Patrick Logan

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.

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.

Histopathological study of 49 cases of keratoconus

Aims: Keratoconus is a bilateral and asymmetrical corneal ectasia. The pathophysiology of this disorder has yet to be fully elucidated. The purpose of our study was to document the prevalence of the most common morphological features of keratoconic corneas. Methods: A retrospective analysis of 49 cases diagnosed as keratoconus between 2001 and 2006 was undertaken. Histopathological reports were reviewed to obtain data such as age and gender. Specimens were fixed in 10% buffered paraformaldehyde solution for 24 h, bisected through the centre of the button, and embedded in paraffin. Sections were stained with haematoxylin and eosin (H&E) and periodic acid‐Schiff (PAS) for light microscopic examination. Results: The studied group was composed of 29 men and 20 women. Age at the time of the penetrating keratoplasty was 39 ± 14 years (mean ± standard deviation). Forty of the 49 specimens (82%) presented with epithelial thinning. Other common features of keratoconus included breaks in Bowmans layer in 35 (71%), compaction of the stromal collagen fibres in 31 (63%), and folds in Descemets membrane in 31 (63%) cases. Other less common histopathological findings were: presence of superficial iron deposits in 14 (29%), deep stromal scarring in 12 (24%), epithelial scarring in 11 (22%), endothelial cell loss in 11 (22%), and breaks in Descemets membrane in nine (18%) cases. Conclusions: Some of the histopathological findings associated with keratoconus are subtle. It is important to be aware of them in order to properly confirm the clinical diagnosis.

Evidence for the Role of Blue Light in the Development of Uveal Melanoma

Uveal melanoma is the most common malignancy of the adult eye. Although it is a relatively infrequent tumor, clinical prognosis is often poor owing to a high incidence of aggressive metastatic disease, for which there are limited treatment options. Little is known about the etiology of this condition, although several risk factors have been identified. Unlike cutaneous melanoma, however, ultraviolet radiation does not figure prominently among these risk factors. In this review, we focus on an associated form of visible electromagnetic radiation, high-energy short-wave (blue) light, a causative agent in various forms of age-related retina damage, as a previously overlooked risk factor in uveal melanoma development and progression. Finally, we discuss the impact of these data on contemporary ocular therapy, particularly the debate surrounding the filtering capabilities of intraocular lenses used to replace dysfunctional crystalline lenses during cataract surgery.

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.

Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines

Background: Uveal melanoma (UM) is a disease that affects approximately five people per million in the United States. This disease metastasises predominantly to the liver, and treatment options following the clinical detection of these sequelae are limited. Vascular endothelial growth factor-A (VEGF-A) is the primary activator of tumour angiogenesis and functions by binding to VEGF-Receptor 2 (VEGF-R2) and is often required for tumour growth beyond 2–3 mm. The purpose of this study was to investigate the expression of VEGF-A and the primary VEGF-R2 in three UM cell lines. Furthermore, we investigated the effects of VEGF-A inhibition on receptor activation and production of other cytokines. Finally, the effects of VEGF-A inhibition on the proliferation, migration, and invasion in the cell lines were ascertained. Materials: Three UM cell lines (92.1, OCM-1, and UW-1) were incubated with and without the addition of 100 μg/mL of bevacizumab. VEGF-A expression under both conditions was determined by sandwich enzyme-linked immunosorbent assay (ELISA), and phosphorylated VEGF-R2 expression was determined using western blot. The effects of VEGF-A inhibition on 20 cytokines (IL-1a, IL-2, IL-5, IL-8, IL-12p70, GM-CSF, IFNy, CCL3, MMP-9, TNF-a, IL-1b, IL-4, IL-6, IL-10, IL-13, GRO, MCP-1, MIP-1b, and RANTES) were determined using a multiplex sandwich ELISA. Proliferation rates before and after treatment were evaluated via sulforhodamine B assay, and migration and invasion assays implementing the Boyden chamber technique, the latter with artificial extracellular matrix, were used to assess their respective abilities. The Student’s t-test was used to compare changes in cytokine expression following VEGF-A inhibition. Analysis of variance was used to compare changes in the functional abilities of three uveal melanoma cell lines following VEGF-A inhibition. A P-value < 0.05 was considered statistically significant. Results: All three cell lines produced copious amounts of VEGF-A in culture (92.1, 11785.5 ± 231.8 pg/μL; OCM-1, 4608.0 ± 324.0 pg/μL; UW-1, 8309.3 ± 634.5 pg/μL), which was reduced to undetectable levels following the administration of bevacizumab (P< 0.05). Similarly, detectable phosphorylated VEGF-R2 was present in all cells, which was reduced significantly in all cell lines following bevacizumab treatment (107525.2 ± 8602.0 versus 1024.5 ± 98.2, 46587.3 ± 4192.9 versus 12821.1 ± 1666.7, and 60394.3 ± 4026.4 versus 6908.2 ± 607.2; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Of the cytokines investigated, only MMP-9 and CCL3 were ubiquitously altered across all three cell lines following bevacizumab treatment; they were upregulated (CCL3: 1072.50 ± 18.77 pg/mL versus 1281.00 ± 72.34 pg/mL; 22.5 ± 7.85 pg/mL versus 62.00 ± 9.16 pg/mL; 20.33 ± 6.35 pg/mL versus 35.00 ± 6.22 pg/mL; control versus bevacizumab; MMP-9: 25.50 ± 5.47 pg/mL versus 88.25 ± 13.38 pg/mL; 19.75 ± 4.14 pg/mL versus 45.25 ± 8.36 pg/mL; 3.25 ± 1.09 pg/mL versus 19.25 ± 3.77 pg/mL; control versus bevacizumab; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Bevacizumab significantly reduced the proliferation of one cell line (92.1: 0.405 ± 0.012 versus 0.509 ± 0.033; bevacizumab versus control; values OD; P< 0.05), the migration of two cell lines (92.1: 0.071 ± 0.003 versus 0.115 ± 0.003; OCM-1: 0.049 ± 0.005 versus 0.117 ± 0.014; bevacizumab versus control; values OD; P< 0.05), and did not significantly affect invasion. Conclusion: Despite the significant reduction in phosphorylated VEGF-R2 levels, bevacizumab did not have a dramatic impact on the functional abilities of the three UM cell lines studied. Our results indicate that compensatory mechanisms, such as the upregulation of MMP-9 and CCL-3, following bevacizumab administration may mitigate its effects on these abilities.

