Shawn C. Maloney

Molecular Pathways Mediating Liver Metastasis in Patients with Uveal Melanoma

Uveal melanoma arises from melanocytes located in the uveal tract of the eye and is the most common primary intraocular tumor in adults. Metastatic liver disease is the overwhelming cause of death in uveal melanoma patients, with almost 50% of patients developing liver metastases up to 15 years after diagnosis. Most of these patients do not present with any evidence of overt metastasis at the time of initial diagnosis although it is assumed that they have undetectable micrometastases. Currently, there are no therapeutic modalities to prevent or efficiently treat the metastatic disease in uveal melanoma patients. Recent discoveries have shed light on the molecular pathways that may contribute to the progression of liver metastasis. The aim of this review is to describe new insights into the genetic and molecular pathways that may play a role in the development of liver metastases in uveal melanoma patients.

Expression of cyclooxygenase-2 in choroidal neovascular membranes from age-related macular degeneration patients.

Purpose: The objective of this study was to investigate the expression of cyclooxygenase (COX)-2 in human choroidal neovascular membranes. Methods: Paraffin-embedded sections of choroidal neovascular membranes excised from 16 patients with wet age-related macular degeneration were used for this study. Sections were subjected to immunohistochemistry using a monoclonal mouse antihuman COX-2 antibody. Staining was classified as either negative or positive in retinal pigment epithelial cells, vascular endothelial cells, and fibroblasts. Serial sections were stained for vimentin expression to confirm tissue antigenicity. Results: Eleven of 16 (69%) choroidal neovascular membranes stained positive for COX-2 in retinal pigment epithelial cells, with 6 (38%) of these also expressing COX-2 in vascular endothelial cells and 6 (38%) in fibroblasts. None of the sections that were negative for COX-2 in the retinal pigment epithelial cells showed COX-2 expression in the other cell types assessed. There was a statistically significant difference (P = 0.0097) in the mean ages between the COX-2 positive group (65.6 years) and COX-2 negative group (76.8 years) as determined by a two-tailed, unpaired Student’s t-test. Conclusion: The expression of COX-2 in human choroidal neovascular membranes suggests a possible role for this modulator in age-related macular degeneration pathogenesis. The age-dependent expression observed is novel and warrants further investigation.

Current and emerging treatment options for uveal melanoma

Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults, with a 10-year cumulative metastatic rate of 34%. The most common site of metastasis is the liver (95%). Unfortunately, the current treatment of metastatic UM is limited by the lack of effective systemic therapy. Options for the management of the primary intraocular tumor include radical surgery as well as conservative treatments in order to preserve visual acuity. For metastatic disease, several approaches have been described with no standard method. Nevertheless, median survival after liver metastasis is poor, being around 4–6 months, with a 1-year survival of 10%–15%. In this review, the authors summarize current and promising new treatments for UM.

The effect of blue light exposure in an ocular melanoma animal model

BackgroundUveal melanoma (UM) cell lines, when exposed to blue light in vitro, show a significant increase in proliferation. In order to determine if similar effects could be seen in vivo, we investigated the effect of blue light exposure in a xenograft animal model of UM.MethodsTwenty New Zealand albino rabbits were injected with 1.0 × 106 human UM cells (92.1) in the suprachoroidal space of the right eye. Animals were equally divided into two groups; the experimental group was exposed to blue light, while the control group was protected from blue light exposure. The eyes were enucleated after sacrifice and the proliferation rates of the re-cultured tumor cells were assessed using a Sulforhodamine-B assay. Cells were re-cultured for 1 passage only in order to maintain any in vivo cellular changes. Furthermore, Proliferating Cell Nuclear Antigen (PCNA) protein expression was used to ascertain differences in cellular proliferation between both groups in formalin-fixed, paraffin-embedded eyes (FFPE).ResultsBlue light exposure led to a statistically significant increase in proliferation for cell lines derived from intraocular tumors (p < 0.01). PCNA expression was significantly higher in the FFPE blue light treated group when compared to controls (p = 0.0096).ConclusionThere is an increasing amount of data suggesting that blue light exposure may influence the progression of UM. Our results support this notion and warrant further studies to evaluate the ability of blue light filtering lenses to slow disease progression in UM patients.

