MiR-181a-5p inhibits uveal melanoma development by targeting GNAQ and AKT3.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor type in adults. Even after the treatment of the ocular tumor, the prognosis of patients with metastasis remains poor. Hence, an urgent unmet need exists to identify novel approaches to treat advanced UM. Previous studies have revealed G subunit alpha Q and alpha 11 (GNAQ/11) mutations in more than 85% of patients with UM, thus indicating the importance of GNAQ and downstream signaling pathways in UM occurrence. Here, we demonstrate that microRNA (miR)-181a-5p, a small non-coding RNA, effectively inhibited the viability, proliferation, and colony formation but induced apoptosis of UM cells. Furthermore, silencing GNAQ or AKT3 mimicked the anti-UM effects of miR-181a-5p, whereas overexpression of GNAQ or AKT3 rescued the anti-UM effects induced by miR-181a-5p. In addition, miR-181a-5p had a stronger effect in decreasing the viability of GNAQ mutant than GNAQ wild-type cells. Moreover, miR-181a-5p suppressed the total expression and phosphorylation of members of the ERK and PI3K/AKT/mTOR signaling pathways. Importantly, miR-181a-5p potently inhibited the growth of UM xenografts in nude mice. MiR-181a-5p also decreased the expression of Ki67, GNAQ, and AKT3, and induced the expression of cleaved-caspase3 in UM tumors. These results suggest that miR-181a-5p inhibits UM development by targeting GNAQ and AKT3.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Trends in Radiation Practices for Female Ocular Oncologists in North America: A Collaborative Study of the International Society of Ocular Oncology.

BACKGROUND: The aim of this study was to determine the known radiation exposure, attitudes, and consequent risk modifications among female ocular oncologists in North America who routinely administer radioactive plaque brachytherapy treatment and are members of the International Society of Ocular Oncology. METHODS: Nineteen female ocular oncologists completed an anonymous 17-question radiation exposure survey. RESULTS: Eleven of the participants chose to routinely wear lead protection during surgery; 8 did not. Fifteen of 19 participants reported using an unloaded "nonactive" template to prepare for plaque implantation. During pregnancy, 11 of 13 participants continued to perform plaque brachytherapy. Eight of these 11 undertook measures to decrease radiation exposure self-reported as lead wear and other. The average reported anxiety regarding fertility was 2.1 (SD, 2.2) on a scale from 1 to 10. CONCLUSION: This study corroborates prior literature that surgeons' exposure to radiation during plaque brachytherapy is minimal. Nonetheless, there remains some anxiety regarding exposure risk to women, due to potential effects on fertility and fetal health. We found variability in exposure monitoring, required training, and precautions during pregnancy amongst this group of surgeons. Improved education and clearer pregnancy guidelines may equip female ocular oncologists with optimal knowledge regarding risk of radiation exposure.

Choroidal extranodal marginal zone lymphoma diagnosed by full-thickness retinochoroidal biopsy: case report and review of the literature.

The case of an 89-year-old man who was referred for a painless decrease of vision in his right eye (RE) is reported. Fundus examination of the RE showed an elevated amelanotic lesion located in the posterior pole with an adjacent focal round pigmented lesion. There was also a more peripheral amelanotic lesion extending from 6 to 9 o'clock clockwise inferotemporally. Uveitis workup and imaging studies of brain and orbits were normal. A retinochoroidal biopsy was done and showed the presence of choroidal extranodal marginal zone lymphoma. The patient was treated with external beam radiotherapy. This report presents a review of the literature of all reported cases of choroidal extranodal marginal zone lymphoma.

Technique for Robotic Stereotactic Irradiation of Choroidal Melanoma.

Radiotherapy has a long history in the organ-sparing management of choroidal melanoma. Joining plaque radiotherapy and proton irradiation, stereotactic robotic photon irradiation is a new tool in the radiation oncologist's armamentarium for ocular tumors. The non-coplanar fields with steep dose gradients are well suited to spare uninvolved retina, anterior chamber, and the optic nerve. In our practice, it is the preferred treatment for melanomas that are non-amenable to standard plaque brachytherapy. Since late 2010, we have treated more than 40 patients with our robotic linear accelerator. This case-based technical note outlines the technique used at the University of Montreal, Montreal, Canada.

Conjunctival Involvement of T-Cell Lymphoma in a Patient with Mycosis Fungoides.

