American Joint Committee on Cancer Tumor Staging System Predicts the Outcome and Metastasis Pattern in Conjunctival Melanoma.

PURPOSE: To assess the predictive value of the tumor staging system in the AJCC Cancer Staging Manual, Eighth Edition, and histologic features for outcomes and metastasis patterns in conjunctival melanoma (CM). DESIGN: Retrospective, single-center cohort study. PARTICIPANTS: Eighty-three patients with CM were treated at Shanghai Ninth People's Hospital between 2000 and 2021. METHODS: We reviewed the clinical and histologic parameters and used Kaplan-Meier survival curves and Cox proportional hazards regression models for risk factor analyses. MAIN OUTCOME MEASURES: Time to nodal/distant metastasis, disease-specific survival, metastatic pattern, and metastatic site. RESULTS: At presentation, 5 patients (6%) had clinical tumor (cT)1 disease, 34 patients (41%) had cT2 disease, and 44 patients (53%) had cT3 disease. Four patients (5%) had nodal metastasis (N1), and none had distant metastasis (M1). During follow-up, 12 patients (14%) developed nodal metastasis, 29 patients (35%) developed distant metastasis, and 26 patients (31%) died of disease. The brain, liver, and lung were common distant metastasis sites. Higher cT category was associated with increased risks of distant metastasis (P < 0.001) and disease-specific death (P = 0.002). The separate analysis of primary and recurrent tumors at presentation showed that the patients with cT3 tumors had a higher risk of distant metastasis than those with cT2 tumors. Greater tumor thickness, ulceration, and the presence of regression were correlated with distant metastasis. Previously unreported mutations were detected in the tumor suppressor genes FAT atypical cadherin 4 (FAT4) and spleen associated tyrosine kinase (SYK). Among the 29 patients who developed distant metastasis, we analyzed 2 patterns of metastasis: Eleven patients (38%) developed nodal metastasis before distant metastasis, and 18 patients (62%) developed distant metastasis without previously known nodal metastasis. The patients with cT3 tumors were more likely to follow the latter metastasis pattern (P = 0.02). CONCLUSIONS: Conjunctival melanoma presented with mostly advanced stages and high rates of distant metastasis in the current Chinese cohort. This study confirmed the prognostic value of the tumor staging system in the AJCC Cancer Staging Manual, Eighth Edition, for Chinese patients. Histologic features, such as tumor thickness and ulceration, should be emphasized when assessing prognosis and guiding the treatment of CM.

m6A RNA hypermethylation-induced BACE2 boosts intracellular calcium release and accelerates tumorigenesis of ocular melanoma.

Ocular melanoma, including uveal melanoma (UM) and conjunctival melanoma (CM), is the most common and deadly eye cancer in adults. Both UM and CM originate from melanocytes and exhibit an aggressive growth pattern with high rates of metastasis and mortality. The integral membrane glycoprotein beta-secretase 2 (BACE2), an enzyme that cleaves amyloid precursor protein into amyloid beta peptide, has been reported to play a vital role in vertebrate pigmentation and metastatic melanoma. However, the role of BACE2 in ocular melanoma remains unclear. In this study, we showed that BACE2 was significantly upregulated in ocular melanoma, and inhibition of BACE2 significantly impaired tumor progression both in vitro and in vivo. Notably, we identified that transmembrane protein 38B (TMEM38B), whose expression was highly dependent on BACE2, modulated calcium release from endoplasmic reticulum (ER). Inhibition of the BACE2/TMEM38B axis could trigger exhaustion of intracellular calcium release and inhibit tumor progression. We further demonstrated that BACE2 presented an increased level of N6-methyladenosine (m6A) RNA methylation, which led to the upregulation of BACE2 mRNA. To our knowledge, this study provides a novel pattern of BACE2-mediated intracellular calcium release in ocular melanoma progression, and our findings suggest that m6A/BACE2/TMEM38b could be a potential therapeutic axis for ocular melanoma.

Novel insight into the regulatory roles of diverse RNA modifications: Re-defining the bridge between transcription and translation.

