GATA6 identifies an immune-enriched phenotype linked to favorable outcomes in patients with pancreatic cancer undergoing upfront surgery.

This study underscores GATA6's role in distinguishing classical and basal-like pancreatic ductal adenocarcinoma (PDAC) phenotypes. Retrospective studies associate GATA6 immunohistochemistry (IHC) expression with survival outcomes, warranting prospective validation. In a prospective treatment-naive cohort of patients with resected PDAC, GATA6 IHC proves a prognostic discriminator, associating high GATA6 expression with extended survival and the classical PDAC phenotype. However, GATA6's prognostic significance is numerically lower after gemcitabine-based neoadjuvant chemoradiotherapy compared to its significance in patients treated with upfront surgery. Furthermore, GATA6 is implicated in immunomodulation, although a comprehensive investigation of its immunological role is lacking. Treatment-naive PDAC tumors with varying GATA6 expression yield distinct immunological landscapes. Tumors highly expressing GATA6 show reduced infiltration of immunosuppressive regulatory T cells and M2 macrophages but increased infiltration of immune-stimulating, antigen-presenting, and activated T cells. Our findings caution against solely relying on GATA6 for molecular subtyping in clinical trials and open avenues for exploring immune-based combination therapies.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Purpose: Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods: Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results: The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion: The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

Increase in venous thromboembolism in SARS-CoV-2 infected lung tissue: proteome analysis of lung parenchyma, isolated endothelium, and thrombi.

AIMS: COVID-19 pneumonia is characterized by an increased rate of deep venous thrombosis and pulmonary embolism. To better understand the pathophysiology behind thrombosis in COVID-19, we performed proteomics analysis on SARS-CoV-2 infected lung tissue. METHODS: Liquid chromatography mass spectrometry was performed on SARS-CoV-2 infected postmortem lung tissue samples. Five protein profiling analyses were performed: whole slide lung parenchyma analysis, followed by analysis of isolated thrombi and endothelium, both stratified by disease (COVID-19 versus influenza) and thrombus morphology (embolism versus in situ). Influenza autopsy cases with pulmonary thrombi were used as controls. RESULTS: Compared to influenza controls, both analyses of COVID-19 whole-tissue and isolated endothelium showed upregulation of proteins and pathways related to liver metabolism including urea cycle activation, with arginase being among the top upregulated proteins in COVID-19 lung tissue. Analysis of isolated COVID-19 thrombi showed significant downregulation of pathways related to platelet activation compared to influenza thrombi. Analysis of isolated thrombi based on histomorphology shows that in situ thrombi have significant upregulation of coronavirus pathogenesis proteins. CONCLUSIONS: The decrease in platelet activation pathways in severe COVID-19 thrombi suggests a relative increase in venous thromboembolism, as thrombi from venous origin tend to contain fewer platelets than arterial thrombi. Based on histomorphology, in situ thrombi show upregulation of various proteins related to SARS-CoV-2 pathogenesis compared to thromboemboli, which may indicate increased in situ pulmonary thrombosis in COVID-19. Therefore, this study supports the increase of venous thromboembolism without undercutting the involvement of in situ thrombosis in severe COVID-19.

Multiomic profiling of transplant glomerulopathy reveals a novel T-cell dominant subclass.

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype.

B cell immune profiles in dysbiotic vermiform appendixes of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both locally and systemically. Moreover, increasing evidence suggests that microbial compositions inside the tumor, as well as in the oral cavity and the gut, are important factors in shaping the PDAC immune landscape. However, the gut-associated lymphoid tissue (GALT) has not previously been explored in PDAC patients. In this study, we analyzed healthy vermiform appendix (VA) from 20 patients with PDAC and 32 patients with colon diseases by gene expression immune profiling, flow cytometry analysis, and microbiome sequencing. We show that the VA GALT of PDAC patients exhibits markers of increased inflammation and cytotoxic cell activity. In contrast, B cell function is decreased in PDAC VA GALT based on gene expression profiling; B cells express significantly fewer MHC class II surface receptors, whereas plasma cells express the immune checkpoint molecule HLA-G. Additionally, the vermiform appendix microbiome of PDAC patients is enriched with Klebsiella pneumoniae, Bifidobacterium animalis, and Adlercreutzia equolifaciens, while certain commensals are depleted. Our findings may suggest impaired B cell function within the GALT of PDAC patients, which could potentially be linked to microbial dysbiosis. Additional investigations are imperative to validate our observations and explore these potential targets of future therapies.

The origin of intestinal cancer in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males.

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Whole-body bioluminescence imaging of T-cell response in PDAC models.

Introduction: The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Methods: Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Results: Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. Conclusions: In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs.