Background. Ocular involvement in mycosis fungoides (MF) cases occurs in one-third of patients with the eyelid being the most frequent site affected; however, conjunctival involvement is rarely reported. Herein, we report a rare case of conjunctival involvement of MF. Case Presentation. A 66-year-old man who was previously diagnosed with MF in 2010 and was treated presented in 2014 complaining of foreign body sensation and redness in both eyes. Slit lamp examination of both eyes showed erythematous conjunctival growth that extended circumferentially. Physical examination revealed erythematous skin lesions on different body parts. Conjunctival biopsy was performed and revealed a dense, highly polymorphic lymphocytic population. The immunophenotype demonstrated a neoplastic T-cell origin consistent with MF. A diagnosis of conjunctival involvement by MF was made. The conjunctiva was treated with radiotherapy resulting in tumor regression. There were no recurrences at the 6-month follow-up. Conclusion. T-cell lymphoma should be considered in patients with a history of MF presenting with conjunctival and skin lesions.

Cyclooxygenase-2 expression in human irradiated uveal melanomas.

AIM: Previous studies have shown that radiotherapy is a stimulus for cyclooxygenase-2 (COX-2) expression and that use of COX-2 inhibitors enhances the radio sensitivity of tumor cells. The objective of this study was to evaluate COX-2 expression, and its correlation with tumor regrowth after irradiation, in enucleated eyes with uveal melanomas. METHODS: Fifteen tissue samples from patients who underwent enucleation after radiotherapy between 1988 and 2001 were used. Nine cases (60%) were enucleated because of tumor regrowth and six (40%) because of severe complications of radiotherapy. Specimens were immunostained for COX-2, and tumor cells were evaluated for specific cytoplasmic and granular immunostaining. COX-2 expression for these cases was compared with that in the previous study including 40 non-irradiated uveal melanoma cases. COX-2 expression was also correlated with tumor regrowth after radiotherapy. RESULTS: Two cases (13.3%) were positive and thirteen (86.7%) were negative for COX-2 expression. One of the positive cases had been enucleated because of tumor regrowth and one because of radiotherapy complications. There was no relationship between tumor regrowth and COX-2 expression. COX-2 expression was significantly lower in irradiated cases than in non-irradiated cases in the previous study (p<0.001). CONCLUSIONS: In contrast with studies showing an increase of COX-2 expression in other irradiated malignancies, irradiation was not a factor inducing COX-2 in uveal melanomas. Radiotherapy may, moreover, be a factor that reduces COX-2 expression in uveal melanomas.

Immunohistochemical panel of undifferentiated orbital metastatic carcinomas.

PURPOSE: To examine the applicability of an immunohistochemical panel of seven monoclonal antibodies to identify the primary site of poorly differentiated orbital metastatic carcinomas. MATERIAL AND METHODS: Immunohistochemistry was performed to detect cytokeratin (CK) 7, CK20, thyroid transcription factor-1 (TTF-1), BRST1, BRST2, carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) in seven cases of poorly differentiated orbital metastases. Of the seven cases, four were female and three male. The youngest patient was thirty-six while the oldest was eighty-eight years of age. RESULTS: The immunohistochemical panel alone was helpful to identify the primary source of the metastatic lesion in three out of the seven cases. Two of them were metastatic breast carcinomas (BRST1, BRST2 positive) and one was a prostate carcinoma (PSA positive). By correlating the immunohistochemical results with the previous clinical history, the primary site could be identified in two more cases. In those metastatic lesions, the positive staining for CK7, CK20, and CEA, associated with negative staining for BRST1, BRST2, PSA and TTF-1, indicated bladder as the probable primary site. In two out of seven cases, the metastatic tumor was only positive for CEA, therefore a primary site could not be identified. CONCLUSIONS: An immunohistochemical panel of poorly differentiated orbital metastases is helpful in the identification of the primary tumor site. The association of seven markers with the patient's clinical history allowed for the positive identification of the primary tumor in the majority of these cases.

Characterization of ocular and metastatic uveal melanoma in an animal model.

