Review: Characteristics and Outcomes of Patients with Early or Late Uveal Melanoma Metastasis

Purpose: The purpose of this study was to determine whether the metastatic rates in patients with gene expression profile (GEP) class 1A versus 1B posterior uveal malignant melanoma supported or contradicted predictions of very low metastatic rate in GEP 1A cases and moderate rate in GEP 1B cases. Patients/Methods: 164 patients with a cytopathologically confirmed primary posterior uveal malignant melanoma classified by GEP testing as class 1 (100 GEP 1A, 64 GEP 1B) were evaluated. Kaplan-Meier rates of metastasis were computed and plotted for the GEP class 1 subgroups. Median follow-up of patients who were still alive without metastasis on the date of data analysis was 100.5 months for the GEP 1A patients and 97.2 months for the GEP 1B patients. Results: The actuarial 5-year rate of uveal melanoma metastasis was 10.8% (std. error = 3.2%) in the GEP 1A patients versus 0% in the GEP 1B patients, and the actuarial 10-year rate of metastasis was 12.2% (std. error = 3.5%) in the GEP 1A patients versus 2.1% (std. error 2.1%) in the GEP 1B patients. Conclusion: The results of this retrospective single-center study cast doubt on the validity of the prognostic stratification of GEP class 1 posterior uveal malignant melanomas into very low risk (GEP 1A) versus intermediate risk (GEP 1B) of metastasis subgroups provided by the commercially available GEP test.

Background Osteopontin is highly expressed in many metastatic tumors,but its expression in uveal melanoma and the relationship of osteopontin with metastasis is still under investigation. Objective This study was to investigate the relationship between osteopontin expression and uveal melanoma metastatic potential.Methods Fifty samples of uveal melanoma were collected in Beijing Tongren Hospital.The expressions of osteopontin in the samples were detected using immunohistochemistry,and expressions of osteopontin mRNA in different metastatic potential uveal melanoma cell lines (MU M-2B,C918,OCM-1A) were analyzed using quantitative polymerase chain reaction (Q-PCR). Results Osteopontin was positively expressed in 10 samples of 13 patients with metastasis and 14 samples of 37 patients without metastasis,showing a significant difference between them (x2=5.888,P=0.000).There were 11 samples and 13 samples exhibiting the positive expression of osteopontin in 15 epithelial type and 35 non-epithelial type of uveal melanoma respectively with the significant difference (x2=5.510,P =0.000).The positive expressing numbers of osteopontin were 20 tumors of 30 affected tumors and 4 tumors of 20unaffected ciliary tumors with the significant difference (x2 =10.470,P =0.000 ).No significant differences in the expression of osteopontige were found between different gender,age,eyes,largest basal diameters and invasion of scleral canal( P =0.536,0.256,0.802,0.848,0.555 ).The relative expression value of osteopontin mRNA was in turn 1.00±0.04,0.91 ±0.03,0.08±0.01 in MUM-2B,C918,OCM-1A with the significant difference among three cell lines( F=33.135,P＜0.05).The osteopontige mRNA expression was significant enhanced in high metastatic potentialcell lines( MUM-2B,C918 ) compared with low metastatic potential cell lines( OCM-1 A) (P＜0.05 ),but no significant difference was seen between two high metastatic potential cell lines(MUM-2B,C918) (P=0.804). Conclusions Osteopontige expression is associated with the uveal melanoma cell line invasion and metastasis potential.These results imply that osteopontige expression maybe an indicator of uveal melanoma invasion and metastasis ability. Key words: Uveal melanoma; Osteopontin; Invasion and metastasis

