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Background and Aim of the Study: Mutations in the Gα-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms., Methods: We analyzed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation., Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1-associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High-risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression., Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Toxic tumour syndrome (TTS) is a particularly aggressive form of secondary vasculopathy occurring after radiation therapy of uveal melanoma due to the persistence of the necrotic tumour mass inside the eye. The development of TTS confers a particularly unfavourable functional and anatomical ocular prognosis, ultimately requiring enucleation in most cases if untreated. Vitreoretinal (VR) surgery has been successfully applied for treatment and prevention of TTS using both resecting and non-resecting techniques. In this systematic review, we aim to define characteristics of uveal melanomas benefiting the most from secondary VR surgery and to outline the optimal type and timing of VR intervention in such cases. Analysis of the literature reveals that endoresection should be performed within 3 months after radiotherapy to tumours thicker than 7 mm and with a largest basal diameter between 8 mm and 15 mm with post-equatorial location, especially after proton beam treatment. Alternatively, endodrainage remains a valid therapeutic option in eyes with macula-off retinal detachment, tumour diameter larger than 15 mm or ciliary body involvement. VR surgery can be successful in the management of TTS following radiotherapy for uveal melanoma when timing and indication are appropriately evaluated.

Background: Prognostic parameters in sentinel node (SN)-positive melanoma are important indicators to identify patients at high risk of recurrence who should be candidates for adjuvant therapy. We aimed to evaluate the presence of melanoma cells beyond the SN capsule-extranodal extension (ENE)-as a prognostic factor in patients with positive SNs., Methods: Data from 1,047 patients with melanoma and positive SNs treated from 2001 to 2020 at the Istituto Nazionale dei Tumori in Milano, Italy, were retrospectively investigated. Kaplan-Meier survival and crude cumulative incidence of recurrence curves were estimated. A multivariable logistic model was used to investigate the association between ENE and selected predictive factors. Cox models estimated the effect of the selected predictors on survival endpoints., Results: Median follow-up was 69 months. The 5-year overall survival rate was 62.5% and 71.7% for patients with positive SNs with and without ENE, respectively. The 5-year disease-free survival rate was 54.0% and 64.0% for patients with positive SNs with and without ENE, respectively. The multivariable logistic model showed that age, size of the main metastatic focus in the SN, and numbers of metastatic non-SNs were associated with ENE (all P<.0001). The multivariable Cox regression models showed the estimated prognostic effects of ENE associated with age, ulceration, size of the main metastatic focus in the SN, and number of metastatic non-SNs (all P<.0001) on disease-free survival and overall survival., Conclusions: ENE was a significant prognostic factor in patients with positive-SN melanoma. This parameter may be useful in clinical practice as a selection criterion for adjuvant treatment in patients with stage IIIA disease with a tumor burden <1 mm in the SN. We recommend its inclusion as an independent prognostic determinant in future updates of melanoma guidelines.

Relatively little is known about the genetic aberrations of conjunctival melanomas (CoM) and their correlation with clinical and histomorphological features as well as prognosis. The aim of this large collaborative multicenter study was to determine potential key biomarkers for metastatic risk and any druggable targets for high metastatic risk CoM. Using Affymetrix single nucleotide polymorphism genotyping arrays on 59 CoM, we detected frequent amplifications on chromosome (chr) 6p and deletions on 7q, and characterized mutation-specific copy number alterations. Deletions on chr 10q11.21-26.2, a region harboring the tumor suppressor genes, PDCD4, SUFU, NEURL1, PTEN, RASSF4, DMBT1, and C10orf90 and C10orf99, significantly correlated with metastasis (Fisher's exact, p ≤ 0.04), lymphatic invasion (Fisher's exact, p ≤ 0.02), increasing tumor thickness (Mann-Whitney, p ≤ 0.02), and BRAF mutation (Fisher's exact, p ≤ 0.05). This enhanced insight into CoM biology is a step toward identifying patients at risk of metastasis and potential therapeutic targets for systemic disease., (© 2019 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons Ltd.)

Uveal melanoma (UM) exhibits recurring chromosomal abnormalities and gene driver mutations, which are related to tumor evolution/progression. Almost half of the patients with UM develop distant metastases, predominantly to the liver, and so far there are no effective adjuvant therapies. An accurate UM genetic profile could assess the individual patient's metastatic risk, and provide the basis to determine an individualized targeted therapeutic strategy for each UM patient. To investigate the presence of specific chromosomal and gene alterations, BAP1 protein expression, and their relationship with distant progression free survival (DPFS), we analyzed tumor samples from 63 UM patients (40 men and 23 women, with a median age of 64 years), who underwent eye enucleation by a single cancer ophthalmologist from December 2005 to June 2016. UM samples were screened for the presence of losses/gains in chromosomes 1p, 3, 6p, and 8q, and for mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX. BAP1 protein expression was detected by immunohistochemistry (IHC). Multivariate analysis showed that the presence of monosomy 3, 8q gain, and loss of BAP1 protein were significantly associated to DPFS, while BAP1 gene mutation was not, mainly due to the presence of metastatic UM cases with negative BAP1 IHC and no BAP1 mutation detected by Sanger sequencing. Loss of BAP1 protein expression and monosomy 3 represent the strongest predictors of metastases, and may have important implications for implementation of patient surveillance, properly designed clinical trials enrollment, and adjuvant therapy., (© 2018 Wiley Periodicals, Inc.)