Choroidal Neovascular Membranes Express Toll-Like Receptor 3

Background: Recent evidence has suggested a role for toll-like receptor 3 (TLR3) in experimental models of age-related macular degeneration (AMD). To date, however, few data exist about TLR3 in human AMD. The purpose of this study was to investigate the expression of TLR3 in human choroidal neovascular (CNV) membranes. Methods: Immunostaining for TLR3 was performed on sections of CNV membranes from 8 AMD patients and eyes from 4 donors without CNV. Results: All CNV membranes expressed TLR3 in retinal pigment epithelial (RPE) cells. One was classified as having strong intensity, 5 as having moderate intensity and 2 as having weak intensity. All cases had ≧30% of the RPE cells staining for TLR3, ranging from 30 to 90%. No expression of TLR3 was observed in vascular endothelial cells or fibroblasts in any CNV membrane. In the donor eyes, the RPE cells near the ora serrata stained stronger than those at the posterior pole, where no staining was observed in 3 out of 4 cases. Conclusion: TLR3 was found in all CNV membranes and was expressed exclusively in RPE cells. The observed difference in RPE staining for TLR3 in donor eyes and CNV membranes suggests a possible role for this receptor in human neovascular AMD.

Amfenac increases the radiosensitivity of uveal melanoma cell lines

PurposeTo evaluate the proliferation rates of five human uveal melanoma (UM) cell lines after treatment with amfenac, a cyclooxygenase (COX)-2 inhibitor, and subsequent radiation exposure.MethodsFive human UM cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1) and one human fibroblast cell line (BJ) were incubated with amfenac. Treated and non-treated cell lines were then exposed to various doses of γradiation: 0, 2, 4, 6, and 8 Gy. Sulphorhodamine-B assay was used to assess proliferation rates 48 h post-radiation.ResultsTreatment of UM cell lines with amfenac prior to radiation led to a marked reduction in proliferation rates. This difference was statistically significant in all cell lines at every radiation dose (P<0.005), with the exception of 92.1 at 2 Gy (P=0.157). Fibroblasts treated with amfenac showed significantly higher proliferation rates after 2 and 8 Gy, with no significant differences at 0, 4, and 6 Gy.ConclusionsThe radiosensitivity of UM cell lines was increased by the administration of amfenac, the active metabolite of nepafenac. There appears to be a radioprotective effect of amfenac on human fibroblasts. The topical administration of nepafenac may decrease tumour recurrence and radiation-induced complications while broadening the indications for radiotherapy by treating larger tumours.

Expression of SIRT1 and DBC1 in Developing and Adult Retinas

Sirtuin 1 (SIRT1) is a deacetylase that can regulate various biological processes via repression of transcription. Its activity has been linked to the differentiation of neural progenitor cells, although little is known about its function during retinal development. The study described herein was undertaken to evaluate the expression of SIRT1 and its innate inhibitor, DBC1, in retinal tissues and progenitor cells. We found both SIRT1 and DBC1 to be widely expressed in mouse and human retinas, with subtle differences in subcellular distribution of each protein. We further demonstrate that nuclear-localized SIRT1 is only seen in human-derived retinal progenitor cells and not in adult retinas, suggesting that this nuclear localization may be important in retinal development. Moreover, we observed cytoplasmic DBC1 in a subset of progenitor cells as well as in mature ganglion cells, indicating that the progenitor cell subset, which was comprised predominantly of small cells, may represent a population of ganglion cell precursors. Collectively, the data presented in this study provide support for SIRT1 and DBC1 as regulators of retinal development and normal retinal physiology.

Imatinib Mesylate Alters the Expression of Genes Related to Disease Progression in an Animal Model of Uveal Melanoma