Uveal Melanoma Risk Factors: A Systematic Review of Meta-Analyses

ABSTRACT Purpose: There is currently no clinical risk-assessment tool allowing identification of patients at risk for developing uveal melanoma (UM) who might benefit from regular screening. As a first step toward the elaboration of such a tool, we systematically reviewed UM risk factors already established by meta-analysis. Methods: Two reviewers independently screened Pubmed, Medline, Embase, and Web of Science from their respective inception dates until July 2016 using a combination of keywords and MeSH terms. Eligible studies were meta-analyses or systematic reviews providing pooled odds ratios (ORs) of risk factors for UM development or sufficient information to calculate them. Methodological quality was evaluated using the Assessment of Multiple Systematic Reviews tool. Results: Four meta-analyses with a mean methodological quality score of 65.9% (min: 54.5%; max: 72.7%) were included. The following significant risk factors were identified: atypical cutaneous nevi (OR 2.82, 95% CI 1.10–7.26), welding (OR 2.05, 95% CI 1.20–3.51), occupational cooking (OR 1.81, 95% CI 1.33–2.46), fair skin color (OR 1.80, 95% CI 1.31–2.47), light eye color (OR 1.75, 95% CI 1.31–2.34), common cutaneous nevi (OR 1.74, 95% CI 1.27–2.39), propensity to sunburn (OR 1.64, 95% CI 1.29–2.09), iris nevi (OR 1.53, 95% CI 1.03–2.27), and cutaneous freckles (OR 1.27, 95% CI 1.09–1.49). Non-significant factors included outdoor leisure activity, occupational sunlight exposure, latitude of birth, and hair color. Conclusion: Moderate quality of evidence determined nine significant risk factors for developing UM. Knowledge of these variables will assist researchers in the elaboration of a formal risk-assessment tool allowing clinicians to estimate susceptibility to the disease and necessity of regular screening.

Sebaceous adenomas of the eyelid and Muir-Torre Syndrome

Background/aims Sebaceous adenomas (SAs) are rare, benign sebaceous gland tumours of the eyelid. SAs may be associated with primary internal malignancies. This association is known as Muir-Torre Syndrome (MTS). The purpose of this study was to approximate the prevalence of SAs, to determine the reliability of the clinical diagnosis of SAs and to demonstrate immunohistochemical staining of DNA mismatch repair proteins mutL homologue 1 (MLH1) and mutS homologue 2 (MSH2) for a case of MTS. Methods We reviewed the histopathology reports from all eyelid specimens collected between 1993 and 2013 at the Henry C Witelson Ocular Pathology Laboratory to determine the proportion of SAs. For the SAs identified on histopathology, we looked at patient charts to see what diagnosis was originally suspected on clinical examination. Immunohistochemical staining for MLH1 and MSH2 was performed on all SAs to screen for MTS. Results Of the 5884 eyelid specimens collected, 9 were SAs (6 women, 3 men; 42–72 years old). The diagnosis of SA was suspected clinically in only one of the nine cases based on the gross appearance of the eyelid lesion. Immunohistochemistry revealed one SA case with positive MLH1 expression and negative MSH2 expression. These findings prompted systemic work-up and this patient was diagnosed with MTS after discovery of a colon adenocarcinoma T2M0N0. Conclusions The diagnosis of eyelid SA is rare. The importance of this benign eyelid tumour stems from its association with internal malignancies in MTS. Immunohistochemical staining of mismatch repair proteins MLH1 and MSH2 is a valid and accessible strategy for investigating MTS in patients with SAs.