Density of PAS positive patterns in uveal melanoma: Correlation with vasculogenic mimicry, gene expression class, BAP-1 expression, macrophage infiltration, and risk for metastasis.

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

Ultrastructural Organization of Uveal Melanoma Stromal Cells.

Typical blood capillaries and vessels in uveal melanoma were shown and different types of uveal melanoma stromal cells were determined by electron microscopy and immunohistochemical analysis. Macrophages, fibroblasts of varying degrees of differentiation and endothelial-like cells with numerous caveolae in the cytoplasm were found in the channels of the extracellular matrix surrounding accumulations of tumor cells. The presence local structures positively stained for markers of the blood and lymphatic vessels (CD31 and podoplanin) in channels of the extracellular matrix suggests that the described endothelial-like cells can be the structural basis for blood and lymphatic vessels of the tumor.

Clinical Significance of Nucleoli Cytomorphology Assessment in Patients With Uveal Melanoma.

AIM: To assess the prognostic significance of nucleolar morphological parameters in a large cohort of patients with uveal melanoma. PATIENTS AND METHODS: The presence, size and number of nucleoli of cancer cells were assessed in haematoxylin and eosin (HE)-stained slides of 164 formalin-fixed paraffin-embedded primary uveal melanoma tissue specimens. The results were correlated with clinicopathological features and patient survival. RESULTS: The presence of macronucleoli and multiple nucleoli significantly correlated with the epithelioid type of uveal melanoma, high mitotic rate, and marked pleomorphism. There was a positive correlation between the presence of macronucleoli as well as the number of nucleoli and the largest tumour basal diameter. The increased nucleolus count in tumour cells positively correlated with primary tumour (pT) staging. The presence of both prominent and multiple nucleoli was associated with significantly reduced overall and disease-free survival. CONCLUSION: Histological assessment of nucleolar morphology in routine HE staining would be a helpful low-cost method to obtain reliable prognostic information.

Molecular regulation of vasculogenic mimicry in human uveal melanoma cells: role of helix-loop-helix Id2 (inhibitor of DNA binding 2).

BACKGROUND: Vasculogenic mimicry (VM) is a tumor angiogenesis process in which highly aggressive melanoma cells form patterned, tubular networks in an in vitro, three-dimensional culture that mimics vasculogenic networks formed by endothelial cells. These cells also express endothelial cell-associated genes such as vascular endothelial-cadherin (VE-cadherin) and are correlated with poor clinical prognosis in patients. However, the molecular underpinnings of this phenomenon remain elusive. METHODS: Three-dimensional cultures of highly and poorly aggressive uveal melanoma cells were observed by inverted light microscope and scanning electronic microscope for VM. RNAi (RNA interference) technology was used to examine whether inhibitor of DNA binding 2 (Id2) was involved in the uveal melanoma vasculogenic mimicry. Western blot analysis showed changes of Id2 and VE-cadherin expression in highly and poorly aggressive melanoma cells in vitro. Migration analysis of highly and poorly aggressive uveal melanoma cells in vitro illuminated the role of Id2 in tumor cells migration. RESULTS: We show here that a transient knockdown of Id2 by RNA interference abrogates VM and VE-cadherin expression in highly aggressive uveal melanoma cells. Furthermore, inhibition of Id2 changes cellular stability and creates a more dynamic condition. Transfected cells also migrate better than untransfected cells. CONCLUSIONS: This study shows that Id2 is an important regulator of VM. Specifically, Id2 affects VE-cadherin expression, and is critical for the formation of vasculogenic-like networks.

Ultrastructural alterations in extraocular muscles following iodine-125 brachytherapy for uveal melanoma.

PURPOSE: This study investigated the ultrastructural changes in extraocular muscles under which radioactive plaques had been placed for the treatment of uveal melanoma. METHODS: At the time of plaque removal, biopsies were taken from four horizontal recti that had been left in situ over plaques and from one lateral rectus muscle that had been disinserted before brachytherapy. Normal lateral recti from enucleated eyes were used as controls. Iodine-125 seeds were used with a mean total activity of 54.04 mCi, remaining for an average of 149.62 hours over the sclera. RESULTS: Muscles that had been left in situ over the radioactive plaques demonstrated a focal decrease in muscular tissue and increased fibroblasts and collagen. Electron microscopy showed increased collagen, loss of sarcoplasmic reticulum and swollen mitochondria. The disinserted muscle in the plaque group appeared unaffected. CONCLUSIONS: Despite the theoretical shielding properties of plaques, leaving an extraocular muscle over the plaque may lead to several non-specific ultrastructural changes.

