RETINAL OXIMETRY IS ALTERED IN EYES WITH CHOROIDAL MELANOMA BUT NOT IN EYES WITH CHOROIDAL NEVI.

PURPOSE: To compare retinal vessel oxygenation in eyes with an untreated choroidal nevus or choroidal melanoma. METHODS: The affected and fellow eye of patients with an untreated choroidal nevus (n = 42) or choroidal melanoma (n = 45) were investigated using noninvasive retinal oximetry (Oxymap T1). Oxygen saturation of arterioles (ArtSat) and venules (VenSat) was determined, together with the arteriovenous difference (AV-difference). RESULTS: In choroidal nevus patients, retinal oximetry did not differ between the affected and fellow eye: the mean ArtSat was 94.5% and 94.2% (P = 0.56), the VenSat was 60.5% and 61.3% (P = 0.35), and the AV-difference was 34.0% and 32.9% (P = 0.18), respectively. In choroidal melanoma patients, alterations were detected: the mean ArtSat was 94.8% and 93.2% (P = 0.006), the VenSat was 58.0% and 60.0% (P = 0.014), and the AV-difference was 36.8% and 33.2% (P < 0.001), respectively. The largest increase in AV-difference was observed between the retinal halves without the lesion in melanoma eyes compared with the corresponding half in the fellow eye (37.5% vs. 32.1%, P < 0.001). CONCLUSION: Although retinal oximetry was not significantly altered in eyes with a choroidal nevus, eyes with choroidal melanoma showed an increased ArtSat and decreased VenSat, leading to an increased AV-difference. These changes may be caused by inflammation and a higher metabolism, with larger oxygen consumption, leading to altered blood flow and intraocular oxygen relocation.

Bevacizumab and intraocular tumors: an intriguing paradox.

PURPOSE: Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro. METHODS: B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated. RESULTS: Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures. CONCLUSIONS: In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

Triamcinolone acetonide and anecortave acetate do not stimulate uveal melanoma cell growth.

PURPOSE: Radiotherapy-induced radiation retinopathy can develop in over 40% of eyes treated for uveal melanoma. Triamcinolone acetonide (TA) and anecortave acetate (AA) can be used to treat radiation retinopathy. It is not known whether TA or AA has any effect on potentially still viable uveal melanoma cells in the choroid after radiotherapy. We therefore studied the effect of these drugs on the proliferation of uveal melanoma cell lines in vitro. Furthermore, as these drugs are supposed to counteract vascular leakage, we determined their effect on the expression and production of the proangiogenic vascular endothelial growth factor-A (VEGF-A), the antiangiogenic pigment epithelium-derived factor (PEDF), and thrombospondin-1 (TSP-1) in uveal melanoma cells. METHODS: Three uveal melanoma cell lines were treated in vitro with TA or AA. Cell proliferation was measured by counting cells and using the Water-Soluble Tetrazolium Salt-1 (WST-1) assay. VEGF-A and PEDF production was measured by ELISA, and intracellular expression of angiogenic-associated genes including VEGF-A, PEDF, and TSP-1 was determined by real-time quantitative RT-PCR. RESULTS: We found no effect of TA or AA on tumor cell growth or production of VEGF-A and PEDF in any of the three uveal melanoma cell lines tested. Regarding expression as measured by RT-PCR, TA had an inhibiting effect on TSP-1 in only one cell line, and no effect on VEGF-A or PEDF. AA showed a similar lack of effect. CONCLUSIONS: Since TA and AA do not stimulate uveal melanoma cell growth, it seems to be safe to use these drugs to treat radiation retinopathy after irradiation for uveal melanoma. Additional experiments using more cell lines or primary tumor cell cultures are needed to validate this conclusion. Furthermore, the results of our study suggest that TA does not exert its antileakage effect through downregulation of VEGF-A or upregulation of TSP-1 or PEDF in uveal melanoma cell lines. It is possible that TA and AA influence these pro- and antiangiogenic factors only under hypoxic circumstances. Further investigation is needed.

Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker.