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1-3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

Mast Cells in Kidney Transplant Biopsies With Borderline T Cell-mediated Rejection and Their Relation to Chronicity.

Mast cells are potential contributors to chronic changes in kidney transplants (KTx). Here, the role of mast cells (MCs) in KTx is investigated in patients with minimal inflammatory lesions. Methods: Fourty-seven KTx biopsies (2009-2018) with borderline pathological evidence for T cell-mediated rejection according to the Banff'17 Update were retrospectively included and corresponding clinical data was collected. Immunohistochemistry for tryptase was performed on formalin-fixed paraffin-embedded sections. Cortical MCs were counted and corrected for area (MC/mm²). Interstitial fibrosis was assessed by Sirius Red staining and quantified using digital image analysis (QuPath). Results: Increased MC number was correlated to donor age (spearman's r = 0.35, P = 0.022), deceased donor kidneys (mean difference = 0.74, t [32.5] = 2.21, P = 0.035), and delayed graft function (MD = 0.78, t [33.9] = 2.43, P = 0.020). Increased MC number was also correlated to the amount of interstitial fibrosis (r = 0.42, P = 0.003) but did not correlate with transplant function over time (r = -0.14, P = 0.36). Additionally, transplant survival 2 y post-biopsy was not correlated to MC number (mean difference = -0.02, t [15.36] = -0.06, P = 0.96). Conclusions: MC number in suspicious (borderline) for acute T cell-mediated rejection is correlated to interstitial fibrosis and time post-transplantation, suggesting MCs to be a marker for cumulative burden of tissue injury. There was no association between MCs and transplant function over time or transplant survival 2 y post-biopsy. It remains unclear whether MCs are just a bystander or have pro-inflammatory or anti-inflammatory effects in the KTx with minimal lesions.

Issues with RNF43 antibodies to reliably detect intracellular location.

RNF43 is an important negative regulator of ß-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of ß-catenin. RNF43 has also been suggested to regulate ß-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/ß-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.

Transcriptome analysis reveals tumor microenvironment changes in glioblastoma.

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

Scleral Proteome in Noninfectious Scleritis Unravels Upregulation of Filaggrin-2 and Signs of Neovascularization.

Purpose: Scleritis is a severe inflammatory ocular disorder with unknown pathogenesis. We investigated healthy sclera as well as sclera affected by noninfectious scleritis for differentially expressed proteins using a mass spectrometry approach. Methods: We collected scleral samples of enucleated eyes due to severe noninfectious scleritis (n = 3), and control scleral tissues (n = 5), all exenterated eyes for eyelid carcinomas (n = 4), or choroidal melanoma (n = 1) without scleral invasion. Samples were prepared for the nano liquid-chromatography mass spectrometer (LC-MS), data were analyzed using proteomics software (Scaffold), and is available via ProteomeXchange (identifier PXD038727). Samples were also stained for immuno-histopathological evaluation. Results: Mass spectrometry identified 629 proteins within the healthy and diseased scleral tissues, whereof collagen type XII, VI, and I were the most abundantly expressed protein. Collagen type II-XII was also present. Filaggrin-2, a protein that plays a crucial role in epidermal barrier function, was found upregulated in all scleritis cases. In addition, other epithelial associated proteins were upregulated (such as keratin 33b, 34, and 85, epiplakin, transglutaminase-3, galectin 7, and caspase-14) in scleritis. Further, upregulated proteins involved in regulation of the cytoskeleton (vinculin and myosin 9), and housekeeping proteins were found (elongation factor-2 and cytoplasmic dynein 1) in our study. Upregulation of filaggrin-2 and myosin-9 was confirmed with immunohistochemistry, the latter protein showing co-localization with the endothelial cell marker ETC-related gene (ERG), indicating neovascularization in scleral tissue affected by scleritis. Conclusions: We found upregulation of filaggrin-2 and signs of neovascularization in scleral tissue of patients with noninfectious scleritis. Further research, ideally including more scleritis cases, is needed to validate our findings.

Modeling bile duct ischemia and reoxygenation injury in human cholangiocyte organoids for screening of novel cholangio-protective agents.