Purpose: To report on the clinical characteristics and outcomes for patients with iris melanoma using proton therapy., Design: Retrospective study., Participants: One hundred seven patients with iris melanoma from 3 regional ophthalmologic centers., Methods: A retrospective study was conducted for iris melanoma patients from 3 regional ophthalmologic centers referred to and treated at a single proton therapy facility between 1996 and 2015., Main Outcome Measures: At each follow-up visit, examinations included measurement of best-corrected VA, slit-lamp, examination, indirect ophthalmoscopy, and ultrasound biomicroscopy., Results: With a median follow-up of 49.5 months, 5 of 107 patients experienced a local relapse within a median of 36.3 months. The cumulative incidence of relapse was 7.5% at 5 years. All 5 patients showed involvement of the iridocorneal angle (P = 0.056). Diffuse iris melanoma showed a higher risk of relapse (P = 0.044). Four patients showed out-of-field relapse and 1 showed angular relapse. Three patients were retreated with proton therapy, whereas 2 other patients, one with T1b disease and another with diffuse T3 disease, underwent secondary enucleation. None of the patients experienced metastases nor died of iris melanoma. Vision improved in 59.4% of patients (n = 60/101). However, cataracts occurred in 57.4% of the 54 patients (n = 31) without cataract or implant at diagnosis. Secondary glaucoma was reported in 7.6% of the patients (n = 8), uveitis in 4.7% (n = 5), and hyphema in 3.7% (n = 4). All but 5 cases of complications were mild, transient, and not sight limiting after treatment. Five cases of glaucoma, including 1 with uveitis, were severe and associated with visual deterioration., Conclusions: Proton therapy showed efficacy and limited morbidity in iris melanomas., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Importance: Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases., Objective: To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM., Design, Setting, and Participants: In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015., Main Outcomes and Measures: Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival., Results: We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival., Conclusions and Relevance: Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.

Purpose: In parapapillary melanoma patients, radiation-induced optic complications are frequent and visual acuity is often compromised. We investigated dose-effect relationships for the optic nerve with respect to visual acuity after proton therapy., Methods and Materials: Of 5205 patients treated between 1991 and 2014, those treated using computed tomography (CT)-based planning to 52 Gy (prescribed dose, not accounting for relative biologic effectiveness correction of 1.1) in 4 fractions, with minimal 6-month follow-up and documented initial and last visual acuity, were included. Deterioration of ≥0.3 logMAR between initial and last visual acuity results was reported., Results: A total of 865 consecutive patients were included. Median follow-up was 69 months, mean age was 61.7 years, tumor abutted the papilla in 35.1% of patients, and tumor-to-fovea distance was ≤3 mm in 74.2% of patients. Five-year relapse-free survival rate was 92.7%. Visual acuity was ≥20/200 in 72.6% of patients initially and 47.2% at last follow-up. A wedge filter was used in 47.8% of the patients, with a positive impact on vision and no impact on relapse. Glaucoma, radiation-induced optic neuropathy, maculopathy were reported in 17.9%, 47.5%, and 33.6% of patients, respectively. On multivariate analysis, age, diabetes, thickness, initial visual acuity and percentage of macula receiving 26 Gy were predictive of visual acuity. Furthermore, patients irradiated to ≥80% of their papilla had better visual acuity when limiting the 50% (30-Gy) and 20% (12-Gy) isodoses to ≤2 mm and 6 mm of optic nerve length, respectively., Conclusions: A personalized proton therapy plan with optic nerve and macular sparing can be used efficiently with good oncological and functional results in parapapillary melanoma patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To describe the dynamics of thickness and internal reflectivity after proton beam therapy (PBT) in uveal melanoma., Participants: One hundred and ninety-eight consecutive patients with choroidal or ciliary body melanoma treated by PBT were retrospectively considered., Methods: The post-PBT follow-up included ophthalmologic examination, retinography, and B and A modes of standardized echography every 6 months. A total of 1393 examinations were performed. We take into account 4 tumour categories according to the seventh TNM classification., Results: Before PBT, tumour thickness ranged from 1.5 to 12.5 mm with a mean of 5.9 mm. Its decrease after radiotherapy was best fitted by the sum of a first-order exponential decay and a constant with a decay half-life of 15 months. Based on the fit, tumour thickness stabilized on a constant value representing, on average, 47% of the initial value. Mean internal reflectivity before PBT was 68%. The dynamics of the reflectivity were best fitted by an exponential and a constant, with rise half-life of 11 months, and stability value of 87%., Conclusions: We found that ultrasonographic dynamics of uveal melanoma treated by PBT resembles a function composed of the sum of a constant and a first-order exponential, as previously noted in studies on brachytherapy. Interestingly, after PBT, because of its shorter half-life, internal reflectivity has a faster dynamic response than thickness in large tumours, suggesting that increase of internal reflectivity is a more sensitive indicator of early response to therapy in larger tumours., (© 2013 Canadian Ophthalmological Society Published by Canadian Ophthalmological Society All rights reserved.)

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported. Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab. Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α. EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate survival and clinical outcome for patients with a large uveal melanoma treated by either enucleation or proton beam radiotherapy (PBRT)., Procedures: This retrospective non-randomized study evaluated 132 consecutive patients with T3 and T4 choroidal melanoma classified according to TNM stage grouping., Results: Cumulative all-cause mortality, melanoma-related mortality and metastasis-free survival were not statistically different between the two groups (log-rank test, p = 0.56, p = 0.99 and p = 0.25, respectively). Eye retention of the tumours treated with PBRT at 5 years was 74% (SD 6.2%). In these patients at diagnosis, 73% of eyes had a best-corrected visual acuity (BCVA) of 0.1 or better. After 12 and 60 months, BCVA of 0.1 or better was observed in 47.5 and 32%, respectively., Conclusion and Message: Although enucleation is the most common primary treatment for large uveal melanomas, PBRT is an eye-preserving option that may be considered for some patients., (Copyright © 2012 S. Karger AG, Basel.)

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.