INTRODUCTION: Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. MATERIALS AND METHODS: UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. RESULTS: Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. CONCLUSIONS: Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM.

Anti-angiogenic therapy in uveal melanoma.

For several decades, targeting of tumor-related vessels has been regarded as a potential anticancer therapy. Such anti-angiogenic therapy is based on the assumption that a tumor cannot grow beyond the limits of diffusion (about 1-2 mm) of oxygen and nutrients from capillaries, unless angiogenesis takes place. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, regulating vasopermeability as well as the proliferation and migration of endothelial cells. In several types of cancer (colon carcinoma, soft tissue sarcomas and gastric cancer), serum VEGF levels are a marker for disease stage and an indicator of metastasis. VEGF levels are significantly elevated in uveal melanoma patients with metastatic disease compared to patients without metastases. Anti-angiogenic therapy, such as bevacizumab, is currently used for the treatment of metastases of several malignancies. Anti-angiogenic therapy has not yet been tested for the treatment of primary uveal melanoma or related metastatic disease. Clinicians, however, have a broad experience with anti-angiogenic agents in patients with uveal melanoma by treating the complications of radiation therapy. We will discuss tumor angiogenic processes and related molecular pathways in uveal melanoma. The role of VEGF and the potential use of current commercially and experimentally available anti-angiogenic drugs for the treatment of primary uveal melanoma and/or metastatic disease will be explained below.

Macrophages in uveal melanoma and in experimental ocular tumor models: Friends or foes?

Macrophages belong to the innate immune system and as such constitute one of the first barriers against infection. They play an important role in wound healing, in inflammation and in angiogenesis, but are also essential in the first stage of a "danger response". After scavenging debris, they can digest cellular proteins into smaller pieces, and protein-derived peptides can subsequently be presented to the immune system. Depending on the activation state of the macrophage, this antigen presentation may trigger a full-blown active immune response, or may suppress a potential immune reaction. Macrophages constitute a heterogeneous cell population described by many names, with varying phenotypic characteristics, depending on their tissue location and state of activation. They play important roles in different ocular tissues, including the cornea and the choroid, and have been found to be involved in anti-tumor immune responses in mouse ocular tumor models. One would thus expect macrophages to belong to the "good guys" that help to protect our body against dangers such as cancer. In human uveal melanoma however, a high density of macrophages is associated with a poor prognosis for the patient. Macrophages play a role in promoting angiogenesis, and thus may stimulate tumor growth; in addition, macrophages have also been found to suppress anti-melanoma immune responses. These functions may shift during aging. Taken together, these new observations extend our understanding of the diverse functions of macrophages and show us their different faces, making them either "friends or foes" in human uveal melanoma. A better understanding of these multifaceted cells will help in developing new treatments to prevent the growth of metastases in uveal melanoma patients.

Regulation of VEGF-A in uveal melanoma.

PURPOSE: Blood vessels are important constituents of intraocular uveal melanoma (UM), but whether angiogenesis is regulated by environmental factors such as ischemia or by genetic mechanisms is not known. This study was undertaken to examine the regulation of the proangiogenic factor vascular endothelial growth factor (VEGF-A). METHODS: Cell lines and primary tumors were tested for expression of VEGF-A, under normoxic and hypoxic conditions, using quantitative PCR, ELISA, WST-1 viability, and in-cell Western experiments. VEGF-A serum levels were determined by ELISA. RESULTS: Hypoxia induced expression of HIF-1alpha and VEGF-A in UM cell lines and primary tumor cultures, but it did not influence proliferation. VEGF-A expression in primary tumors was variable, demonstrating no correlation with specific histologic markers or prognosis. However, VEGF-A levels were significantly raised in UM patients with metastases compared with those without metastases (P < 0.001). CONCLUSIONS: VEGF-A expression by UM cells is mainly controlled by hypoxia and involves the HIF-1alpha pathway, thus indicating an important role for the tumor cell environment. Metastases led to increased serum VEGF-A levels, indicating that VEGF-A may be involved in the growth of metastases.