BAP1 Loss Promotes Suppressive Tumor Immune Microenvironment via Upregulation of PROS1 in Class 2 Uveal Melanomas.

Uveal melanoma (UM) is the most common primary cancer of the eye and is associated with a high rate of metastatic death. UM can be stratified into two main classes based on metastatic risk, with class 1 UM having a low metastatic risk and class 2 UM having a high metastatic risk. Class 2 UM have a distinctive genomic, transcriptomic, histopathologic, and clinical phenotype characterized by biallelic inactivation of the BAP1 tumor-suppressor gene, an immune-suppressive microenvironment enriched for M2-polarized macrophages, and poor response to checkpoint-inhibitor immunotherapy. To identify potential mechanistic links between BAP1 loss and immune suppression in class 2 UM, we performed an integrated analysis of UM samples, as well as genetically engineered UM cell lines and uveal melanocytes (UMC). Using RNA sequencing (RNA-seq), we found that the most highly upregulated gene associated with BAP1 loss across these datasets was PROS1, which encodes a ligand that triggers phosphorylation and activation of the immunosuppressive macrophage receptor MERTK. The inverse association between BAP1 and PROS1 in class 2 UM was confirmed by single-cell RNA-seq, which also revealed that MERTK was upregulated in CD163+ macrophages in class 2 UM. Using ChIP-seq, BAP1 knockdown in UM cells resulted in an accumulation of H3K27ac at the PROS1 locus, suggesting epigenetic regulation of PROS1 by BAP1. Phosphorylation of MERTK in RAW 264.7 monocyte-macrophage cells was increased upon coculture with BAP1-/- UMCs, and this phosphorylation was blocked by depletion of PROS1 in the UMCs. These findings were corroborated by multicolor immunohistochemistry, where class 2/BAP1-mutant UMs demonstrated increased PROS1 expression in tumor cells and increased MERTK phosphorylation in CD163+ macrophages compared with class 1/BAP1-wildtype UMs. Taken together, these findings provide a mechanistic link between BAP1 loss and the suppression of the tumor immune microenvironment in class 2 UMs, and they implicate the PROS1-MERTK pathway as a potential target for immunotherapy in UM.

RB1 loss triggers dependence on ESRRG in retinoblastoma.

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

PieParty: visualizing cells from scRNA-seq data as pie charts.

Single-cell RNA sequencing (scRNA-seq) has been a transformative technology in many research fields. Dimensional reduction techniques such as UMAP and tSNE are used to visualize scRNA-seq data in two or three dimensions for cells to be clustered in biologically meaningful ways. Subsequently, gene expression is frequently mapped onto these plots to show the distribution of gene expression across the plots, for instance to distinguish cell types. However, plotting each cell with only a single color leads to repetitive and unintuitive representations. Here, we present PieParty, which allows scRNA-seq data to be plotted such that every cell is represented as a pie chart, and every slice in the pie charts corresponds to the gene expression of a single gene. This allows for the simultaneous visualization of the expression of multiple genes and gene networks. The resulting figures are information dense, space efficient, and highly intuitive. PieParty is publicly available on GitHub at https://github.com/harbourlab/PieParty.

Analytical Validation and Performance of a 7-Gene Next-Generation Sequencing Panel in Uveal Melanoma.

INTRODUCTION: Gene expression profiling (GEP) is widely used for prognostication in patients with uveal melanoma (UM). Because biopsy tissue is limited, it is critical to obtain as much genomic information as possible from each sample. Combined application of both GEP and next-generation sequencing (NGS) allows for analysis of RNA and DNA from a single biopsy sample, offers additional prognostic information, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of 7 genes commonly mutated in UM. METHODS: One hundred five primary UM tumors were analyzed, including 37 formalin-fixed paraffin-embedded (FFPE) and 68 fine-needle aspiration biopsy specimens. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of >500X per region of interest. RESULTS: The 7-gene panel achieved a positive percent agreement of 100% for detection of both single-nucleotide variants and insertions/deletions, with a technical positive predictive value of 98.8% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the assay's reproducibility and repeatability. DISCUSSION/CONCLUSION: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small-gauge needle biopsy sample or FFPE specimen.

Impact of Genetic Ancestry on Prognostic Biomarkers in Uveal Melanoma.