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.

Establishment of cell lines of uveal melanoma. Methodology and characteristics.

Six continuous cell lines have been established from choroidal and ciliary body melanomas. These lines have been maintained in culture for at least 100 in vitro population doublings for periods over 1 year. They were established initially using a human diploid fibroblast strain MRC-5 as a feeder layer. Cells were grown in Ham's F-12 medium containing fetal bovine and horse sera and supplemented with glucose, cholera toxin, and epidermal growth factor. Culture doubling times ranged from 72-96 hr; cloning efficiencies ranged from 1-5% in the absence of a feeder layer. Six cell lines were studied in detail by electron microscopy, and all were found to have evidence of melanosomes and/or premelanosomes. The morphology of the cells was characteristic of melanomas as defined by the Callender classification, with cell types ranging from spindle A to epithelioid. Karyotypic studies revealed the presence of only human chromosomes with modal numbers ranging from 48-54 in the different lines.

Characterization of uveal melanoma cell lines that grow as xenografts in rabbit eyes.

Two cell lines, OCM-1 and OCM-2, were established from biopsied specimens of choroidal melanomas of spindle B and mixed cell type morphologies. Both cell lines were phenotypically malignant. Karyotypic analyses revealed human chromosomes with a modal number of 95 and 85, respectively. These cell lines have been passaged for over 2 years and are essentially immortal. The cells grew without contact inhibition as monolayers in liquid culture or as clones in soft agar. Electron microscopy revealed melanoma cells containing premelanosomes and cultures free from contamination by fibroblasts. To categorize the morphologies of these cultured cells better by the Callender classification, they were grown as xenografts in the anterior chambers of rabbits immunosuppressed with daily i.m. doses of Cyclosporin A (10 mg/kg). Tumor plaques were detected after 10 days. The eyes were enucleated and fixed in formalin and stained with hematoxylin and eosin for histopathological evaluation. The xenograft from OCM-1 was found to consist predominantly of spindle B-type melanoma cells. In contrast, the xenograft from OCM-2 contained epithelioid, spindle B and clear cell ("balloon") melanoma cells. The ability of these cell lines to grow as xenografts confirmed their neoplastic origin. In fact, the types of the uveal melanoma cells in the xenografts resembled those in the original biopsied tumors. This suggests that the morphology of human uveal melanoma cells is an inherited trait and may be genetically fairly stable.

Prediction of metastasis of uveal melanoma: comparison of morphometric determination of nucleolar size and spectrophotometric determination of DNA.

We studied 95 cases of uveal melanoma using cytomorphometry to measure the standard deviation of nucleolar area (SD-NA) based on 200 cells per tumor and microspectrophotometry to determine the quantity of DNA in the nucleus of 100 cells per tumor. The 95 cases of uveal melanoma, in which the eye was enucleated between 1970 and 1973, were selected from the files of the REgistry of Ophthalmic Pathology. Forty-nine patients survived with a median follow-up of 15 years without evidence of metastasis at the time of the last follow-up, and 46 patients died of metastatic melanoma. Statistical analysis indicated that SD-NA was a better predictor of metastasis than DNA determination in this data set.

Ultrastructural changes of human trabecular meshwork after photocoagulation with a diode laser.

A diode laser, which emitted infrared radiation at a wavelength of 810 nm, was used to perform trabecular photocoagulation in a human eye due for enucleation for malignant melanoma. For comparison, burns were applied with an argon blue-green laser (488-514.5 nm). With each laser, the treatment spot size was 100 microns and the pulse duration was 0.20 sec. Visible lesions were produced with a power of between 750 mW and 1.2 W with the diode laser, and 500-900 mW with the argon laser. The pattern of damage produced by both modalities was similar and essentially consisted of contraction or expansion of trabecular beams, with trabecular destruction occurring only in relation to high power exposures. These findings confirm that trabecular photocoagulation is not a process that depends upon the wavelength of the incident energy at the two spectral extremes of 488 nm and 810 nm.