RNA modifications can be added or removed by a variety of enzymes that catalyse the necessary reactions, and these modifications play roles in essential molecular mechanisms. The prevalent modifications on mRNA include N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), pseudouridine (Ψ), inosine (I), uridine (U) and ribosemethylation (2'-O-Me). Most of these modifications contribute to pre-mRNA splicing, nuclear export, transcript stability and translation initiation in eukaryotic cells. By participating in various physiological processes, RNA modifications also have regulatory roles in the pathogenesis of tumour and non-tumour diseases. We discussed the physiological roles of RNA modifications and associated these roles with disease pathogenesis. Functioning as the bridge between transcription and translation, RNA modifications are vital for the progression of numerous diseases and can even regulate the fate of cancer cells.

FTO elicits tumor neovascularization in cancer-associated fibroblasts through eliminating m6A modifications of multiple pro-angiogenic factors.

Cancer-associated fibroblasts (CAFs) exhibit notable versatility, plasticity, and robustness, actively participating in cancer progression through intricate interactions within the tumor microenvironment (TME). N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic mRNA, playing essential roles in mRNA metabolism and various biological processes. Howbeit, the precise involvement of m6A in CAF activation remains enigmatic. In this study, we revealed that the m6A demethylase FTO supports CAF-mediated angiogenesis through activation of EGR1 and VEGFA in conjunctival melanoma (CoM). First, single-cell transcriptome analysis revealed that FTO was specifically upregulated in the CAF population, thereby contributing to the hypo-m6A status in the TME of CoM. Moreover, CAFs of CoM displayed extensive proangiogenic potential, which was largely compromised by FTO inhibition, both in vitro and in vivo. By employing multi-omics analysis, we showed that FTO effectively eliminates the m6A modifications of VEGFA and EGR1. This process subsequently disrupts the YTHDF2-dependent mRNA decay pathway, resulting in increased mRNA stability and upregulated expression of these molecules. Collectively, our findings initially indicate that the upregulation of FTO plays a pivotal role in tumor development by promoting CAF-mediated angiogenesis. Therapeutically, targeting FTO may show promise as a potential antiangiogenic strategy to optimize cancer treatment.

Single-cell sequencing depicts tumor architecture and empowers clinical decision in metastatic conjunctival melanoma.

Conjunctival melanoma (CoM) is a potentially devastating tumor that can lead to distant metastasis. Despite various therapeutic strategies for distant metastatic CoM, the clinical outcomes remain unfavorable. Herein, we performed single-cell RNA sequencing (scRNA-seq) of 47,017 cells obtained from normal conjunctival samples (n = 3) and conjunctival melanomas (n = 7). Notably, we noticed a higher abundance of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME), correlated with enhanced angiogenic capacity and increased VEGFR expression in distal metastatic CoM. Additionally, we observed a significant decrease in the proportion of total CD8+ T cells and an increase in the proportion of naive CD8+ T cells, contributing to a relatively quiescent immunological environment in distal metastatic CoM. These findings were confirmed through the analyses of 70,303 single-cell transcriptomes of 7 individual CoM samples, as well as spatially resolved proteomes of an additional 10 samples of CoMs. Due to the increase of VEGFR-mediated angiogenesis and a less active T cell environment in distal metastatic CoMs, a clinical trial (ChiCTR2100045061) has been initiated to evaluate the efficacy of VEGFR blockade in combination with anti-PD1 therapy for patients with distant metastatic CoM, showing promising tumor-inhibitory effects. In conclusion, our study uncovered the landscape and heterogeneity of the TME during CoM tumorigenesis and progression, empowering clinical decisions in the management of distal metastatic CoM. To our knowledge, this is the initial exploration to translate scRNA-seq analysis to a clinical trial dealing with cancer, providing a novel concept by accommodating scRNA-seq data in cancer therapy.

High-risk histopathologic features in local advanced conjunctival melanoma.