Uveal melanoma (UM) is the most common cancer of the eye and leads to metastatic death in up to half of patients. Genomic prognostic biomarkers play an important role in clinical management in UM. However, research has been conducted almost exclusively in patients of European descent, such that the association between genetic admixture and prognostic biomarkers is unknown. In this study, we compiled 1381 control genomes from West African, European, East Asian, and Native American individuals, assembled a bioinformatic pipeline for assessing global and local ancestry, and performed an initial pilot study of 141 UM patients from our international referral center that manages many admixed individuals. Global and local estimates were associated with genomic prognostic determinants. Expression quantitative trait loci (eQTL) analysis was performed on variants found in segments. Globally, after correction for multiple testing, no prognostic variable was significantly enriched in a given ancestral group. However, there was a trend suggesting an increased proportion of European ancestry associated with expression of the PRAME oncogene (q = 0.06). Locally enriched European haplotypes were associated with the poor prognosis class 2 gene expression profile and with genes involved in immune regulation (q = 4.7 × 10-11). These findings reveal potential influences of genetic ancestry on prognostic variables, implicate immune genes in prognostic differences based on ancestry, and provide a basis for future studies of admixed patients with UM using rigorous genetic ancestry methodology.

Single-cell analysis reveals new evolutionary complexity in uveal melanoma.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.

Intraocular Metastasis in Unilateral Multifocal Uveal Melanoma Without Melanocytosis or Germline BAP1 Mutations.

Importance: There has been speculation on the pathogenesis of unilateral multifocal uveal melanoma, but there remains no convincing explanation. Genetic analysis suggests that unilateral multifocal uveal melanoma may represent intraocular metastasis with increased risk of systemic metastasis. Objective: To evaluate the pathogenesis of unilateral multifocal uveal melanoma. Design, Setting, and Participants: This clinical case series was conducted in tertiary academic ocular oncology referral centers and included patients with unilateral multifocal uveal melanoma. Main Outcomes and Measures: Gene expression and mutation profiling of tumor samples. Results: Four patients (all male; age range, 54-77 years) who were diagnosed with uveal melanoma were treated with plaque brachytherapy, and subsequently developed a second discrete uveal melanoma in the same eye were included. None demonstrated ocular or oculodermal melanocytosis. All 8 tumors available for analysis exhibited class 2 gene expression profiles. In all 4 cases, the initial and subsequent tumors were available for targeted DNA sequencing and identical driver mutations were present in both tumors. Data were collected from September 2015 to August 2018. Conclusions and Relevance: Unilateral multifocal uveal melanoma in the absence of ocular melanocytosis appears to occur preferentially in tumors with the class 2 gene expression profile and a BRCA1-associated protein 1 gene (BAP1) mutation. The presence of identical BAP1 mutations in multiple tumors in the same eye in the absence of a germline BAP1 mutation suggests intraocular metastasis rather than independent primary tumors. These findings indicate that the first site of metastasis can be within the eye itself and suggest that patients with unilateral multifocal uveal melanoma may be at increased risk of systemic metastasis.

Intraocular Dissemination of Uveal Melanoma Cells Following Radiotherapy: Evolving Management Over the Past Decade.

BACKGROUND AND OBJECTIVE: To describe the presentation and the authors' evolving management strategy for intraocular dissemination of uveal melanoma cells following radiotherapy during the past decade. PATIENTS AND METHODS: Patients with uveal melanoma who developed intraocular dissemination of pigmented cells following radiotherapy. Histopathology was available in two cases. RESULTS: Four patients underwent treatment for progressive intraocular dissemination of uveal melanoma cells at 9 to 41 months following I-125 plaque radiotherapy (three patients) or proton beam radiotherapy (one patient). Treatments included primary enucleation (one patient), vitrectomy followed later by enucleation (one patient), and vitrectomy followed by intravitreal chemotherapy (two patients). Enucleated eyes demonstrated diffuse invasion of intraocular tissues by viable melanoma cells. No patient has developed systemic metastasis to date. CONCLUSIONS: Intraocular dissemination of pigmented cells following radiotherapy for uveal melanoma should raise suspicion for viable invasive melanoma cells. Prompt vitrectomy with intravitreal chemotherapy can be effective in avoiding enucleation in selected cases. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:573-579.].

BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas.

PURPOSE: The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells. RESULTS: Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development. CONCLUSIONS: This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.

Genomic evolution of uveal melanoma arising in ocular melanocytosis.

