Bruno F. Fernandes

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.

Incidence of Melanocytic Lesions of the Conjunctiva in a Review of 10 675 Ophthalmic Specimens

During the study period, 10 675 human ophthalmic specimens were received at The Henry C. Witelson Ophthalmic Pathology Laboratory and Registry, McGill University, Montreal, Canada. Of those, 271 were conjunctival lesions (2.5%), with 101 being classified as melanocytic: 50 (49.5%) nevi, 36 (35.6%) primary acquired melanoses, and 15 (14.9%) melanomas. After exclusion of referred cases, 85 lesions were included in the study: 44 (51.7%) nevi, 33 (38.8%) primary acquired melanoses, and 8 (9.4%) melanomas. The most prevalent location was the bulbar conjunctiva. Conjunctival melanomas were most commonly found in an older age group than primary acquired melanosis or nevi. Conjunctival nevi were subdivided into compound (32.9%), subepithelial (16.4%), and junctional (2.3%). Primary acquired melanosis were further classified into primary acquired melanosis with atypia (8.2%) and primary acquired melanosis without atypia (30.5%). Primary acquired melanoses was the predisposing lesion in 75% of the cases of melanoma. In our sample, referral bias could alter the distribution of conjunctival pigmented lesions, with a shift toward the malignant end.

Expression of the metastasis suppressor gene KISS1 in uveal melanoma

PurposeUveal melanoma (UM) is the most common primary malignant intraocular tumour in adults. Forty-five percent of UM patients develop metastasis within 15 years of initial diagnosis. KISS1, a human metastasis suppressor gene, has been reported to play a role in various human malignancies. The purpose of this study was to investigate the expression of KISS1 in UM and its potential value as a prognostic marker.MethodsThirty-seven cases of paraffin-embedded human UM specimens were immunostained with a KISS1 antibody. Clinical–pathological data were obtained. The relationship between the clinical–pathological data and the expression of KISS1 was evaluated. Moreover, the survival rates of the patients were also assessed. Five UM cell lines (92.1, OCM-1, MKTBR, UW1 and SP6.5) were assayed for KISS1 expression. In addition, real-time PCR was used to determine mRNA levels of KISS1and its receptor GPR54in these cell lines.ResultsThe immunohistochemical results of KISS1 expression displayed cytoplasmic staining in 84% of UM specimens. Low KISS1 expression was associated with a higher risk of metastatic disease (P<0.05). Furthermore, we found that KISS1 was expressed in all five UM cells lines. Real-time PCR analysis confirmed the presence of both KISS1and its receptor GPR54in all five human UM cell lines.ConclusionsTo the best of our knowledge, this is the first time that KISS1has been characterized in UM. The correlation between KISS1 expression and UM survival rate suggests an important role for KISS1as a prognostic marker in this particular tumour.

Ciliary body medulloepithelioma: clinical, ultrasound biomicroscopic and histopathologic correlation

A 3‐year‐old girl presented with a distorted pupil and decrease of visual acuity. A ciliary body mass at the superior‐temporal quadrant could be seen at slit‐lamp examination. Ultrasound biomicroscopy revealed a lesion with lobulated surface, arising at the ciliary body, composed of compact and cystic areas. An iridocyclectomy was performed but the tumour recurred after a few months. The eye was then enucleated. Histopathologic evaluation showed sheets and cords of proliferated medullary epithelium. Cystic spaces and Flexner‐Wintersteiner rosettes were also seen. Those findings correlated well with ultrasound biomicroscopic images. The knowledge of echographic characteristics can significantly assist in the diagnosis of medulloepithelioma of the ciliary body.

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.

Immunohistochemical expression of melan-A and tyrosinase in uveal melanoma

Background Melan-A and tyrosinase are new immunohistochemical markers that can be used in the diagnosis of melanocytic lesions. The aim of this study was to investigate the correlation between radiotherapy or clinicohistopathological parameters and the expression of melan-A and tyrosinase in uveal melanoma. Methods Thirty-six enucleated cases of uveal melanoma were studied. The formalin-fixed, paraffin-embedded specimens were immunostained with monoclonal antibodies against melan-A and tyrosinase. The samples were classified as either positive or negative. The chi-square or the Student-t tests were used to test for the correlation of the expression rates of melan-A and tyrosinase with clinico-pathological parameters. Results Melan-A and tyrosinase were positive in 33 (91.7%) and 35 (97.2%) of the specimens, respectively. There was no significant association between the expression of melan-A or tyrosinase and radiotherapy or any clinico-pathological parameter. All specimens were positive for at least one of the immunohistochemical markers. Conclusion To the best of our knowledge this is the first study concluding that the expression of melanocytic markers such as melan-A and tyrosinase is not influenced by radiotherapy or any clinico-pathological parameter. Moreover, when tyrosinase and melan-A are used together, 100% of the formalin-fixed, paraffin-embedded uveal melanoma samples tested positive for one of those markers.

In vitro characterization and inhibition of the CXCR4/CXCL12 chemokine axis in human uveal melanoma cell lines

PurposeThe CXCR4/CXCL12 chemokine axis may play a critical role in guiding CXCR4+ circulating malignant cells to organ specific locations that actively secrete its ligand CXCL12 (SDF-1) such as bone, brain, liver, and lungs. We sought to characterize the presence of the CXCR4/CXCL12 axis in five uveal melanoma (UM) cell lines in vitro. The ability of TN14003, a synthetic peptide inhibitor that targets the CXCR4 receptor complex, to inhibit this axis was also assessed.MethodsImmunocytochemistry was performed against CXCR4 to confirm expression of this chemokine receptor in all five UM cell lines. Flow cytometry was preformed to evaluate CXCR4 cell surface expression on all five UM cell lines. A proliferation assay was also used to test effects TN14003 would have on cellular proliferation. Inhibition of cellular migration by specifically inhibiting the CXCR4/CXCL12 axis with TN14003 was also investigated. The binding efficacy of TN14003 to the CXCR4 receptor was assessed through flow cytometric methods.ResultsThe CXCR4 receptor was present on all five UM cell lines. All five cell lines expressed different relative levels of surface CXCR4. TN14003 did not affect the proliferation of the five cell lines (p > 0.05). All cell lines migrated towards the chemokine CXCL12 at a level greater than the negative control (p < 0.05). All 5 cell lines pre-incubated with TN14003 prevented cellular migration towards chemokine CXCL12 (p < 0.01). TN14003 preferentially binds CXCR4 to native ligand CXCL12.ConclusionInterfering with the CXCR4/CXCL12 axis, using TN14003 was shown to effectively down regulate UM cell migration in vitro. Knowing that UM expresses the CXCR4 receptor, these CXCR4+ cells may be less likely to colonize distant organs that secrete the CXCL12 ligand, if treated with an inhibitor that binds CXCR4. Further studies should be pursued in order to test TN14003 efficacy in vivo.

Choroidal mass as the presenting sign of small cell lung carcinoma

CASE REPORT To report the case of a 58-year-old man with blurred vision and metamorphopsia who had an amelanotic choroidal mass in the right eye as the presenting sign of a small cell lung carcinoma. Systemic screening failed to reveal a tumor elsewhere, and the lesion was initially treated as a primary ocular tumor. Discovery of the primary site was made 10 months after the ocular diagnosis, and the patient was then treated with systemic chemotherapy and local radiation therapy. COMMENTS The ophthalmologist has a crucial role not only in the management of ocular metastases but also in the diagnosis of the primary nonocular malignancies that present as a choroidal mass. The possibility of ocular metastases in patients with choroidal masses should always be considered whether or not there is a diagnosis of cancer elsewhere.

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.