Uveal melanoma is the most common primary intraocular malignancy, and its metastases are deadly. Significant work has been done to elucidate the molecular framework that causes uveal melanoma development and metastasis. This review is intended to highlight the most recent breakthroughs in the molecular understanding of uveal melanoma. Monosomy of chromosome 3 and class 2 gene-expression profile are well-known indicators of melanoma metastasis. However, some patients with disomy 3 and class 1 gene expression profiling (GEP) still develop metastasis. Disomy 3 tumors may be further classified based upon the presence of an SF3B1 mutation. The role of SF3B1 gene is unclear at this time but may be related to the development of late metastases among disomy 3 uveal melanoma. Class 1 GEP tumors have recently been subdivided into class 1a and class 1b, with class 1b tumors carrying a slightly higher risk of metastasis. Among patients with either class 1 or class 2 GEP, the expression of preferentially expressed antigen in melanoma (PRAME) is an independent risk factor for the development of metastasis. Mutation of GNAQ is the most commonly observed mutation in uveal melanoma, regardless of chromosome 3 status or GEP class. Inhibitors or GNAQ may be targets for therapeutic intervention in uveal melanoma. MicroRNA molecules are small noncoding RNA molecules that have been recently demonstrated to function in RNA silencing and posttranscriptional regulation of gene expression. These molecules may play a role in the development of uveal melanoma metastasis. New findings such as the presence or absence of PRAME, mutations in the SF3B1 gene and microRNA dysregulation have added new layers to our understanding of uveal melanoma. These new concepts will enhance our ability to prognosticate tumor metastasis and may provide targets for therapeutic intervention.

ObjectiveAssess current clinical practices for uveal melanoma (UM) and the impact of molecular prognostic testing on treatment decisions.DesignCross-sectional survey and sequential medical records review.ParticipantsOphthalmologists who treat UM.Methods(A) Medical records review of all Medicare beneficiaries tested by UM gene expression profile in 2012, conducted under an institutional review board-approved protocol. (B) 109 ophthalmologists specializing in the treatment of UM were invited to participate in 24-question survey in 2012; 72 were invited to participate in a 23-question survey in 2014.Main outcome measuresResponses analyzed by descriptive statistics, frequency analyses (percentages, Tukey, histograms), and Fisher’s exact test. Descriptive presentation of essay answers.ResultsThe review of Medicare medical records included 191 evaluable patients, 88 (46%) with documented medical treatment actions or institutional policies related to surveillance plans. Of these 88, all gene expression profiling (GEP) Class 1 UM patients were treated with low-intensity surveillance. All GEP Class 2 UM patients were treated with high-intensity surveillance (P<0.0001 versus Class 1). There were 36 (19%) with information concerning referrals after initial diagnosis. Of these 36, all 23 Class 2 patients were referred to medical oncology; however, none of the 13 Class 1 patients were referred (P<0.0001 versus Class 1). Only Class 2 patients were recommended for adjunctive treatment regimens. 2012 survey: 50 respondents with an annual median of 35 new UM patients. The majority of respondents (82%) performed molecular analysis of UM tumors after fine needle biopsy (FNAB); median: 15 FNAB per year; 2014 survey: 35 respondents with an annual median of 30 new UM patients. The majority offered molecular analyses of UM tumor samples to most patients. Patients with low metastatic risk (disomy 3 or GEP Class 1) were generally assigned to less frequent (every 6 or 12 months) and less intensive clinical visits. Patients with high metastatic risk (monosomy 3 or GEP Class 2) were assigned to more frequent surveillance with hepatic imaging and liver function testing every 3–6 months. High-risk patients were considered more suitable for adjuvant treatment protocols.ConclusionThe majority of ophthalmologists treating UM have adopted molecular diagnostic tests for the purpose of designing risk-appropriate treatment strategies.

Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas. Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells. Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization. Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq. Citation Format: Donghan Shin, Coulson P. Rich, Lehi Acosta, Jackson R. Richards, Jae Hyuk Yoo, Allie H. Grossmann, Zongzhong Tong, Alan L. Mueller, Weiquan Zhu, Dean Y. Li, Shannon J. Odelberg. ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5157.

AimTo determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following...