Imatinib mesylate (IM) is a compound that inhibits both BCR-ABL tyrosine kinase and c-kit receptors. Tyrosine kinases are important in cellular signaling and mediate major cellular processes such as proliferation, differentiation, apoptosis, attachment, and migration. Twenty-six albino rabbits were injected with 1 × 106 human uveal melanoma (UM) cells (92.1) into the suprachoroidal space. Animals were immunosuppressed (cyclosporin A) over the course of the 12-week experiment and divided into two groups (n = 13). The experimental group received IM once daily by gavage while the control group received a placebo. One animal per group was sacrificed every week after the 2nd week. Upon necropsy, organs were harvested for histopathological examination. Cells from the primary tumors were recultured and tested in proliferation and invasion assays. A PCR array was used to investigate the differences in expression of 84 genes related to tumor metastasis. In the treated group, 4 rabbits developed intraocular tumors, with an average largest tumor dimension (LTD) of 2.5 mm and 5 animals reported metastatic disease. Whereas 6 rabbits in the control group developed intraocular tumors, with an average LTD of 5.8 mm and 6 animals reported metastatic disease. The recultured cells from the treated group demonstrated lower proliferation rates and were less invasive (p < 0.001 The PCR array showed differences in expression of genes related to metastasis. Notably, there was 290-fold increase in SERPINB5, a tumor suppressor gene, and a 10-fold higher expression of KISS1, a metastasis suppressor gene, in the treated group. Proangiogenic genes such as VEGFA, PDGFA and PDGFB were downregulated in the treated group. To the best of our knowledge, this is the first report detailing the altered expression of specific genes in UM cells after treatment with IM.

Prostate-specific membrane antigen is undetectable in choroidal neovascular membrane

Background Choroidal neovascular membrane (CNVM) is one of the leading causes of severe visual loss and is often associated with age-related macular degeneration (AMD). Various modalities of treatment, including photocoagulation and surgery, are being considered as options, but with limited success. Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein expressed in benign and malignant prostatic tissues, in some non-prostatic tissues, and in the endothelium of tumor-associated neovasculature of non-prostatic neoplasm. Some studies have suggested that the expression of PSMA is restricted to endothelium from tumor-associated neovasculature and might be stimulated by some tumor-secreted angiogenic factors. However, no previous study demonstrating PSMA expression in non-related tumor neovasculature, such as CNVM, has been performed to date. Furthermore, demonstration of PSMA expression in CNVM in AMD patients could reveal a novel target for antineovascular therapy. The purpose of this study was to evaluate the immunohistochemical expression of PSMA in CNVM from AMD. Methods Immunohistochemical analysis, with a standard avidin-biotin complex technique, was performed using an anti-PSMA mouse monoclonal antibody in 30 specimens of surgically excised CNVM from AMD patients. Antibody to an endothelial cell specific marker, factor VIII, was used to confirm the location of the endothelial cells. Results The angiogenic microvessels of the 30 cases demonstrated negative staining to PSMA while factor VIII was expressed in all cases. Seventy-five percent of the secretory-acinar epithelium of the prostatic hyperplasia specimen stained positive, confirming that the immunohistochemical technique was correctly performed. Conclusion The absence of PSMA expression in non-tumoral neovasculature supports the theory, previously suggested, that endothelial cell PSMA expression may be stimulated by one or more tumor-secreted angiogenic factors. Angiogenesis is very important in neoplasia and the endothelial expression of PSMA in tumor-associated neovasculature may represent a target for antineovasculature-based therapy. The absence of PSMA expression in CNVM suggests that PSMA may not be a potential target for antineovasculature-based therapy.

Expression of SIRT2 and SIRT6 in Retinoblastoma

Purpose: SIRT2 and SIRT6 are members of the sirtuin family and are associated with cancer development and progression in certain tumours, but their expression in retinoblastoma has not been studied. The primary objective of our study was to determine the expression of SIRT2 and SIRT6 in human retinoblastoma cases. Methods: Eighteen formalin-fixed paraffin-embedded blocks of retinoblastoma cases from the Ocular Pathology Registry at the Henry C. Witelson Ocular Pathology Laboratory were obtained, classified and immunostained for SIRT2 and SIRT6 using mouse monoclonal antibodies. Results: Sixteen cases were poorly differentiated retinoblastoma cases. SIRT2 and SIRT6 were expressed in all cases of retinoblastoma although differences in the staining intensity were found between cases. SIRT2 and SIRT6 expression was also observed in various normal structures of the remaining ocular tissue. Conclusions: SIRT2 and SIRT6 are expressed in retinoblastoma, as well as in some normal ocular structures. While precise roles of these proteins must still be determined in retinoblastoma, their expression profiles suggest that further functional studies of both SIRT2 and SIRT6 should be pursued in this cancer.