PURPOSE: To identify high-risk histopathologic and molecular features of local recurrence, nodal metastasis, distant metastasis (DM) and disease-specific death (DSD) in conjunctival melanoma (CoM). METHODS: Ninety patients with pathologically diagnosed CoM between 2008 and 2023 were enrolled. Immunohistochemistry staining of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was performed in 65 and 45 patients, respectively. Cox regression and Kaplan-Meier survival analysis were conducted to identify risk factors for local recurrence, nodal metastasis, DM and DSD. RESULTS: Pathologically, ulceration (hazard ratio [HR]: 3.170; 95% CI: 1.312-7.659; p = 0.01) and regression (HR: 3.196; 95% CI: 1.094-9.335; p = 0.034) were risk factors for DM. Tumour thickness ≥ 4 mm (HR: 4.889; 95% CI: 1.846-12.946; p = 0.001) and regression (HR: 4.011; 95% CI: 1.464-10.991; p = 0.007) were risk factors for DSD. For patients with tumour thickness < 4 mm, the presence of ulceration indicated a higher risk of nodal metastasis (log-rank p = 0.0011), DM (log-rank p = 0.00051) and DSD (log-rank p = 0.02). Patients with regression (+)/tumour-infiltrating lymphocytes (TILs) (+) had a higher risk for DM (log-rank p = 0.011) and DSD (log-rank p = 0.0032). Molecularly, the positive rate of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was 40.00% (26/65), 43.08% (28/65), 70.77% (46/65), 46.67% (21/45) and 28.89% (13/45), respectively. Positive BRAFV600E was identified as an independent risk factor for DM (HR: 2.533; 95% CI: 1.046-6.136, p = 0.039). The expression level of BRAFV600E was positively correlated with vascular invasion (p = 0.01), as well as the expression levels of PD-1 (p = 0.038) and PD-L1 (p = 0.049). CONCLUSIONS: Tumour thickness ≥ 4 mm, ulceration, the coexistence of regression and TILs, and positive BRAFV600E were risk factors for poor prognosis of CoM patients. Besides, expression level of BRAFV600E was positively correlated with the expression levels of PD-1 and PD-L1.

Genetic landscape and prognosis of conjunctival melanoma in Chinese patients.

AIMS: Conjunctival melanoma (CoM) is a rare but highly lethal ocular melanoma and there is limited understanding of its genetic background. To update the genetic landscape of CoM, whole-exome sequencing (WES) and targeted next-generation sequencing (NGS) were performed. METHODS: Among 30 patients who were diagnosed and treated at Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, from January 2018 to January 2023, WES was performed on 16 patients, while targeted NGS was conducted on 14 patients. Samples were analysed to identify the mutated genes, and the potential predictive factors for progression-free survival were evaluated. Furthermore, the expression of the mutated gene was detected and validated in a 30-patient cohort by immunofluorescence. RESULTS: Mutations were verified in classic genes, such as BRAF (n=9), NRAS (n=5) and NF1 (n=6). Mutated FAT4 and BRAF were associated with an increased risk for the progression of CoM. Moreover, decreased expression of FAT4 was detected in CoM patients with a worse prognosis. CONCLUSIONS: The molecular landscape of CoM in Chinese patients was updated with new findings. A relatively high frequency of mutated FAT4 was determined in Chinese CoM patients, and decreased expression of FAT4 was found in patients with worse prognoses. In addition, both BRAF mutations and FAT4 mutations could serve as predictive factors for CoM patients.

Risk factors and prognosis of orbital exenteration in conjunctival melanoma.

OBJECTIVES: To identify the risk factors of orbital exenteration and to evaluate the prognosis of exenterated patients with conjunctival melanoma (CM). METHODS: 79 consecutive CM patients treated at our centre from January 2000 to September 2021 were included. The demographic, clinical and pathological characteristics were compared between eye-sparing patients and orbital exenteration patients. Main outcomes including progression-free survival (PFS), distant metastasis-free survival (DFS) and disease specific survival (DSS) were assessed in exenterated patients. RESULTS: The mean follow-up period was 46 ± 39 months. Risk factors for orbital exenteration were identified as worse cT category (OR, 50.75; 95% CI, 5.40-477.07; P = 0.001) and greater tumour thickness (OR, 1.27; CI, 1.04-1.55; P = 0.02). Of the 32 patients who underwent orbital exenteration, three (9.4%) had local recurrence; six (18.8%) experienced regional metastasis; sixteen (50.0%) suffered distant metastasis and fifteen (46.9%) died of metastatic disease. In patients who received orbital exenteration, palpebral conjunctiva involvement (PFS: P < 0.01; DFS: P < 0.05; DSS: P = 0.04), histological ulceration (PFS: P = 0.03; DFS: P = 0.01; DSS: P = 0.03) and regression (PFS: P = 0.01; DFS: P < 0.01; DSS: P = 0.04) were identified as risk factors for poor prognosis. Caruncle involvement (P = 0.01) was also associated with increased risk of melanoma related mortality in exenterated patients. CONCLUSIONS: Histopathological factors should be taken into account when formulating surgical plans for orbital exenteration and when evaluating patients' prognosis following exenteration. For CM patients with caruncle or palpebral conjunctiva involvement, orbital exenteration should be considered for unresectable disease.