To investigate survival patterns of uveal melanomas involving the anterior chamber by tumor extension and primary tumor location. Retrospective cohort study. Patients diagnosed with uveal melanoma in Denmark between 1943 and 2022 were identified, and cases were reviewed. Patients with tumors involving the anterior chamber were included and categorized into 4 groups based on tumor anatomic extent and configuration: (1) isolated iris melanomas, (2) iris and ciliary body tumors, (3) ring melanomas, and (4) tumors involving iris, ciliary body, and choroid. Patient data were analyzed using survival analysis techniques. Cumulative incidences of metastatic death were calculated using competing risk methods. Cause-specific hazard ratios (HRs) were estimated using Cox proportional hazards regression. Restricted mean survival time and life-years lost were calculated at the 10-year and 20-year follow-ups. Primary outcome was mortality resulting from uveal melanoma metastasis. Secondary measures included cumulative incidences, cause-specific HR, restricted mean survival time, and life-years lost at 10 and 20 years. Among 3859 eligible patients, 789 patients had tumors with anterior chamber involvement, of which 786 patients were included in the analysis. The 20-year cumulative incidence of metastatic death was 0.7% for isolated iris melanomas, 10% for tumors involving the iris and ciliary body, 32% for ring melanomas, and 68% for tumors involving all 3 uveal structures. Compared with tumors involving the iris and ciliary body, the HR was 0.05 (confidence interval [CI] 0.01-0.4) for isolated iris melanomas, 5.0 (CI, 2.5-10.3) for ring melanomas, and 15.3 (CI, 8.3-28.3) for tumors involving all uveal structures. Ring melanomas significantly worsened survival in iris melanomas (HR, 51.7; CI, 11.2- 239.3) but not in ciliary body melanomas. Among tumors involving both the iris and ciliary body, those primarily located in the ciliary body showed substantially higher mortality (HR, 19.1; CI, 2.4-149.5). Survival outcomes of uveal melanomas involving the anterior chamber varies by anatomic extension. Ring melanomas resemble ciliary body melanomas in survival outcomes and drive poor prognosis in iris melanomas. Excluding ring melanomas, tumors primarily located in the iris have very low metastatic potential, even with ciliary body involvement. Tumors involving all 3 uveal structures are associated with extremely poor outcomes. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

uveal melanoma (UM) is the most common primary intraocular tumor in adults, with metastasis being the leading cause of death. However, effective treatments for metastatic UM remain limited. Emerging evidence suggests that cholesterol metabolism plays a role in cancer progression, but its impact on UM metastasis is not well understood. we investigated the effects of miR-181a on UM metastasis using multiple UM cell lines and a suprachoroidal injection mouse model. Functional assays, including migration, invasion, and cancer stem-like cell (CSC) formation, were performed. The target of miR-181a was identified through bioinformatics, luciferase assays, and western blotting. Cholesterol levels were measured, and in vitro and in vivo studies assessed the therapeutic potential of combining miR-181a with crizotinib. miR-181a significantly decreases UM cell migration, invasion, and metastasis. Mechanistically, miR-181a was found to target sterol regulatory element-binding protein 2 (SREBP2), thereby inhibiting cholesterol biosynthesis. This decrease in cholesterol levels hindered reduced epithelial-to-mesenchymal transition (EMT) and led to a decline in cancer stem-like cell (CSC) populations in UM. Furthermore, elevated cholesterol or overexpression of SREBP2 abrogated the anti-metastatic effects of miR-181a. Additionally, a combination of miR-181a and crizotinib significantly inhibited metastasis, both in vitro and in vivo. miR-181a inhibits UM metastasis by targeting SREBP2 and reducing cholesterol biosynthesis. Its combination with crizotinib may provide a promising therapeutic strategy for metastatic UM.

