Uveal Melanoma Research Articles

Cyclin-Dependent Kinase Inhibitors in the Rare Subtypes of Melanoma Therapy

Melanoma occurs in various forms and body areas, not only in the cutis, but also in mucous membranes and the uvea. Rarer subtypes of that cancer differ in genomic aberrations, which cause their minor sensibility to regular cutaneous melanoma therapies. Therefore, it is essential to discover new strategies for treating rare forms of melanoma. In recent years, interest in applying CDK inhibitors (CDKIs) in cancer therapy has grown, as they are able to arrest the cell cycle and inhibit cell proliferation. Current studies highlight selective CDK4/6 inhibitors, like palbociclib or abemaciclib, as a very promising therapeutic option, since they were accepted by the FDA for advanced breast cancer treatment. However, cells of every subtype of melanoma do not react to CDKIs the same way, which is partly because of the genetic differences between them. Herein, we discuss the past and current research relevant to targeting various CDKs in mucosal, uveal and acral melanomas. We also briefly describe the issue of amelanotic and desmoplastic types of melanoma and the need to do more research to discover cell cycle dysregulations, which cause the growth of the mentioned forms of cancer.

Revealing the structural microenvironment of high metastatic risk uveal melanomas following decellularisation

Uveal melanoma (UM) is a rare aggressive intraocular tumour that spreads most commonly to the liver in tumours with loss of one copy of chromosome 3 (HR-M3); current treatments for metastatic disease remain largely ineffective. Pre-clinical research is increasingly using three-dimensional models that better recapitulate the tumour microenvironment (TME). One aspect of the TME is the acellular extracellular matrix (ECM) that influences cell proliferation, migration and response to therapy. Although commercial matrices are used in culture, the composition and biochemical properties may not be representative of the tumour ECM in vivo. This study identifies UM metastatic risk specific ECM proteins by developing methodology for decellularisation of low- and high- metastatic risk tissue samples (LR-D3 vs. HR-M3). Proteomic analysis revealed a matrisome signature of 34 core ECM and ECM-associated proteins upregulated in HR-M3 UM. Combining additional UM secretome and whole cell iTRAQ proteomic datasets revealed enriched GO and KEGG pathways including ‘regulating ECM binding’ and ‘PI3K/Akt signalling’. Structural analyses of decellularised matrices revealed microarchitecture of differing fibre density and expression differences in collagen 4, collagen 6A1 and nidogen 1, between metastatic risk groups. This approach is a powerful tool for the generation of ECM matrices relevant to high metastatic risk UM.

SGSM2 in Uveal Melanoma: Implications for Survival, Immune Infiltration, and Drug Sensitivity.

The abnormal expression of small G protein signaling modulator 2 (SGSM2) is related to the occurrence of thyroid cancer and breast cancer. However, the role of SGSM2 in uveal melanoma (UVM) is unclear. To elucidate this ambiguity, our study utilized bioinformatics analysis and experimental validation. The expression of SGSM2 was detected in UVM cell lines through quantitative real-- time PCR (qRT-PCR). We utilized the Cancer Genome Atlas (TCGA) database to assess the relationship between SGSM2 expression and clinical characteristics, as well as its prognostic significance in UVM. Furthermore, the study examined potential regulatory networks involving SGSM2 in relation to immune infiltration, immune checkpoint genes, microsatellite instability (MSI), and drug sensitivity in UVM. The study also examined SGSM2 expression in UVM single-cell sequencing data. SGSM2 was highly expressed in UVM cell lines. Moreover, elevated levels of SGSM2 in UVM patients were significantly linked to poorer overall survival (OS) (p < 0.001), progress- free survival (PFS) (p < 0.001), and disease-specific survival (DSS) (p < 0.001). Additionally, SGSM2 expression was identified as an independent prognostic factor in UVM patients (p < 0.001). SGSM2 was associated with several pathways, including the calcium signaling pathway, natural killer cell-mediated cytotoxicity, cell adhesion molecules (CAMs), and others. The study revealed that SGSM2 expression in UVM is linked to immune infiltration, immune checkpoint genes, and MSI. Additionally, a significant inverse correlation was observed between SGSM2 expression and the compounds GSK690693, TL-2-105, PHA-793887, Tubastatin A, and SB52334 in UVM patients. SGSM2 may not only serve as an important indicator for prognostic assessment. Still, it may also be a key target for the development of new therapeutic approaches, providing new perspectives on the treatment of UVM patients.

Gadoxetic acid-enhanced MRI for the detection of liver metastases from melanoma.