PURPOSE: To characterize, in detail, tumor development, malignant cell dissemination, and metastasis in a 10-week animal model of uveal melanoma. METHODS: One million 92.1 human primary uveal melanoma cells were injected into the suprachoroidal space of the right eye of 27 immunosuppressed albino rabbits. Intraocular tumor growth was monitored weekly by fundoscopy and by ultrasonography at the end of the experiment. To document the progression of the disease, one animal per week was killed. The enucleated eyes, lungs, and livers were macroscopically examined and histopathologically studied by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemistry. Mononuclear layers isolated from the rabbits' blood samples were cultured. RESULTS: Histopathology showed intraocular tumors in 89% of the animals. Tumor growth was found 1 week after cell inoculation, and by the end of the experiment large tumor masses were observed. Microscopic pulmonary metastatic foci were first observed 4 weeks after cell injection. By the end of the experiment, all the animals had metastasis to the lungs. Interestingly, 18% of the animals also had micrometastasis to the liver. Viable adherent uveal melanoma cells were successfully isolated from peripheral blood and grown in vitro. CONCLUSIONS: In this study, most rabbits developed intraocular tumors followed by lung metastasis, and some of these rabbits later developed liver micrometastases. This novel source of research material warrants a follow-up longer than 10 weeks to further explore the pathophysiologic bases of liver involvement commonly encountered in humans. The success in the isolation and culture of circulating malignant cells in this animal model suggests that it might be worthwhile to explore the application of this technique to the management of patients with primary uveal melanoma.

Cell proliferation profile of five human uveal melanoma cell lines of different metastatic potential.

OBJECTIVE: The aim of this study was to establish a proliferation profile of uveal melanoma cell lines, using different methods, and to compare it with their previously determined metastatic potential (MP). METHODS: Four human uveal melanoma and one transformed human uveal melanocytic cell line were ranked according to proliferation profiles. The proliferation profiles of the cell lines were compared to their MPs, which were previously determined from an immunosuppressed rabbit model. RESULTS: Ranking of the cell lines using pulse labeling with tritiated thymidine was similar to the MP of the cell lines. CONCLUSION: The correlation between the proliferative rate of the uveal melanoma cell lines and their previously determined MP resulted in the proposal of a new classification scheme: high proliferation/high MP, low proliferation/low MP, and high proliferation/no MP. High proliferative capacity of a cell line did not necessarily confer MP; therefore, further cellular functions/adaptations must be required for tumor cell dissemination, survival, and growth at a metastatic site.

The risk of other primary cancer in patients with uveal melanoma: a retrospective cohort study of a Canadian population.

BACKGROUND: The incidence of second primary malignant tumours has doubled during the last 2 decades. These tumours now represent the sixth most common group of cancers. Many authors have described the presence of multiple primary cancers in patients with uveal melanoma. However, no studies have been performed using Canadian data. The purpose of this study was to describe the occurrence of other primary cancers diagnosed before or after uveal melanoma and to calculate the incidence of subsequent primary cancer in a Canadian cohort with uveal melanoma. METHODS: We conducted a retrospective study of a cohort of patients with uveal melanoma diagnosed between 1990 and 2002 at a university-affiliated centre in Montreal. We reviewed medical records to identify patients in whom other, unrelated primary malignant disease had been diagnosed. We used the standardized incidence ratio to calculate the risk of development of a second, unrelated cancer following the diagnosis of uveal melanoma. RESULTS: A total of 129 cases of uveal melanoma were diagnosed. Eighteen patients (14%) also had a diagnosis of an unrelated primary cancer. In nine patients the other cancer had been diagnosed first, and in nine patients the other tumour had been diagnosed after the uveal melanoma. There was no increased risk of development of any particular form of cancer studied for females or males. INTERPRETATION: In our Canadian cohort, statistical analysis showed no increased risk of a second cancer, overall or by organ site, in male or female patients with uveal melanoma. As uveal melanoma is a rare type of cancer, analyses of a much larger cohort may be needed to accurately estimate the risk of development of a second primary cancer in patients with uveal melanoma.

Macrophage-derived soluble factor enhances melanoma inhibitory activity expression by uveal melanoma cells in vitro.