Clinical Characteristics of UM and Association of Metastasis of Uveal Melanoma with Congenital Oculocutaneous Melanosis in Asian Patients: Analysis of 1151 Consecutive Eyes

Introduction: Approximately half of patients with uveal melanoma (UM), a primary eye cancer, will develop metastatic disease. Gene expression profiling (GEP) is clinically validated to stratify risk of metastasis by assigning UM patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy which are limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding or tumor dissemination. Thus, liquid biopsy has emerged as a significantly less invasive alternative. Blood biopsy has largely been unsuccessful for clinical use in UM due to low detection rates in the setting disease limited to the eye, however eye-specific aqueous humor (AH) liquid biopsy may be a better alternative. In this study, we seek to determine the AH proteome related to the advanced GEP class 2 using the diagnostic AH specimens. Method: Twenty UM treatment naive AH were collected before plaque brachytherapy. Patients were sub-grouped by GEP classes into GEP 1 (n=12) and GEP 2 (n=5). Three patients were classified as GEP unknown (GEP NA, n=3) due to the unavailability of tumor biopsy. Ten microliters of AH samples were analyzed by proximity extension assay-derived multiplexed Olink platform. Protein expression levels of 1472 targets were analyzed, compared between GEP classes and correlated with clinical features. Significant differentially expressed proteins (DEPs, fold-change (FC) &amp;gt; 2 or FC &amp;lt; 0.5, P &amp;lt; 0.01) were subjected to Qiagen ingenuity pathway analysis for cellular pathway and upstream regulator identification. Results: GEP 2 class was correlated with AJCC stages (P = 0.012), advanced clinical tumor stages (P = 0.007) and mutated BAP1 (P = 0.018). 45 DEPs were identified when comparing GEP classes. Among them, 31 are up-regulated DEPs [fold-change (FC) &amp;gt;2, P &amp;lt; 0.01] and 14 are down-regulated DEPs (FC &amp;lt; 0.5, P &amp;lt; 0.01) in GEP 2 compared to GEP1. The unsupervised clustering analysis showed that the 45 DEPs well-differentiate AH samples by GEP classes, and the 3 GEP NA samples were clustered with GEP1 class. Pathway analysis showed that 45 DEPs contribute to metastatic-related pathways including cellular movement, cellular proliferation and epithelial to mesenchymal transition. Two upstream regulators, IL1 receptor and SPRY2, were predicted to regulate 8 out of the 45 DEPs. IL1R2 (FC = 3.4, P = 0.021) and SPRY2 (FC = 0.6, P = 0.052) protein expression levels were found matched as prediction in our Olink dataset. Conclusions: 45 AH DEPs could differentiate GEP class 1 and 2 at the diagnostic stage and could be detected even when the tumor was too small to biopsy. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy. Citation Format: Chen-Ching Peng, Liya Xu, Jesse Berry. Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5553.

Independent Prognostic Significance of Gene Expression Profile Class and Largest Basal Diameter of Posterior Uveal Melanomas

Purpose: Uveal melanomas cluster into two molecular groups based on their gene expression profile. Tumors with the class 1 signature rarely metastasize, whereas those with the class 2 signature have a very high rate of metastasis. However, the biological basis for this metastatic propensity of class 2 tumors remains unclear. Towards such an explanation, this study was conducted to determine whether class 2 tumors have a higher proliferative rate than class 1 tumors.Materials and Methods: The study included 28 primary uveal melanomas with extensive clinical, pathologic, and genetic annotation, including age, gender, ciliary body involvement, tumor basal diameter, thickness, cell type, gene expression profile, status of chromosomes 3 and 8p, aneuploidy, and clinical outcome. Immunopositivity for Ki-67 was determined by counting all positive nuclei in representative whole tumor sections.Results: Ki-67 positivity was significantly associated with class 2 gene expression profile, loss of chromosome 3 and increased aneuploidy (P = 0.04, P = 0.004, and P = 0.03, respectively). Ki-67 positivity showed a borderline significant association with epithelioid cell type (P = 0.07). Receiver operating characteristic (ROC) analysis of Ki-67 positivity, using the class 2 signature as an endpoint, identified a Ki-67 score of approximately 20 cells per high power field as the optimal cut-off point between low and high risk for metastasis (log rank test, P = 0.01).Conclusions: On average, class 2 uveal melanomas have a higher proliferative rate than class 1 tumors. Further work is needed to determine whether loss of chromosome 3, increased aneuploidy, or other factors may be responsible for the increased proliferation.

