Purpose: To evaluate outcomes of choroidal melanoma patients treated with 125 I or 103 Pd plaque brachytherapy. Methods and Materials: From 1993 to 2012, our institution treated 160 patients with 103 Pd (56.1%) and 125 patients with 125 I (43.9%) plaque brachytherapy. Tumor outcomes, visual acuity (VA), and toxicity were compared. Multivariate analyses (MVAs) and propensity score analysis were used to help address differences in baseline characteristics. Results: Median followup was longer for 125 I patients, 52.7 vs. 43.5 months (p < 0.01). At baseline, 103 Pd patients had lower rates of VA worse than 20/200 (4.4% vs. 16%, p = 0.002), T3–T4 tumors (17.5% vs. 32.8%, p = 0.03), and transpupillary thermotherapy use (3.1% vs. 9.6%, p = 0.001). Both 103 Pd and 125 I provided > 90% 3-year overall survival and > 93% 5-year secondary enucleation-free survival. On MVA, radionuclide was not predictive for tumor outcomes. A higher percentage maintained vision better than 20/40 with 103 Pd (63% vs. 35%, p = 0.007) at 3 years. MVA demonstrated 103 Pd radionuclide (odds ratio [OR]: 2.12, p = 0.028) and tumor height ≤5 mm (OR: 2.78, p = 0.017) were associated with VA better than 20/40. Propensity score analysis matched 23 125 I with 107 103 Pd patients. 103 Pd continued to predict better VA at 3 years (OR: 8.10, p = 0.014). On MVA for the development of VA worse than 20/200 or degree of vision loss, radionuclide was not significant. Lower rates of radiation retinopathy were seen with 103 Pd than 125 I (3 years: 47.3% vs. 63.9%, p = 0.016), with radionuclide significant in MVA. Conclusions: Both 125 I and 103 Pd achieve excellent tumor control. An increased probability of long-term VA better than 20/40 and reduced risk of radiation retinopathy is associated with 103 Pd.

Integrin αν β3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αν β3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αν β3 -expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αν β3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics.

Background Although thyroid eye disease is a common complication of Graves’ disease, the pathogenesis of the orbital disease is poorly understood. Most authorities implicate the immune response as an important causal factor. We sought to clarify pathogenesis by using gene expression microarray. Methods An international consortium of ocular pathologists and orbital surgeons contributed formalin fixed orbital biopsies. RNA was extracted from orbital tissue from 20 healthy controls, 25 patients with thyroid eye disease (TED), 25 patients with nonspecific orbital inflammation (NSOI), 7 patients with sarcoidosis and 6 patients with granulomatosis with polyangiitis (GPA). Tissue was divided into a discovery set and a validation set. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays which include 54,000 probe sets. Results Principal component analysis showed that gene expression from tissue from patients with TED more closely resembled gene expression from healthy control tissue in comparison to gene expression characteristic of sarcoidosis, NSOI, or granulomatosis with polyangiitis. Unsupervised cluster dendrograms further indicated the similarity between TED and healthy controls. Heat maps based on gene expression for cytokines, chemokines, or their receptors showed that these inflammatory markers were associated with NSOI, sarcoidosis, or GPA much more frequently than with TED. Conclusion This is the first study to compare gene expression in TED to gene expression associated with other causes of exophthalmos. The juxtaposition shows that inflammatory markers are far less characteristic of TED relative to other orbital inflammatory diseases.