The Use of rAAV2-RB1-Mediated Gene Therapy in Retinoblastoma.

Purpose: Retinoblastoma (RB) is a life-threatening malignancy that arises from the retina and is activated upon homozygous inactivation of the tumor suppressor RB1. Gene therapy targeting RB1 is an effective strategy to treat RB. However, it is difficult to target the RB1 gene by site-specific repair, with up to 3366 gene mutation sites identified in RB1. Thus, it is necessary to construct a promising and efficacious gene therapeutic strategy for patients with RB. Methods: To recover the function of the RB1 protein, we constructed a recombinant adeno-associated virus 2 (rAAV2) expressing RB1 that can restore RB1 function and significantly inhibit RB progression. To confirm the clinical feasibility of rAAV2-RB1, the RB1 protein was validated in vitro and in vivo after transfection. To further evaluate the clinical efficacy, RB patient-derived xenograft models were established and applied. The biosafety of rAAV2-RB1 was also validated in immunocompetent mice. Results: rAAV2-RB1 was a rAAV2 expressing the RB1 protein, which was validated in vitro and in vivo. In vitro, rAAV2-RB1 was effectively expressed in patient-derived RB cells. In mice, intravitreal administration of rAAV2-RB1 in a population-based patient-derived xenograft trial induced limited tumor growth. Moreover, after transfection of rAAV2-RB1 in immunocompetent mice, rAAV2-RB1 did not replicate and was expressed in other important organs, except retinas, inducing minor local side effects. Conclusions: Our study suggested a promising efficacy gene therapeutic strategy, which might provide a chemotherapy-independent treatment option for RB.

Biophysics Role and Biomimetic Culture Systems of ECM Stiffness in Cancer EMT.

Oncological diseases have become the second leading cause of death from noncommunicable diseases worldwide and a major threat to human health. With the continuous progress in cancer research, the mechanical cues from the tumor microenvironment environment (TME) have been found to play an irreplaceable role in the progression of many cancers. As the main extracellular mechanical signal carrier, extracellular matrix (ECM) stiffness may influence cancer progression through biomechanical transduction to modify downstream gene expression, promote epithelial-mesenchymal transition (EMT), and regulate the stemness of cancer cells. EMT is an important mechanism that induces cancer cell metastasis and is closely influenced by ECM stiffness, either independently or in conjunction with other molecules. In this review, the unique role of ECM stiffness in EMT in different kinds of cancers is first summarized. By continually examining the significance of ECM stiffness in cancer progression, a biomimetic culture system based on 3D manufacturing and novel material technologies is developed to mimic ECM stiffness. The authors then look back on the novel development of the ECM stiffness biomimetic culture systems and finally provide new insights into ECM stiffness in cancer progression which can broaden the fields' horizons with a view toward developing new cancer diagnosis methods and therapies.

Methylation-driven gene DLL3 is a potential prognostic biomarker in ocular melanoma correlating with metastasis.