Uveal melanoma (UM) is the most frequent primary malignant intraocular tumor in adults with an estimated incidence of 4-5 per million per year in western countries [1]. About 50% of UM patients eventually develop metastasis. In a previous study, the prognosis of Chinese UM was mainly correlated with visual clinical features and gene sequencing results. Models designed to predict UM prognosis have been previously described [2], but these studies were based on Caucasians, not Chinese. Further, our previous study [3] determined that the clinical characteristics were different between Chinese and Caucasian UM patients. Therefore, in this study, we analyzed the clinical characteristics and survival status of 1553 patients diagnosed with UM over a period of 15 years in China, and also included factors that were not included in our previous study, such as the largest basal diameter, thickness, tumor size after updating the criteria, pigmentation, whether complicated with intraocular hemorrhage, ciliary body involvement, extraocular extension and TNM stage. We also constructed comprehensive prognostic models to predict the risk of metastasis within 2 years and death of UM after 2 years following treatment according to clinical characteristics using machine learning. The methods of this study are described in the Supplementary File. The clinical data of the UM patients are shown in Supplementary Table S1. The mean age of subjects was 47.2 ± 12.5 years (median, 48.0 years; range, 5-85 years), indicating that the onset age is much younger than Caucasians [4] for whom it usually occurs in the fifth to sixth decades. This demonstrates one of the racial variations of the disease. Nearly 70% of patients' visual acuity was worse than the 0.30 logarithm of the minimum angle of resolution (LogMAR). Intraocular pressure was within the normal range of 10-21 mmHg in 1279 (85.6%) patients, while it was lower than 10 mmHg in 148 (9.9%) patients and higher than 21 mmHg in 67 (4.5%) patients. The mean largest basal diameter was 12.0 ± 3.6 mm (median, 11.7 mm; range, 1.1-23.0 mm), and the mean tumor thickness was 7.1 ± 3.3 mm (median, 6.9 mm; range, 0.4-23.0 mm). According to the criteria of the American Joint Committee on Cancer classification (7th edition), there were 198 (12.9%) patients at Stage I, 1089 (71.1%) at Stage II, and 243 (15.8%) at Stage III. It was reported that the mixed cell-type tumor is the most common UM [5], but in the present study, the spindle cell-type UM was the most common pathological type (44.6%). The mean follow-up time was 49.5 ± 32.5 months (median, 43 months; range, 0-197 months). We followed 78.4% of patients for more than 2 years, 32.5% for more than 5 years and 3.2% for more than 10 years. The estimated 5-, 10-, and 15-year overall survival rates after treatment initiation were 84.0%, 72.0%, and 66.7% (Figure 1A). Shields et al [4] published a cohort study of 8100 UM patients, demonstrating UM-related survival rates of 91.4%, 86.5% and 83.8% and metastasis rates of 15%, 25% and 31%. Our 5-, 10- and 15-year UM-related survival rates were 85.6%, 75.5% and 69.5%, and were thus lower than Shields et al results [4]. However, the metastasis rates in our study were higher at 18.9%, 27.2% and 31.4% (Figure 1B). Similarly, our UM-related survival rates were lower than those reported in Scotland (92.3%, 87.4% and 83.8%) [6]. A total of 183 patients in our study died of UM metastasis, and the median duration from metastasis to death was 7 months. 75.5% of these patients died within 1 year of metastasis. A previous study showed that 80% of UM patients died within 1 year of metastasis and 92% within 2 years [7]. The most common site of metastasis observed was the liver (Supplementary Table S2). Multiple-site metastasis occurred in 70 patients. Survival analysis and predicting models of 1553 UM patients. A. Kaplan-Meier curve of overall survival. B. Kaplan-Meier curve of metastasis. C. ROC curve of model for predicting death after 2 years of treatment (UMDeath model). D. ROC curve of model for predicting metastasis within 2 years of treatment (UMMetastasis model). Abbreviations: UM, Uveal Melanoma; AUC, Area Under Curve; ROC, Receiver Operating Characteristic We assessed the variables with significant differences in univariate analysis for Cox multivariate regression analysis (Supplementary Table S3). There were few patients with known pathological types (n = 386), including spindle cell-type (n = 172), mixed cell-type (n = 131), and epithelioid cell-type (n = 83). Thus, pathological information was not included in the multivariate analysis. Using the log-rank test, we found that the prognosis of patients with epithelioid cell-type UM was significantly worse than spindle cell-type UM (Bonferroni corrected P < 0.001); which was consistent with another study [8]. Cox multivariate regression analysis of some other possible prognostic factors showed that older age, larger basal diameter and presence of subretinal fluid were associated with high mortality and metastasis, while hemisphere tumor was associated with better prognosis (Supplementary Figures S1 and S2). In addition, tumor pigmentation and position were only related to death (Supplementary Figure S1). We used random forest [9, 10] to construct two models (UMDeath and UMMetastasis): whether a