We aimed to assess imaging findings and detection sensitivity for melanoma liver metastases on gadoxetic acid-enhanced magnetic resonance imaging (MRI). This retrospective study included patients with melanoma liver metastasis who underwent gadoxetic acid-enhanced MRI. Two abdominal radiologists independently evaluated signal characteristics of liver metastases on morphologic imaging (precontrast T1- and T2-weighted imaging), diffusion-weighted imaging (DWI), dynamic imaging, and hepatobiliary phase (HBP). Imaging findings were compared according to detection on HBP and the primary site of the melanoma using logistic regression with the generalized estimating equation (GEE). Detection sensitivity for metastases was compared among different MR imaging sets using logistic regression with GEE. A total of 67 patients with 254 liver metastases were included (44 women; mean age ± standard deviation, 65.6 ± 13.0 years). On HBP, 76.0% of metastases were detected, and 55.5% (141/254) showed hypointensity. Most of the metastases that were not detected on HBP originated from ocular melanomas (98.4%, 60/61), ≤1 cm (90.2%, 55/61) and showed T1 hyperintensity (98.4%, 60/61). Metastases from non-ocular melanomas more frequently showed T1 hypointensity, T2 hyperintensity, diffusion restriction, arterial enhancement, and HBP hypointensity than those from ocular melanomas (Ps ≤ 0.019). The detection sensitivity of HBP (76.0%) was significantly higher than DWI (65.7%, P = 0.006), but lower than morphologic imaging (98.8%, P < 0.001) and dynamic imaging (97.6%, P < 0.001). The detection sensitivity of HBP for melanoma liver metastasis was 76.0%, which was lower than that of morphologic or dynamic imaging. HBP of gadoxetic acid-enhanced MRI has little advantage in detecting melanoma liver metastases.

Treatment of Conjunctival Melanoma Cell Lines With a Light-Activated Virus-Like Drug Conjugate Induces Immunogenic Cell Death.

Conjunctival melanoma (CJM) is a rare malignant ocular surface tumor, which often leads to local recurrences and metastases. In murine models of subcutaneous tumors, treatment with a novel virus-like drug conjugate (VDC; Bel-sar) showed a dual mechanism of action with direct tumor cell killing as well as stimulation of an antitumoral immune response. Bel-sar is currently being evaluated for the treatment of primary uveal melanoma and indeterminate nevi in a phase III clinical trial. We determined whether Bel-sar also has direct antitumor efficiency and a potential immunostimulatory capacity in CJM cells. Three human tumor-derived CJM lines were used. Bel-sar's subcellular and intracellular locations were determined with tracers. Following light activation of Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were assessed. Treated tumor cells were co-cultured with THP-1 derived macrophages to assess tumor-cell phagocytosis. Bel-sar was bound and internalized by CJM cells and subsequently found in the cell membrane, lysosome, Golgi apparatus, and mitochondria. Bel-sar activation induced near complete cell death with half-maximal inhibitory concentration (IC50) values between 30 pM and 60 pM. Finally, light-activated Bel-sar enhanced exposure of DAMPs, including calreticulin, heat shock protein 90, and stimulated phagocytosis by macrophages. Treatment with a novel VDC (Bel-sar) induced pro-immunogenic cell death in all three CJM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs, suggesting Bel-sar is a potential treatment for CJM by a dual mechanism of action. This dual mechanism may provide a targeted and direct killing of tumor cells and induce an immune response which might decrease local recurrences and metastasis.

Injectable nanocomposite hydrogel with cascade drug release for treatment of uveal melanoma

Uveal melanoma (UM) is the most common malignant intraocular tumor with the trait of distant metastases. Currently, the standard clinical therapy results in suboptimal outcomes due to ineffective inhibition of tumor metastasis. Thus, developing novel therapeutic modalities for UM remains a critical priority. Herein, we have developed an injectable nanocomposite hydrogel (HA-DOX/LAP gel) through integrating hyaluronic acid-based drug-loaded nanoparticles into an alginate-dopamine gel, delivering the chemotherapeutic drugs, lapatinib and doxorubicin for combinational treatment of UM. HA-DOX/LAP gel is fabricated in situ by a simple injection of the mixed precursor solution into tumor sites and maintains in vivo for more than 21 days. The entrapped drug-loaded nanoparticles can gradually release from HA-DOX/LAP gel, enhancing tumor targeting and penetration, and synchronously releasing lapatinib and doxorubicin into the acidic intracellular environment to synergistically destroy UM cells. In vivo anti-tumor studies conducted in MuM-2B tumor models demonstrated that HA-DOX/LAP gel significantly impedes tumor growth, diminishes postoperative recurrence, and prolongs overall survivals of UM tumor-bearing mice through only single injection. Remarkably, the escaped drug-loaded nanoparticles effectively reduce the risk of tumor metastases. Our findings provide new insights for the development of multifunctional nanocomposite-incorporating combination therapy against UM by targeting tumor recurrence and metastases.