Melanoma inhibitory activity (MIA) is correlated with tumour progression and development of metastatic disease. Melanoma inhibitory activity, secreted by melanoma cells, is known to inhibit tumour cell attachment to the extracellular matrix enhancing their invasive potential. The regulatory pathways that lead to MIA expression have not yet been elucidated. It is well established that tumour cells and macrophages interact through soluble factors, preventing or enhancing tumour growth. The purpose of the present study was to determine whether soluble factor(s) derived from macrophages lead to the up-regulation of MIA production by human uveal melanoma cell lines (HUMCL) and whether MIA contributes to an increase in the invasive behaviour of HUMCL in vitro. Baseline MIA levels were measured by enzyme-linked immunosorbent assay in five HUMCL of known metastatic potential (92.1>SP6.5>OCM-1>MKT-BR>UW-1). Macrophage conditioned medium (MaCM) was placed on top of the HUMCL and MIA levels were measured at 6, 12, 24, and 36 h. The HUMCL were also seeded in a Matrigel chamber for 72 h and then cells invading the Matrigel were counted. The assay was repeated adding recombinant human MIA to the top layer of each well. All HUMCL expressed MIA at baseline (average of 31 ng/ml at 36 h). Following exposure to MaCM, MIA levels increased to an average of 45.2 ng/ml, with the 92.1 and SP6.5 cell lines expressing the highest MIA levels and UW-1 cell line expressing the lowest level. During the baseline invasion assay, the vast majority of cells (>95%) were found to adhere to the upper surface of the Matrigel. When MIA was added to the invasion chamber, no adhesion or invasion was observed. The results suggest, for the first time, that macrophages secrete a soluble factor(s) that may stimulate nearby melanoma cells to enhance their production of MIA in vitro. Furthermore, increased MIA production may, in turn, increase the invasive properties of the cells by modulating the attachment of HUMCL to the extracellular matrix.

Cyclooxygenase-2 expression in uveal melanoma: novel classification of mixed-cell-type tumours.

BACKGROUND: The expression of cyclooxygenase-2 (COX-2), an inducible prostaglandin (PG) synthase, has been investigated in various human malignant diseases, such as cutaneous melanoma. We investigated the expression of COX-2 in uveal melanoma and related the findings to prognostic factors. METHODS: In 40 cases of uveal melanoma, immunostaining for COX-2 was done. COX-2 expression was related to histopathological prognostic markers, such as cell type, the presence of lymphocytic infiltration and vascular closed loops in the tumour, and cytomorphometry results. RESULTS: COX-2 expression was found in 58% of the cases, and it correlated with markers of poor prognosis, such as epithelioid cell type and the presence of lymphocytic infiltration and vascular closed loops. The uveal melanomas expressing COX-2 had larger nuclei, as determined by cytomorphometry. INTERPRETATION: Whereas epithelioid tumours carry a worse prognosis than spindle cell tumours, until now it has not been possible to give a strong indication of prognosis in mixed-cell tumours. This study showed that mixed-cell tumours, representing the majority of uveal melanomas, may be further subclassified according to COX-2 expression, which serves as a marker of poor prognosis. The role of COX-2 in uveal melanoma should be further elucidated, and the use of COX-2 inhibitors warrants investigation as adjuvant treatment for this life-threatening malignant disease.

Benign ocular adnexal tumours of apocrine, eccrine or hair follicle origin.

BACKGROUND: The differential diagnosis of malignant eyelid tumours, particularly basal cell carcinoma (BCC), includes tumours of skin appendages. The incidence of these adnexal tumours has not been well established. This study aimed to determine the incidence and review the main clinicopathological features of benign tumours arising from skin appendages of the eyelid with apocrine, eccrine or hair follicle differentiation. METHODS: The histopathological diagnoses of 7751 ophthalmic specimens retrieved from 6967 patients between September 1993 and March 2002 at the Henry C. Witelson Ophthalmic Pathology Laboratory and Registry, McGill University, Montreal, were retrospectively reviewed. Clinical data and histopathological diagnoses were obtained for 228 benign adnexal tumours of apocrine, eccrine or hair follicle origin. New histopathological slides were made from the paraffin-embedded specimens and stained with hematoxylin-eosin and periodic acid-Schiffs reagent. RESULTS: Of the 228 benign adnexal tumours, 182 were diagnosed as apocrine or eccrine hydrocystoma (79.8%), 12 pilomatrixoma (5.3%), 12 syringoma (5.3%), 11 trichilemmoma (4.8%), 5 syringocystadenoma papilliferum (2.2%), 3 trichoepithelioma (1.3%) and 3 trichofolliculoma (1.3%). Discrepancies between clinical and histopathological diagnoses were noted in 22 cases (9.6%). INTERPRETATION: Benign tumours originating from skin appendages of the eyelid are rare and frequently have apocrine or eccrine differentiation. These tumours, particularly those originating from the hair follicle, should be considered in the differential diagnosis of BCC of the eyelid.