patient will survive for more than 2 years after treatment and whether the tumor will metastasize within 2 years of treatment using demographic attributes, general ocular features and tumor-specific features, which are listed in Supplementary Table S3. The imbalanced ratio (the ratio between the numbers of majority samples and minority samples) was different when we chose a different censored time, and the ratio was abnormal when the censored time was 3 years. Thus, we chose to predict the prognosis of patients within 2 years. Moreover, four-fold cross-validation was used to fairly assess the performance of random forest, where the training dataset and the testing dataset are subject-independent (each sample was collected from one patient and all samples were split into training and testing datasets in cross-validation, therefore, one sample were used only once in the training or testing dataset). Also, 0.5 was selected as the threshold for both classification problems, on which all statistical results were based. Finally, we applied genetic feature selection to investigate which features were more related to the two classification problems (whether a patient would die after 2 years or have metastasis within 2 years after treatment) (Supplementary Material). When the model for the prediction of death was established, we found that the largest basal diameter, thickness, size, intraocular pressure and initial treatment were related to death. The boxplots show the results of four-fold cross-validation before and after feature selection (Supplementary Figure S3A and S3B). The sensitivity, specificity, accuracy, area under the curve (AUC) and their 95% confidence intervals are shown in Supplementary Table S4. PRiMeUM [2], a model for predicting the risk of metastatic UM, used the clinical characteristics and chromosomal information to determine the risk of metastasis within 2 years after the primary treatment; and showed that the accuracy of risk prediction could reach 83% using only clinical characteristics. Moreover, we established a model for predicting metastasis within 2 years after treatment. It was found that the largest basal diameter, size, stage, age, intraocular pressure and gender were associated with metastasis. The boxplots show four-fold cross-validation results before and after feature selection (Supplementary Figure S4A and S4B). The sensitivity, specificity, accuracy, AUC and their 95% confidence intervals are shown in Supplementary Table S5. Figures 1C and 1D, Supplementary Figures S3C and S4C depict the receiver operating characteristic and precision-recall curves of one-fold out of four-fold cross-validation for predicting death and metastasis, respectively. Two predictive models each found that intraocular pressure was an influencing factor for UM metastasis and death, and intraocular pressure and prognosis were related to tumor volume. The larger the tumor, the greater the impact on the eyeball structure and physiological conditions, and thus is more likely to cause changes in intraocular pressure. This present study is currently the largest retrospective study on UM in China. The age of onset for Chinese UM patients was earlier and the prognosis was worse than Western patients. The largest basal diameter and age were found to be related to prognosis in both statistical results and predictive models. Our predictive models found that intraocular pressure was related to both death and metastasis. These models achieved a high sensitivity and specificity for predicting survival and can predict metastasis to a certain extent. The authors thank those who participated in the present study. Supported by The Capital Health Research and Development of Special (2020-1-2052), Science & Technology Project of Beijing Municipal Science & Technology Commission (Z201100005520045, Z181100001818003), The Beijing Municipal Administration of Hospitals' Ascent Plan (DFL20150201), The National Natural Science Foundation of China (82101180), Beijing Natural Science Foundation (7204245), Scientific Research Common Program of Beijing Municipal Commission of Education (KM202010025018), Beijing Municipal Administration of Hospitals' Youth Programme (QMS20190203), Beijing Dongcheng District Outstanding Talents Cultivating Plan(2018). No conflicting relationship exists for any author. This research adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Beijing Tongren Hospital. We obtained informed consent from all of these patients. YML, YL, and WBW contributed to the study conception. YNC, YNW and MXC designed the current study. HHZ, YNH, YF,YJL and JTL collected the data and implemented the follow-up. KZ, YNC and YNW performed the analyses and verified the underlying data. RTC, RF and HW arranged and checked the consistency of results. YNC and YNW wrote the initial draft of the manuscript. WBW and KZ commented on the manuscript and gave final approval of the version to be published. Written informed consent was obtained from the patients for participation in the study and publication of this article. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Correspondence and requests for materials and code should be addressed to WBW ([email protected]), YL ([email protected]) or YML ([email protected]). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