Deciphering the intricate relationship between macrophages, pigmentation, and prognosis in uveal melanoma

High pigmentation and the abundance of M2 macrophages have been identified as negative predictors in uveal melanoma (UM). Risk factors associated with UM that are prevalent in high-risk white populations are still present, though less common, in relatively low-risk Asian population. Research shows that proangiogenic M2 macrophages and monosomy 3 play a significant role in UM progression. Our aim is to investigate the impact of tumor-associated macrophages in UM and examine their correlation with monosomy 3 & pigmentation. TEM was used to analyze the morphology of macrophages in UM. Forty UM samples underwent FISH for monosomy 3 identification. Immunohistochemistry was done to assess M2/M1 macrophages on 82 UM tissue samples. IL-10 and IL-12 expression was quantified in UM serum samples by ELISA. Expression of all markers was correlated with pigmentation markers (TYRP1, TYRP2, SILV & MITF). Prognostic outcomes were determined using the Cox proportional hazard model & log-rank test. Increased expression of M2/M1 macrophages was observed in 31 UM cases, which correlated with high expression of pigmentation markers. IL-10 concentration was high in UM cases. Monosomy 3 was evident in 50% of UM cases and significantly associated with increased immunoexpression of M2/M1 macrophages and pigmentation markers. Reduced MFS was observed in UM patients with high M2/M1 macrophage expression (p=0.001). High pigmentation and increased M2 macrophage density could impact the tumor microenvironment in UM. This could contribute to ineffective antitumor immune responses in UM patients. Our findings suggest avenues for developing novel therapeutic approaches to counteract these immunosuppressive effects in UM.

GNAQ/GNA11-Related Benign and Malignant Entities—A Common Histoembriologic Origin or a Tissue-Dependent Coincidence

Uveal melanoma (UM), recognized as the most prevalent primary intraocular malignancy in adults, is primarily driven by mutations in the GNAQ and GNA11 genes. These genetic alterations are also implicated in other conditions, which exhibit distinct morphological characteristics. In this article, we investigate the role of GNAQ and GNA11 mutations across varied disorders (e.g., UM, skin blue nevi, and hemangiomas), emphasizing the shared pathogenic mechanisms that connect them despite their differing clinical manifestations. By investigating the molecular pathways affected by these mutations, we provide insights into the potential for targeted therapies that could address not only UM but also other disorders associated with GNAQ/GNA11 mutations. Moreover, we discuss the role of SOX10-positive perivascular cells that may be implicated in the complex pathophysiology of GNAQ/GNA11-related entities. Understanding the common molecular foundation of these conditions opens new ways for research and treatment opportunities, potentially leading to more effective, personalized therapeutic strategies.

The effect of intra- and inter-fractional motion on target coverage and margins in proton therapy for uveal melanoma

Introduction.This study aims to assess the effective lateral margin requirements for target coverage in ocular proton therapy (OPT), considering the unique challenges posed by eye motion and hypofractionation. It specifically addresses the previously unaccounted-for uncertainty contribution of intra-fractional motion, in conjunction with setup uncertainties, on dosimetric determination of lateral margin requirements.Method.The methodology integrates dose calculations from the in-house developed treatment planning system OCULARIS with measured intra-fractional motion, patient models from EyePlan and Monte Carlo (MC) sampling of setup uncertainties. The study is conducted on 16 uveal melanoma patients previously treated in the OPTIS2 treatment room at the Paul Scherrer Institute (PSI).Result.The retrospective simulation analysis highlights a significant impact of non-systematic factors on lateral margin requirements in OPT. Simulations indicate that reducing the 2.5 mm clinical lateral margin, represented by a 2.1 mm margin in this work, would have resulted in inadequate target coverage for two patients, revealing a greater impact of non-systematic factors on lateral margin requirements.Conclusions.This work characterizes intra-fractional motion in 16 OPT patients and identifies limitations of clinical margin selection protocols for OPT applications. A novel framework was introduced to assess margin sufficiency for target coverage. The findings suggest that prior research underestimated non-systematic factors and overestimated systematic contributions to lateral margin components. This re-evaluation highlights the critical need to prioritize the management of non-systematic uncertainty contributions in OPT.