Treatment of many posterior-segment ocular indications would benefit from improved targeting of drug delivery to the back of the eye. Here, we propose the use of iontophoresis to direct delivery of negatively charged nanoparticles through the suprachoroidal space (SCS) toward the posterior pole of the eye. Injection of nanoparticles into the SCS of the rabbit eye ex vivo without iontophoresis led to a nanoparticle distribution mostly localized at the site of injection near the limbus and <15% of nanoparticles delivered to the most posterior region of SCS (>9 mm from the limbus). Iontophoresis using a novel microneedle-based device increased posterior targeting with >30% of nanoparticles in the most posterior region of SCS. Posterior targeting increased with increasing iontophoresis current and increasing application time up to 3 min, but further increasing to 5 min was not better, probably due to the observed collapse of the SCS within 5 min after injection ex vivo. Reversing the direction of iontophoretic flow inhibited posterior targeting, with just ~5% of nanoparticles reaching the most posterior region of SCS. In the rabbit eye in vivo, iontophoresis at 0.14 mA for 3 min after injection of a 100 μL suspension of nanoparticles resulted in ~30% of nanoparticles delivered to the most posterior region of the SCS, which was consistent with ex vivo findings. The procedure was well tolerated, with only mild, transient tissue effects at the site of injection. We conclude that iontophoresis in the SCS using a microneedle has promise as a method to target ocular drug delivery within the eye, especially toward the posterior pole.

BACKGROUND: Metastases account for 90% of all cancer-related deaths, becoming a therapeutic problem. Approximately 50% of all uveal melanoma (UM) patients will develop metastases, mainly in the liver. Post-mortem analyses of livers from metastatic UM patients showed two different metastatic growth patterns: infiltrative and nodular. The infiltrative pattern exhibits tumor infiltration directly to the hepatic lobule and minimal angiogenesis. The nodular pattern shows clusters of tumor cells around the portal venules that efface the liver parenchyma. We recently demonstrated Natural Killer (NK) cells play a pivotal role in the control of hepatic metastases and the pigment epithelial-derived factor (PEDF) controls angiogenesis in the liver using our established ocular melanoma animal model. In this study we investigated the role of NK cells and PEDF in the development of metastatic growth patterns, as this can contribute to the development of novel therapeutics specific towards each growth pattern. METHODS: We utilize our established ocular melanoma animal model by inoculation of B16-LS9 melanoma cells into C57BL/6 J mice (WT), anti-asialo GM1-treated C57BL/6 J mice (NK-depleted), and PEDF-/- C57BL/6 J mice. Three weeks after inoculation we evaluated the metastatic growth patterns and stratified them based of the numbers of tumor cells. To evaluate angiogenesis the mean vascular density (MVD) was calculated. The immune compartment of the liver was analyzed by flow cytometry. RESULTS: Our in vivo work showed two distinct metastatic growth patterns, the infiltrative and nodular, recapitulating the post-mortem analyses on human liver tissue. We discovered NK cells control the infiltrative growth. In contrast, PEDF controlled anti-angiogenic responses, showing higher MVD values compared to NK-depleted and WT animals. The myeloid lineage, comprised of monocytes, macrophages, and myeloid-derived suppressor cells, was reduced in the absence of NK cells or PEDF. CONCLUSIONS: Our animal model recapitulates the metastatic growth patterns observed in the human disease. We demonstrated a role for NK cells in the development of the infiltrative growth pattern, and a role for PEDF in the development of the nodular pattern. The understanding of the complexity associated with the metastatic progression has profound clinical implications in the diagnostic and disease-management as we can develop and direct more effective therapies.

Purpose: Reduced nuclear expression of BRCA1 associated protein 1 (BAP-1) is associated with a high risk for metastasis in uveal melanoma. Manual assessment of the expression level may face issues with interobserver reproducibility. This could be improved with digital image analysis (DIA). Methods: Thirty enucleated eyes with uveal melanoma from the Emory Eye Center (Atlanta, GA; years 2009–2017) were included and stained with BAP-1. Retrospective data on patient and tumor characteristics were retrieved. Patients were randomized to a training or validation cohort. Their tumor sections were digitally scanned and scored for percentage of BAP-1–positive cells with the QuPath Bioimage analysis software. Results: Interobserver concordance was 75% (Cohen’s k 0.52) with manual BAP-1 scoring and 88% to 94% with DIA (Cohen’s k 0.75–0.88). Positive and negative predictive values for metastasis were 90% and 100% with DIA, 80% and 86% with manual scoring, and 78% and 88% with gene expression class 2. In binary logistic regression, manual and DIA of BAP-1 and gene expression class 2 were associated with metastasis, but none retained significance in multiple regression. Metastasis-free survival was significantly shorter with low BAP-1 expression as defined by DIA (log-rank P = 0.02), but not with manual scoring (log-rank P = 0.36) or with gene expression class 2 (log-rank P = 0.17). Conclusions: DIA of BAP-1 is a competitive alternative to manual assessment as well as gene expression profiling in prognostication of enucleated specimens with uveal melanoma. Translational Relevance: The emerging scope for automatization of qualified diagnostic tasks is applied to uveal melanoma.

