increased flow of glucose through the impaired blood-aqueous barrier in the affected eye, resulting in additional glycation of corneal proteins and hence in increased autofluorescence. The corneal autofluorescence is not increased in patients with a choroidal naevus, because the blood-aqueous barrier is not impaired in the affected eye in these patients. Measurement of corneal autofluorescence is simple, fast, and non-invasive, and might be helpful to distinguish between patients with choroidal melanoma and those with choroidal naevus.

Purpose: To investigate the prognostic significance of the expression of epidermal growth factor receptor (EGFR) in uveal melanoma. EGFR is a transmembrane glycoprotein, and its expression has been correlated with the development of metastases in various malignancies., Methods: Frozen sections from 22 primary uveal melanomas were examined for EGFR expression by a three-step immunoperoxidase staining, using a mouse anti-human EGFR IgG2b monoclonal antibody. The results were compared with patient survival and clinical and histopathologic parameters., Results: EGFR expression could not be determined on one tumor due to excessive pigmentation. Two patients died of causes unrelated to melanoma, and two patients were lost to follow-up. Out of 21 tumors, six tumors showed immunoreactivity for EGFR. Five of these six patients (83%) died due to metastases, compared with 2 (17%) of 12 patients with no EGFR expression (Kaplan-Meier analysis P = 0.0004). EGFR-positive tumors tended to have a greater tumor prominence and a higher mitotic rate., Conclusions: The expression of EGFR was significantly correlated with death due to metastatic disease and therefore can be regarded as an important prognostic factor in human uveal melanoma.

Transpupillary thermotherapy (TTT) is a new treatment modality for uveal melanoma. We studied whether application of TTT influences the immunogenicity of the tumour cells in vivo or the expression of molecules related to apoptosis. Immunohistochemistry using monoclonal antibodies directed against HLA molecules, HMB45, P53, Fas ligand (FasL), Fas, Bcl-2 and tumour-infiltrating cells was applied to sections of an enucleated eye containing a uveal melanoma that received TTT 1 week before enucleation. The innermost part of the tumour which had been exposed directly to the laser treatment showed no staining for HLA antigens, nor for Fas or FasL epitopes. The intermediate part of the tumour showed a wet necrosis and HLA expression similar to the expression in the peripheral tumour. A large number of macrophages were observed in the necrotic as well as the intact tumour tissue, especially bordering the wet necrotic area. FasL and Bcl-2 were only expressed in the viable, outer part of the tumour. This immunological evaluation of one case of uveal melanoma treated with TTT revealed that TTT may not only have a direct destructive effect on the primary tumour, but may also influence the immunogenicity of uveal melanoma cells, induce infiltration of macrophages into the tumour, and induce apoptosis. The presence of many macrophages suggests that they play a role in the removal of the TTT-treated tumour tissue by phagocytosis.

Objective: To evaluate the efficacy and safety of transpupillary thermotherapy in treating choroidal melanoma., Methods and Patients: To perform transpupillary thermotherapy, infrared diode laser energy at 810 nm was used with a beam diameter of about 3 mm and 1-minute exposure time. All 50 patients had choroidal melanoma. We performed transpupillary thermotherapy in 21 tumors that had responded insufficiently to 800 Gy ruthenium 106 brachytherapy; it was combined with 800 Gy106Ru brachytherapy for 10 tumors greater than 5 mm in height and with 600 Gy for 19 tumors 5 mm or less in height., Results: All but 1 tumor exhibited reduction in tumor height within a mean follow-up of 20.5 months (range, 6-49 months). In 41 eyes (82%), the tumor flattened completely. Visual acuity was 20/60 or better in 43 eyes (86%) before treatment and in 14 eyes (28%) at the last examination because of radiation vasculopathy. Neovascular glaucoma developed in 1 eye, and total retinal detachment developed in 2 eyes. Tumor recurrence was observed in 1 patient., Conclusions: Although long-term results are necessary to properly appraise this new therapy, transpupillary thermotherapy may be useful as a complementary modality to brachytherapy.

We studied the effect of transpupillary thermotherapy (TTT) by a diode laser at 810 nm combined with episcleral ruthenium-106 plaque treatment (106Ru) on lens transparency in patients with choroidal melanoma. Lens transmission of blue-green light was measured by fluorophotometry in 17 patients treated with 106Ru treatment and TTT (measured 0.36 years after treatment), 12 patients treated with 106Ru alone (measured 19 years after treatment) and 25 age-matched healthy controls. Differences in lens transmission were not significant between treated and untreated fellow eyes (p > 0.15) nor between patient and control eyes (p > 0.25). TTT of choroidal melanoma combined with 106Ru plaque irradiation did not have a significant effect on the lens transparency up to 6 years after treatment.

Background: The level of HLA expression on a tumour may influence the immunological response against this tumour, and vice versa. HLA expression was determined in a primary uveal melanoma, its metastases, and on a cell line derived from this melanoma, and the presence and type of infiltrate in tissue sections were also studied., Methods: Immunohistochemistry with monoclonal antibodies (MAbs) against HLA class I and II, T cells, NK cells, and macrophages., Results: Primary and metastatic lesions, as well as the cell line showed high levels of expression of the monomorphic determinants of HLA class I. Expression of the polymorphic HLA-A2 and HLA-A3 antigens was decreased on metastases to the skin and liver. HLA-Bw4 expression was low on all lesions, as well as expression of HLA class II. Tumour infiltrating cells consisted mainly of CD3, CD4, and CD8 positive cells. Expression on the cell line corresponded to expression on the primary tumour., Conclusion: The primary uveal melanoma as well as the cell line showed a high expression of monomorphic and polymorphic HLA-A antigens, while metastases showed a high expression of monomorphic and a lower expression of polymorphic antigens. This variation in expression may support tumour cell escape from NK cells as well as CTL mediated lysis.

Over 100 new cases of uveal melanoma are diagnosed annually in the Netherlands. In the absence of metastases, eye-preserving treatment is possible in principle. Since 1984, Dutch patients are referred for eye-preserving treatment to Leiden University Hospital which consequently has gained great experience. Brachytherapy (with Ruthenium-106 applicators) is only suitable for uveal melanomas with a diameter < or = 15 mm and a prominence (thickness perpendicular to the sclera) < or = 5 mm; after 10 years, the eye was preserved in 80% of the patients; 75% retained a visual acuity > or = 0.25. Proton irradiation is being applied for large, strongly prominent melanomas in patients for whom enucleation is not acceptable, for instance with a melanoma in the only eye still functioning. The eyes could be preserved in 83.3% over a period of 5 years. Transpupillary thermotherapy combined with episcleral Ruthenium-106 application is called sandwich therapy; in a study involving 50 patients, 49 melanomas (98%) began to regress within 4 weeks and after a follow-up period of 12 months all tumours with a prominence < 5 mm had regressed completely.