Background: Ocular melanoma is an aggressive malignancy with a high rate of metastasis and poor prognosis. Increasing evidence indicated that DNA methylation plays an important role in the occurrence and development of ocular melanoma. Hence, exploring new diagnostic and prognostic biomarkers at the genetic level may be beneficial to the prognosis of patients with ocular melanoma. Methods: We collected DNA methylation and gene expression profiles of human UM (uveal melanoma) and CM (conjunctival melanoma) samples from various datasets. We conducted differential methylation and expression analyses to screen the potential biomarkers. Correlation analysis was performed to investigate the relationships between the expression level of DLL3 (delta-like protein 3) and the methylation level of its corresponding CpGs. We explored the prognostic and diagnostic value of DLL3 in UM and CM. Functional annotation and GSEA (gene set enrichment analysis) were applied to get insight into the possible biological roles of DLL3. A cohort of 60 ocular melanoma patients as well as UM and CM cell lines were used to validate our findings in bioinformatic analyses. Results: We found that DLL3 was a methylation-driven gene correlating with UM metastasis. The CpGs of DLL3 are mainly located in the gene body and their methylation level positively correlated to DLL3 expression. Multivariate Cox regression analysis revealed that DLL3 was an independent protective factor for UM patients. High DLL3 expression significantly prolonged the overall survival and disease-free survival of UM patients. DLL3 also showed a promising power to distinguish CM from normal tissues. Functional annotation exhibited that DLL3 may suppress UM progression through modulating immune activities and down-regulating various signaling pathways. External datasets, biospecimens, and cell lines further validated the aberrant expression and prognostic role of DLL3 in ocular melanoma. Conclusion: Methylation-driven gene DLL3 could serve as a new potential diagnostic and prognostic biomarker in ocular melanoma. Our findings may contribute to improving the clinical outcomes of patients with UM or CM.

Regulation of epigenetic homeostasis in uveal melanoma and retinoblastoma.

Uveal melanoma (UM) and retinoblastoma (RB), which cause blindness and even death, are the most frequently observed primary intraocular malignancies in adults and children, respectively. Epigenetic studies have shown that changes in the epigenome contribute to the rapid progression of both UM and RB following classic genetic changes. The loss of epigenetic homeostasis plays an important role in oncogenesis by disrupting the normal patterns of gene expression. The targetable nature of epigenetic modifications provides a unique opportunity to optimize treatment paradigms and establish new therapeutic options for both UM and RB with these aberrant epigenetic modifications. We aimed to review the research findings regarding relevant epigenetic changes in UM and RB. Herein, we 1) summarize the literature, with an emphasis on epigenetic alterations, including DNA methylation, histone modifications, RNA modifications, noncoding RNAs and an abnormal chromosomal architecture; 2) elaborate on the regulatory role of epigenetic modifications in biological processes during tumorigenesis; and 3) propose promising therapeutic candidates for epigenetic targets and update the list of epigenetic drugs for the treatment of UM and RB. In summary, we endeavour to depict the epigenetic landscape of primary intraocular malignancy tumorigenesis and provide potential epigenetic targets in the treatment of these tumours.

Antimicrobial Action of Biocompatible Silver Microspheres and Their Role in the Potential Treatment of Fungal Keratitis.

Silver nanoparticles have drawn considerable attention as excellent antimicrobial agents because of their characteristics, including broad antimicrobial spectrum, durable antimicrobial property, and large specific surface area. However, the toxicity of silver nanoparticles limits the in vivo application in the antimicrobial therapy field. Here, we developed a novel silver-based biomaterial to achieve favorable biocompatibility as well as enhanced antimicrobial activity. Silver microspheres (AgMPs) were synthesized using bovine serum albumin as a template and H2O2 as an activator. Electron microscopy results showed that AgMPs had a honeycombed inner structure with an approximate diameter of 800 nm. The minimum inhibitory concentration results exhibited that AgMPs had effective antimicrobial action against bacteria and fungi when the concentration was greater than 32 and 16 µg/mL, respectively. The cell proliferation results suggested that AgMPs have no influence on corneal epithelial cell growth when the concentration was under 25 µg/mL. The in vivo antifungal therapy experiments demonstrated that 25 µg/mL AgMPs could effectively combat Candida smooth wound infections. Overall, AgMPs exhibited substantial antimicrobial action on fungi in addition to biosafety on corneal epithelial cells at a concentration within 16-25 µg/mL. Our study shows that AgMPs can be used as an ocular surface drop candidate to treat fungal keratitis.