Phenotypic Biomarkers of Aqueous Extracellular Vesicles from Retinoblastoma Eyes

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.

Survival Distinctions for Cases Representing Immunologically Cold Tumors via Intrinsic Disorder Assessments for Blood-Sourced TRB Variable Regions

T cell receptor beta (TRB) sequences were recovered from the Cancer Genome Atlas Uveal Melanoma blood exome files. Intrinsic disorder scores for amino acid (AA) sequences of the entire TRB variable region were obtained and evaluated as potentially representative of overall survival (OS) distinctions, i.e., for cases representing the upper and lower 50th percentiles for intrinsic disorder scores. Analyses using four intrinsic disorder assessment tools indicated that a lower intrinsic disorder of the blood-sourced TRB variable regions, including continuous AA sequences of the V-gene segment, the complementarity-determining region-3, and the J-gene segment, was associated with a better OS probability (with log-rank p-values ranging from 0.002 to 0.014). We further determined that intrinsic disorder assessments could be used for OS stratification for a second, immunologically cold cancer: MYCN amplified neuroblastoma. Thus, intrinsic disorder assessments of blood-sourced, full TRB variable regions may provide a novel patient stratification approach for patients with immunologically cold cancers.

Machine learning and single-cell RNA sequencing reveal relationship between intratumor CD8+ T cells and uveal melanoma metastasis

PurposeUveal melanoma (UM) is adults’ most common primary intraocular malignant tumor. It has been observed that 40% of patients experience distant metastasis during subsequent treatment. While there exist multigene models developed using machine learning methods to assess metastasis and prognosis, the immune microenvironment’s specific mechanisms influencing the tumor microenvironment have not been clarified. Single-cell transcriptome sequencing can accurately identify different types of cells in a tissue for precise analysis. This study aims to develop a model with fewer genes to evaluate metastasis risk in UM patients and provide a theoretical basis for UM immunotherapy.MethodsRNA-seq data and clinical information from 79 μm patients from TCGA were used to construct prognostic models. Mechanisms were probed using two single-cell datasets derived from the GEO database. After screening for metastasis-related genes, enrichment analysis was performed using GO and KEGG. Prognostic genes were screened using log-rank test and one-way Cox regression, and prognostic models were established using LASSO regression analysis and multifactor Cox regression analysis. The TCGA-UVM dataset was used as internal validation and dataset GSE22138 as external validation data. A time-dependent subject work characteristic curve (time-ROC) was established to assess the predictive ability of the model. Subsequently, dimensionality reduction, clustering, pseudo-temporal analysis and cellular communication analysis were performed on GSE138665 and GSE139829 to explore the underlying mechanisms involved. Cellular experiments were also used to validate the relevant findings.ResultsBased on clinical characteristics and RNA-seq transcriptomic data from 79 samples in the TCGA-UVM cohort, 247 metastasis-related genes were identified. Survival models for three genes (SLC25A38, EDNRB, and LURAP1) were then constructed using lasso regression and multifactorial cox regression. Kaplan-Meier survival analysis showed that the high-risk group was associated with poorer overall survival (OS) and metastasis-free survival (MFS) in UM patients. Time-dependent ROC curves demonstrated high predictive performance in 6 m, 18 m, and 30 m prognostic models. Cell scratch assay showed that the 24 h and 48 h migration rates of cells with reduced expression of the three genes were significantly higher than those of the si-NC group. CD8 + T cells may play an important role in tumour metastasis as revealed by immune infiltration analysis. An increase in the percentage of cytotoxic CD8 + T cells in the metastatic high-risk group was found in the exploration of single-cell transcriptome data. The communication intensity of cytotoxic CD8 was significantly enhanced. It was also found that the CD8 + T cells in the two groups were in different states, although the number of CD8 + T cells in the high-risk group increased, they were mostly in the exhausted and undifferentiated state, while in the low-risk group, the CD8 + T cells were mostly in the functional state.ConclusionsWe developed a precise and stable 3-gene model to predict the metastatic risk and prognosis of patients. CD8 + T cells exhaustion in the tumor microenvironment play a crucial role in UM metastasis.