Ocular melanocytosis is the most important predisposing condition for the eye cancer uveal melanoma (UM). Here, we present a patient who developed UM arising within ocular melanocytosis who was treated with enucleation (eye removal), which provided an invaluable opportunity to interrogate both the UM and adjacent uveal tissue containing the melanocytosis using whole-exome and deep-targeted sequencing. This analysis revealed a clonal PLCB4 mutation in the melanocytosis, confirming that this is indeed a neoplastic condition and explaining why it predisposes to UM. This mutation was present in 100% of analyzed UM cells, indicating that a PLCB4-mutant cell gave rise to the UM. The earliest aberrations specific to the tumor were loss of Chromosomes 1p, 3, and 9p, which were present in virtually all tumor cells. A mutation in BAP1 arose later on the other copy of Chromosome 3 in a tumor subclone, followed by a gain of Chromosome 8q. These findings provide a mechanistic explanation for the well-known clinical association between ocular melanocytosis and UM by showing that this predisposing condition introduces the first "hit" and thereby increases the stochastic likelihood of acquiring further aberrations leading to UM.

Gene Expression Profiling and PRAME Status Versus Tumor-Node-Metastasis Staging for Prognostication in Uveal Melanoma.

PURPOSE: To compare the prognostic accuracy of gene expression profiling (GEP) combined with PRAME status vs the clinical Tumor-Node-Metastasis (TNM) staging in patients with uveal melanoma (UM). DESIGN: Retrospective cohort study. METHODS: The study included 240 consecutive patients with UM. Tumors were assessed for GEP status (Class 1 or Class 2) using a validated 15-gene assay and PRAME expression status using quantitative polymerase chain reaction. TNM staging was according to the American Joint Committee on Cancer 8th edition. Statistical analysis included univariate and multivariate Cox proportional hazard models. Metastasis was the primary endpoint. RESULTS: GEP was Class 1 in 128 (53.3%) cases and Class 2 in 112 (46.7%) cases. PRAME status was negative in 157 (65.4%) cases and positive in 83 (34.6%) cases. TNM was stage I in 26 (10.8%) cases, IIA in 67 (27.9%) cases, IIB in 50 (20.8%) cases, IIIA in 59 (24.6%) cases, and IIIB in 38 (15.8%) cases. Metastatic disease was detected in 59 (24.6%) cases after median follow-up of 29 months (mean 42 months; range 1-195 months). Variables associated with metastasis included (in order of decreasing significance): GEP class (P = 1.5 × 10-8), largest basal tumor diameter (P = 2.5 × 10-6), PRAME status (P = 2.6 × 10-6), and TNM stage (P = 3.7 × 10-6). The prognostic accuracy of an optimized 3-category GEP/PRAME model (P = 8.6 × 10-14) was superior to an optimized TNM model (P = 1.3 × 10-5). CONCLUSIONS: In UM, molecular prognostic testing using GEP and PRAME provides prognostic accuracy that is superior to TNM staging.

Punctuated evolution of canonical genomic aberrations in uveal melanoma.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.

Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas.

BACKGROUND: We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. RESULTS: Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS: Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. CONCLUSIONS: PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Driver Mutations in Uveal Melanoma: Associations With Gene Expression Profile and Patient Outcomes.

IMPORTANCE: Frequent mutations have been described in the following 5 genes in uveal melanoma (UM): BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. Understanding the prognostic significance of these mutations could facilitate their use in precision medicine. OBJECTIVE: To determine the associations between driver mutations, gene expression profile (GEP) classification, clinicopathologic features, and patient outcomes in UM. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of patients with UM treated by enucleation by a single ocular oncologist between November 1, 1998, and July 31, 2014. MAIN OUTCOMES AND MEASURES: Clinicopathologic features, patient outcomes, GEP classification (class 1 or class 2), and mutation status were recorded. RESULTS: The study cohort comprised 81 participants. Their mean age was 61.5 years, and 37% (30 of 81) were female. The GEP classification was class 1 in 35 of 81 (43%), class 2 in 42 of 81 (52%), and unknown in 4 of 81 (5%). BAP1 mutations were identified in 29 of 64 (45%), GNAQ mutations in 36 of 81 (44%), GNA11 mutations in 36 of 81 (44%), SF3B1 mutations in 19 of 81 (24%), and EIF1AX mutations in 14 of 81 (17%). Sixteen of the mutations in BAP1 and 6 of the mutations in EIF1AX were previously unreported in UM. GNAQ and GNA11 mutations were mutually exclusive. BAP1, SF3B1, and EIF1AX mutations were almost mutually exclusive with each other. Using multiple regression analysis, BAP1 mutations were associated with class 2 GEP and older patient. EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement. SF3B1 mutations were associated with younger patient age. GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter. GNA11 mutations were not associated with any of the analyzed features. Using Cox proportional hazards modeling, class 2 GEP was the prognostic factor most strongly associated with metastasis (relative risk, 9.4; 95% CI, 3.1-28.5) and melanoma-specific mortality (relative risk, 15.7; 95% CI, 3.6-69.1) (P < .001 for both). After excluding GEP class, the presence of BAP1 mutations was the factor most strongly associated with metastasis (relative risk, 10.6; 95% CI, 3.4-33.5) and melanoma-specific mortality (relative risk, 9.0; 95% CI, 2.8-29.2) (P < .001 for both). CONCLUSIONS AND RELEVANCE: BAP1, SF3B1, and EIF1AX mutations occur during UM tumor progression in an almost mutually exclusive manner and are associated with different levels of metastatic risk. These mutations may have value as prognostic markers in UM.

PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma.

PURPOSE: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors. EXPERIMENTAL DESIGN: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR. RESULTS: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003). CONCLUSIONS: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM.

Microincisional vitrectomy for retinal detachment in I-125 brachytherapy-treated patients with posterior uveal malignant melanoma.

PURPOSE: To analyze functional and anatomical outcomes following 23/25(+) gauge microincisional pars plana vitrectomy surgery (MIVS) in patients with radiation-related retinal detachment after successful 125-iodine (I-125) brachytherapy treatment for malignant uveal melanoma. PATIENTS AND METHODS: Retrospective case series of 102 consecutive eyes of 102 patients with history of uveal melanoma treated with I-125 brachytherapy that underwent MIVS at the Bascom Palmer Eye Institute. All cases were evaluated for surgical complications and local tumor control. Extended follow-up included Snellen's best-corrected visual acuity, intraocular pressure evaluation, quantitative echography, indirect ophthalmoscopy, and fundus imaging with optical coherence tomography/wide-field photography. RESULTS: All patients had radiation-related complications, including retinal detachment (102 eyes), vasculopathy (91 eyes), optic neuropathy (32 eyes), and/or vitreous hemorrhage (8 eyes). Sixty-seven patients had vitreoretinal traction. Average follow-up after MIVS was 19.5 months, and from plaque removal was 57.7 months. Interval from plaque to MIVS was 38.1 months. Initial visual acuity was 20/258, which improved to 20/101 at 1 month, 20/110 at 3 months, 20/116 at 6 months, and 20/113 at 12 months (P < 0.05). No eyes required enucleation. Melanoma-related mortality was 0.9% (1/102). There was no intra- or extraocular tumor dissemination, and no tumor recurrence. CONCLUSION: MIVS was effective in improving visual function and anatomy in patients with radiation-related retinal detachment. Tumors decreased in size and there was no evidence of recurrence or tumor dissemination. This combined procedure addresses the modifiable causes of visual loss in patients with previously treated malignant uveal melanoma and has the potential to enhance their visual function.

Intravitreal injection analysis at the Bascom Palmer Eye Institute: evaluation of clinical indications for the treatment and incidence rates of endophthalmitis.

OBJECTIVE: To report the incidence of endophthalmitis, in addition to its clinical and microbiological aspects, after intravitreal injection of vascular-targeting agents. METHODS: A retrospective review of a consecutive series of 10,142 intravitreal injections of vascular targeting agents (bevacizumab, ranibizumab, triamcinolone acetonide, and preservative-free triamcinolone acetonide) between June 1, 2007 and January 31, 2010, performed by a single service (TGM) at the Bascom Palmer Eye Institute. RESULTS: One case of clinically-suspected endophthalmitis was identified out of a total of 10,142 injections (0.009%), presenting within three days of injection of bevacizumab. The case was culture-positive for Staphylococcus epidermidis. Final visual acuity was 20/40 after pars plana vitrectomy surgery. CONCLUSIONS: In this series, the incidence of culture-positive endophthalmitis after intravitreal injection of vascular agents in an outpatient setting was very low. We believe that following a standardized injection protocol, adherence to sterile techniques and proper patient follow-up are determining factors for low incidence rates.

L-DOPA is an endogenous ligand for OA1.

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.