Purpose: To examine the BRCA1-associated protein-1 (BAP1) expression of primary uveal melanomas without and with metastasis, and to analyze the correlation between the BAP1 immunoreactivity of primary uveal melanoma and other clinicopathologic features. Design: Retrospective case series. Participants: Forty patients with uveal melanoma (mean age, 57.98±14.75 years) were included in this analysis, of whom 20 had no metastatic disease and 20 had metastasis. Methods: Medical records and histology slides of patients with primary uveal melanoma treated by enucleation were reviewed. BAP1 expression was evaluated by immunohistochemical staining of formalin-fixed, paraffin-embedded sections. Immunoreactivity in the nucleus and cytoplasm were graded by estimating the percentage of primary tumor cells showing a positive staining of their nucleus or cytoplasm per 1 high-power field 200× (grades 0–3). Main Outcome Measures: Tumor size, histologic features, nuclear and cytoplasmic BAP1 immunoreactivity grade, and patient outcome, including development of metastasis. Results: Significantly lower (P = 0.025) nuclear BAP1 immunoreactivity was observed in the metastatic melanoma group. Greater tumor thickness, basal diameter, and more advanced TNM stage were associated with an increased odds ratio of developing metastasis (P < 0.05). In addition, tumors with a higher proportion of cells expressing nuclear BAP1 had decreased odds of developing metastatic disease in a multivariate model (P = 0.042). Metastasis-free survival was significantly longer in patients with uveal melanoma with high nuclear BAP1 stain (P = 0.004). Conclusions: Time to metastasis differs in patients with primary uveal melanoma with different grades of nuclear BAP1 immunoreactivity. Nuclear BAP1 stain is the only significant independent predictor of metastatic disease in this study. Our data support the role of BAP1 immunohistochemical staining of primary uveal melanoma to evaluate metastatic risk.

PURPOSE. To evaluate BRAF, NRAS, and GNAQ mutations in surgical specimens of common and blue conjunctival melanocytic nevi. METHODS. Surgical specimens from 25 conjunctival melanocytic nevi (23 common and 2 blue) of 25 patients were evaluated. All common nevi were analyzed immunohistochemically for the expression of BRAF V600E or NRAS Q61R. One lesion with negative immunoreactivity and for all blue nevi, a hybridization capture-based next-generation sequencing method was employed for mutation analysis. For common nevi, genetic features were compared with clinical and histopathologic findings. Continuous variables (age at excision and largest basal diameter) were compared with a Students’s t-test and all categoric variables were compared with Fisher’s Exact Test. RESULTS. Of common melanocytic nevi, 9 (39.1%) were immunoreactive for NRASQ61R and 13 (56.5%) were immunoreactive for BRAFV600E. One common nevus, which was immunonegative for both BRAFV600E and NRASQ61R was found to harbor an NRASQ61K mutation by sequence analysis. Patients with NRAS-mutated nevi were more likely to report occurrence of the lesion prior to 18-years old and more likely to have intrinsic cysts. The mean largest basal diameter was 6.0 and 3.5 mm for NRAS- and BRAF-immunoreactive lesions, respectively (P = 0.003). GNAQ mutations were identified in each of the two blue nevi of this study. CONCLUSIONS. These findings document that common conjunctival melanocytic nevi have mutually exclusive mutations in BRAF and NRAS. The two conjunctival blue nevi harbored GNAQ mutations. This suggests the driver mutations of conjunctival nevi are similar to those of nevi of the skin. At the molecular level, conjunctival nevi appear more like cutaneous nevi than choroidal nevi.

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd3+ binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM-1s-1 at 1.5 T and 25 °C) and strong Gd3+ selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.