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.

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.

© 2017 American Academy of Ophthalmology 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 describe the natural history and management of a rare case of iris melanoma in a pediatric patient. Observations: A Caucasian female presented with left pupillary abnormalities at age 7, progressive iris changes at age 9, and markedly elevated intraocular pressure with advanced optic nerve cupping at 11 years of age. She was found to have a pigmented lesion overlying her iris and invading her angle. Trans-corneal fine needle aspirate biopsy demonstrated malignant melanoma of the iris. The patient subsequently underwent Iodine-125 plaque brachytherapy for the tumor. Conclusions: and Importance: Early identification and treatment of iris melanoma may be associated with decreased risk of local progression and metastatic disease. Treatment of glaucoma in conjunction with uveal melanoma is complicated by tumor specific considerations, including treatment of the tumor and prevention of metastasis.

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.

We examined liver specimens from 15 patients with uveal melanoma (UM) who had died of their disseminated disease. We found 2 distinct growth patterns of UM metastasis: infiltrative (n = 12) and nodular (n = 3). In the infiltrative pattern, individual UM cells with a CD133+ cancer stem cell–like phenotype were present and formed aggregates of stage I < 50-μm-diameter micrometastases in the sinusoidal spaces. These micrometastases appeared to expand, destroy adjacent hepatocytes, and form stage II 51- to 500-μm-diameter and then stage III > 500μm-diameter metastases, which were encapsulated by collagenized fibrous septae. In the nodular growth pattern, CD133+ melanoma cells aggregated adjacent to portal venules and subsequently appeared to grow and efface the adjacent hepatocytes to form stage II 51- to 500-μm-diameter nodules that surrounded the portal venu le. These avascular nodules appeared to further expand to form stage III > 500-μm-diameter nodules that exhibited vascularization with minimal fibrosis. The tumor stem cell–like phenotype seen in individual UM cells was lost as the tumors progressed. There were CD56+ natural killer cells in sinusoidal spaces and CD3+ lymphocytes in periportal areas. The nodular growth pattern showed UM cells expressing MMP9 and VEGF. UM cells in both above-described growth patterns exhibited variable BAP1 expression. We propose that changes in the liver microenvironment are related to metastatic UM growth. We hypothesize that these changes include immune regulation within the sinusoidal space for the infiltrative pattern and changes in the VEGF/PEDF ratio for the nodular pattern.

Background/Aims: To clarify the pathogenesis of fibrosis in inflammatory orbital diseases, we analysed the gene expression in orbital biopsies and compared our Results: with those reported for idiopathic pulmonary fibrosis. Methods: We collected 140 biopsies from 138 patients (58 lacrimal glands; 82 orbital fat). Diagnoses included healthy controls (n=27), non-specific orbital inflammation (NSOI) (n=61), thyroid eye disease (TED) (n=29), sarcoidosis (n=14) and granulomatosis with polyangiitis (GPA) (n=7). Fibrosis was scored on a 0-3 scale by two experts, ophthalmic pathologists. Gene expression was quantified using Affymetrix U133 plus 2.0 microarray. Results: Within orbital fat, fibrosis was greatest among subjects with GPA (2.75±0.46) and significantly increased in tissue from subjects with GPA, NSOI or sarcoidosis ( p<0.01), but not for TED, compared with healthy controls (1.13±0.69). For lacrimal gland, the average score among controls (1.36±0.48) did not differ statistically from any of the four disease groups. Seventy three probe sets identified transcripts correlating with fibrosis in orbital fat (false discovery rate <0.05) after accounting for batch effects, disease type, age and sex. Transcripts with increased expression included fibronectin, lumican, thrombospondin and collagen types I and VIII, each of which has been reported upregulated in pulmonary fibrosis. Conclusions: A pathologist's recognition of fibrosis in orbital tissue correlates well with increased expression of transcripts that are considered essential in fibrosis. Many transcripts implicated in orbital fibrosis have been previously implicated in pulmonary fibrosis. TED differs from other causes of orbital fat inflammation because fibrosis is not a major component. Marked fibrosis is less common in the lacrimal gland compared with orbital adipose tissue.