BACKGROUND: Ischemia of the bile duct is a common feature in liver disease and transplantation, which represents a major cause of morbidity and mortality, especially after liver transplantation. Detailed knowledge of its pathogenesis remains incomplete due to the lack of appropriate in vitro models. METHODS: To recapitulate biliary damage induced by ischemia and reperfusion in vitro, human intrahepatic cholangiocyte organoids (ICOs) were grown at low oxygen levels of 1% up to 72 h, followed by re-oxygenation at normal levels. FINDINGS: ICOs stressed by ischemia and subsequent re-oxygenation represented the dynamic change in biliary cell proliferation, upregulation of epithelial-mesenchymal transition (EMT)-associated markers, and the evocation of phase-dependent cell death programs similar to what is described in patients. Clinical-grade alpha-1 antitrypsin was identified as a potent inhibitor of both ischemia-induced apoptosis and necroptosis. INTERPRETATION: These findings demonstrate that ICOs recapitulate ischemic cholangiopathy in vitro and enable drug assessment studies for the discovery of new therapeutics for ischemic cholangiopathies. FUNDING: Dutch Digestive FoundationMLDS D16-26; TKI-LSH (Topconsortium Kennis en Innovatie-Life Sciences & Health) grant RELOAD, EMC-LSH19002; Medical Delta program "Regenerative Medicine 4D"; China Scholarship Council No. 201706230252.

Successful pharmacological intervention at different levels of the complement system in an in vitro complement fixation model for bullous pemphigoid.

Bullous pemphigoid (BP) is characterized by deposition of immunoglobulins and complement along the epidermal basement membrane (BM). In humans, there is a lack of functional studies targeting the complement system (CS). This study investigates activation of all complement pathways in BP skin biopsies. Moreover, pharmacological inhibition at different levels of the CS was investigated using anti-complement compounds in a complement fixation BP assay. In this retrospective study, 21 frozen biopsies from BP patients were stained by direct immunofluorescence for C1q, MBL, ficolin-2, C4d, properdin, C3c and C5b-9. Sera from 10 patients were analysed in a complement fixation assay in the presence of C1 inhibitor, anti-factor B monoclonal antibody (mAb), anti-C3 mAb and anti-C5 mAb and compared with dexamethasone. The two readouts were the quantity of complement deposited along the BM and the release of sC5b-9 in the supernatant. Our results show classical and alternative complement pathway activation in BP skin biopsies, but could not demonstrate significant lectin pathway activation. In contrast to dexamethasone, complement deposition along the BM could be selectively inhibited by anti-C1 and anti- factor B. More downstream, selective intervention at the level of C3 and C5 could effectively reduce complement deposition along the BM and the release of sC5b-9 in the supernatant. This study shows that selective intervention in either the classical, alternative or terminal pathway prevented deposition of complement along the BM in an in vitro BP model. The results of our study greatly encourage the clinical development of complement inhibitors for the treatment of BP.

Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma.

Background: Only a small group of patients with glioblastoma multiforme (GBM) survives more than 36 months, so-called long-term survivors. Recent studies have shown that chromosomal instability (CIN) plays a prognostic and predictive role among different cancer types. Here, we compared histological (chromosome missegregation) and bioinformatic metrics (CIN signatures) of CIN in tumors of GBM typical survivors (≤36 months overall survival), GBM long-term survivors and isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytomas. Methods: Tumor sections of all gliomas were examined for anaphases and chromosome missegregation. Further CIN signature activity analysis in the The Cancer Genome Atlas (TCGA)-GBM cohort was performed. Results: Our data show that chromosome missegregation is pervasive in high grade gliomas and is not different between the 3 groups. We find only limited evidence of altered CIN levels in tumors of GBM long-term survivors relative to the other groups, since a significant depletion in CIN signature 11 relative to GBM typical survivors was the only alteration detected. In contrast, within IDH-mutant grade 4 astrocytomas we detected a significant enrichment of CIN signature 5 and 10 activities and a depletion of CIN signature 1 activity relative to tumors of GBM typical survivors. Conclusions: Our data suggest that CIN is pervasive in high grade gliomas, however this is unlikely to be a major contributor to the phenomenon of long-term survivorship in GBM. Nevertheless, further evaluation of specific types of CIN (signatures) could have prognostic value in patients suffering from grade 4 gliomas.

FOXD1 Is a Transcription Factor Important for Uveal Melanocyte Development and Associated with High-Risk Uveal Melanoma.

Uveal melanoma (UM) is a deadly ocular malignancy, originating from uveal melanocytes. Although much is known regarding prognostication in UM, the exact mechanism of metastasis is mostly unknown. Metastatic tumor cells are known to express a more stem-like RNA profile which is seen often in cell-specific embryonic development to induce tumor progression. Here, we identified novel transcription regulators by reanalyzing publicly available single cell RNA sequencing experiments. We identified five transcription regulators of interest: ELL2, KDM5B, REXO4, RBFOX2 and FOXD1. Our most significant finding is FOXD1, as this gene is nearly exclusively expressed in high-risk UM and its expression is associated with a poor prognosis. Even within the BAP1-mutated UM, the expression of FOXD1 is correlated with poor survival. FOXD1 is a novel factor which could potentially be involved in the metastatic capacity of high-risk UM. Elucidating the function of FOXD1 in UM could provide insight into the malignant transformation of uveal melanocytes, especially in high-risk UM.