PurposeTo screen the microarray expression of CDH1, ECM1, EIF1B, FXR1, HTR2B, ID2, LMCD1, LTA4H, MTUS1, RAB31, ROBO1, and SATB1 genes which are predictive of primary uveal melanoma metastasis, and NFKB2, PTPN18, MTSS1, GADD45B, SNCG, HHIP, IL12B, CDK4, RPLP0, RPS17, RPS12 genes that are differentially expressed in metastatic uveal melanoma in normal whole human blood and tissues prone to metastatic involvement by uveal melanoma. MethodsWe screened the GeneNote and GNF BioGPS databases for microarray analysis of genes predictive of primary uveal melanoma metastasis and those differentially expressed in metastatic uveal melanoma in normal whole blood, liver, lung and skin. ResultsMicroarray analysis showed expression of all 22 genes in normal whole blood, liver, lung and skin, which are the most common sites of metastases. In the GNF BioGPS database, data for expression of the HHIP gene in normal whole blood and skin was not complete.ConclusionsMicroarray analysis of genes predicting systemic metastasis of uveal melanoma and genes differentially expressed in metastatic uveal melanoma may not be used as a biomarker for metastasis in whole blood, liver, lung, and skin. Their expression in tissues prone to metastasis may suggest that they play a role in tropism of uveal melanoma metastasis to these tissues.

Risk of metastasis is increased by the presence of chromosome 3 monosomy in uveal melanoma (UM). This study aimed to identify more accurate biomarker for risk of metastasis in UM. A total of 80 patients with UM from TCGA were assigned to two groups based on the metastatic status, and bioinformatic analyses were performed to search for critical genes for risk of metastasis. SLC25A38, located on chromosome 3, was the dominant downregulated gene in metastatic UM patients. Low expression of SLC25A38 was an independent predictive and prognostic factor in UM. The predictive potential of SLC25A38 expression was superior to that of pervious reported biomarkers in both TCGA cohort and GSE22138 cohort. Subsequently, its role in promoting metastasis was explored in vitro and in vivo. Knock-out of SLC25A38 could enhance the migration ability of UM cells, and promote distant metastasis in mice models. Through the inhibition of CBP/HIF-mediated pathway followed by the suppression of pro-angiogenic factors, SLC25A38 was situated upstream of metastasis-related pathways, especially angiogenesis. Low expression of SLC25A38 promotes angiogenesis and metastasis, and identifies increased metastatic risk and worse survival in UM patients. This finding may further improve the accuracy of prognostic prediction for UM.