PO0306: Ocular Brachytherapy with I-125 an Increasingly Important Option in Ocular Melanoma: A Case Series from a Low Middle Income Country, Latin American Experience

PO0306: Ocular Brachytherapy with I-125 an Increasingly Important Option in Ocular Melanoma: A Case Series from a Low Middle Income Country, Latin American Experience

Expression of Markers Associated with Epithelial-Mesenchymal Transition and Extracellular Matrix Degradation in Human Uveal Melanoma.

The expression of markers associated with epithelial-mesenchymal transition (EMT) and extracellular matrix degradation in human uveal melanoma tissue samples and postequatorial zone of the choroid was assessed by immunohistochemical staining. Increased expression of EMT markers E-cadherin and vimentin was observed in the tumor. The ratio of MMP-9 to TIMP-1 proteins related to the extracellular matrix degradation was higher in the tumor. These results may indicate activation of EMT-like process in the uveal melanoma cells and degradation of the extracellular matrix, which can contribute to the development of collective invasion in uveal melanoma.

Genetic Features of Uveal Melanoma

Background/Objectives: Although it comprises only 5% of all melanomas, uveal melanoma (UM) is the most commonly observed primary intraocular cancer. Methods: Poor patient survival persists in spite of innovative systemic therapies. In fact, approximately fifty percent of UM patients develop metastases from micro-metastases that remain undetected at the exact time of diagnosis. Results: The molecular understanding of UM is significantly enhanced by the recent identification of several mutations that are responsible for the metastasis, growth, and survival of UM. The crucial point is a more accurate genetic analysis for patient follow-up and metastatic risk prediction. Conclusions: This review provides a brief summary of the molecular features of UM that are recently discovered, as well as cytogenetic markers and biochemical pathways that are associated with the development of UM metastases.

Response to comment on: "Efficacy and safety of tebentafusp in patients with metastatic uveal melanoma: A systematic review and meta-analysis".

Response to comment on: "Efficacy and safety of tebentafusp in patients with metastatic uveal melanoma: A systematic review and meta-analysis".

Comment on: "Efficacy and safety of tebentafusp in patients with metastatic uveal melanoma: A systematic review and meta-analysis".

Comment on: "Efficacy and safety of tebentafusp in patients with metastatic uveal melanoma: A systematic review and meta-analysis".

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines

BackgroundUveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines.MethodsFour primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared.ResultsIn all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA.ConclusionsThis data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Neuropeptide Precursor VGF Promotes Liver Metastatic Colonization of Gαq Mutant Uveal Melanoma by Facilitating Tumor Microenvironment via Paracrine Loops.

Uveal melanoma (UM), the predominant primary ocular malignancy, often progresses to liver metastasis with limited therapeutic options. The interplay of the tumor microenvironment, encompassing secreted soluble factors, plays a crucial role in facilitating liver metastasis. In this study, the role is elucidated of the neural growth factor-inducible gene (VGF), a secreted neuropeptide precursor, in Gαq mutant UM. Employing a multiomics approach, encompassing transcriptomic and secretomic analyses, the intricate involvement of VGF in UM progression is unveiled. VGF is upregulated in Gαq mutant UM cells and associated with poor prognosis of UM patients. Targeting VGF significantly suppressed the growth of UM in vitro and in vivo. Further evidence shows that VGF is regulated by Gαq through MAPK/CREB pathway. Mechanistically, CREB modulates VGF expression by directly binding to consensus DNA response elements in the promoters of the VGF gene. Combined inhibition of Gαq and MEK remarkably reduces tumor burden in the UM xenograft model. Notably, VGF triggers liver metastatic colonization of UM and activates the fibrosis of hepatic stellate cells (HSCs), creating a favorable microenvironment, through an autocrine and paracrine loop. Furthermore, VGF directly binds to TGFBR2 and regulates TGF-β-SMAD signaling pathway, thereby regulating genes associated with endothelial-mesenchymal transition (EMT) to promote metastasis. Taken together, these findings identify VGF as a pivotal driver in the progression and metastasis of Gαq mutant UM and confers a promising therapeutic target and strategy for UM patients.

Overcoming Barriers: Diagnosis of High-Grade Melanoma: Perspective from a Clinical Case

We present the case of a 30-year-old male patient with loss of visual acuity in the left eye. The imaging study detected a lesion of high suspicion of tumor in the left eye. Enucleation of the left eye was performed, confirming the diagnosis of choroidal melanoma by histopathological study. Subsequently, the disease progressed with neoplastic activity in the lungs, liver, lymph nodes and central nervous system. A case description is provided along with a bibliographic review, focusing on the approach and prognostic factors in patients with metastatic disease.