Differential expression of chemokine receptors on uveal melanoma cells and their metastases

PURPOSE: To determine the expression of the chemokine receptors CXCR4 and CCR7 on human uveal melanoma cells and their metastases and the effect of liver-borne factors on the chemotactic responses of uveal melanoma cells. METHODS: Four human uveal melanoma cell lines and three cell lines of uveal melanoma metastases were examined by RT-PCR and flow cytometry for their constitutive expression of CXCR4 and CCR7. The effect of the liver and liver-borne factors on the expression of CXCR4 and CCR7 was determined after intracameral, intrasplenic, and subcutaneous transplantation of uveal melanoma cells in nude mice. Chemotactic responses of melanoma cells to liver-borne factors were determined by in vitro chemotaxis assays using protein extracts of hepatocytes and striated muscle tissue. RESULTS: All the primary uveal melanoma cell lines expressed CXCR4 and CCR7 message and protein, whereas the metastases cell lines expressed little or no chemokine receptor. Extracts of human liver cells stimulated chemotaxis of uveal melanoma cells, which could be inhibited by anti-CXCR4 antibody. Liver-borne factors also induced the downregulation of CXCR4 and CCR7 on uveal melanoma cells. Uveal melanoma cells maintained their high expression of CXCR4 and CCR7 after intracameral transplantation. However, CXCR4 and CCR7 expression was sharply reduced in liver metastases arising from intraocular melanomas. CONCLUSIONS: CXCR4 and CCR7 provide directional migration of uveal melanoma cells toward the liver, the most common site for the formation of uveal melanoma metastases. However, soluble factors elaborated by hepatocytes induce the downregulation of CXCR4 and CCR7 on metastatic uveal melanoma cells.     

Apoptosis inhibitor, survivin, in posterior uveal melanoma: comparison among primary tumors, tumors resistant to brachytherapy, tumors with liver metastases, and liver metastases

PURPOSE: To study the expression of survivin, an apoptosis inhibitor protein, in human posterior uveal melanoma. METHODS: Specimens were divided according to eyes with tumors that were enucleated primarily, those resistant to brachytherapy, eyes from patients with known liver metastases, and liver metastases. RESULTS: There was only low expression of survivin in uveal melanoma. No difference in survivin positive cell counts per high power field (PCC/HPF) were found among tumors that were enucleated primarily (n = 33), tumors with previous brachytherapy (n = 29), tumors with liver metastases (n = 12) or liver metastases (n = 18). Corresponding counts were 11.8 (+/-14.3), 11.8 (+/-16.8), 7.1 (+/-11.2), and 4.7 (+/-8.8) in the four groups, respectively (p > 0.05). Half of the liver metastases showed no staining for survivin. Twenty patients (24%) had tumor-related death at the end of follow-up. CONCLUSIONS: Survivin is expressed in posterior uveal melanomas that were treated by enucleation, as well as in tumors that were previously treated with brachytherapy or liver metastases; however, its expression by immunostaining did not seem with correlate with the tumor biological activity.     

Liver function tests in metastatic uveal melanoma

PURPOSE: To evaluate trends in liver function tests before detection of liver metastases from uveal melanoma. DESIGN: Retrospective, comparative, observational case control study. METHODS: Setting: The Israel uveal melanoma center at the Hadassah University Hospital. Patient Population: A total of 307 uveal melanoma patients who were diagnosed with uveal melanoma and followed between the years 1988 and 1998. Of them, 30 metastatic patients who had regular follow-up by liver function tests (LFTs) and liver imaging were included in this study. Eighty nonmetastatic patients were randomly chosen as controls. Observation Procedure: The medical records of the metastatic and control groups were reviewed documenting LFTs and liver imaging results. Main Outcome Measures: The mean level of each LFT, its sensitivity, specificity, and likelihood ratio at various time periods before the detection of metastases by liver imaging. RESULTS: At the time of diagnosis of liver metastases by imaging, 50% of patients had at least one abnormal LFT (compared with only 5% of the control group). While no change was noted in the mean serum levels of bilirubin, mean lactate dehydrogenase (LDH), alkaline-phosphatase, gamma glutamyl transpeptidase (gamma GTP) aspartate-aminotrasferase, and alanine-aminotrasferase levels seem to rise, even within normal limits, during the 6 months before the detection of metastases. Based on likelihood ratios, alkaline-phosphatase and lactate dehydrogenase were the most predictive tests. Lactate dehydrogenase and aspartate-aminotransferase were already predictive at 80% of the upper normal limit, whereas alkaline-phosphatase and gamma-glutamyltransferase were most predictive at the upper normal limit. CONCLUSIONS: Monitoring the changes in selected LFTs, even within normal limits, can help predict metastatic uveal melanoma.     

Prognostic factors of liver metastases from uveal melanoma

Objectives This study was designed to assess survival and identify prognostic factors for liver metastases diagnosed by systematic screening in uveal melanoma patients. Methods Among 602 consecutive patients treated over 10 years for uveal melanoma and followed by systematic semi-annual hepatic screening (abdominal ultrasonography), 63 (10.5%) developed liver metastases; these patients form the basis of this study. Factors including patient demographics, characteristics of the uveal tumor, metastasis-free interval, severity of liver metastatic involvement, and treatments of metastases were studied retrospectively regarding their prognostic value, using univariate (Kaplan-Meier method) and multivariate (Cox model) analyses. Results Thirty-five patients (55.6% of the metastatic population) received systemic chemotherapy or best supportive care only; 14 patients (22.2% of the metastatic population) diagnosed with diffuse liver involvement had cytoreductive surgery and intra-arterial chemotherapy; 14 (22.2% of the metastatic population) had complete surgical removal of liver metastases followed by postoperative intra-arterial chemotherapy. No significant surgical complications were experienced. The median overall survival after diagnosis of liver metastases was 15 months. It reached 25 months for selected patients with complete resection (P=0.0002). In this cohort of 63 patients, ten or fewer preoperatively diagnosed metastases and primary uveal melanoma not involving the ciliary body were independently associated with better prognosis. Conclusions This study suggests that selected patients with screened liver metastases from uveal melanoma may benefit from aggressive treatment, including surgery. The two independent favorable prognostic factors are fewer than ten metastases at screening and the absence of ciliary body involvement. Laurent Kodjikian had full access to all the data and takes responsibility for the integrity of the data in the study and the accuracy of the data analysis A summary of this work was presented at the 27th congress of the “Societa Italiana di Chirurgia Oncologica” (May 29–31, 2003) and published as an abstract This work was presented at the 24th Meeting of the Club Jules Gonin, September 2004, Athens (Greece)     

Modeling the behavior of uveal melanoma in the liver

PURPOSE: To model the behavior of uveal melanoma in the liver. METHODS: A 15-muL suspension of metastatic MUM2B or either primary OCM1 or M619 uveal melanoma cells was injected into the liver parenchyma of 105 CB17 SCID mice through a 1-cm abdominal incision. Animals were killed at 2, 4, 6, or 8 weeks after injection. Before euthanatization, 3% FITC-BSA buffer was injected into the retro-orbital plexus of one eye of three mice. Liver tissues were examined by light and fluorescence microscopy, and were stained with human anti-laminin. Vasculogenic mimicry patterns were reconstructed from serial laser scanning confocal microscopic stacks. RESULTS: OCM1a cells formed microscopic nodules in the mouse liver within 2 weeks after injection and metastasized to the lung 6 weeks later. By contrast, M619 and MUM2B cells formed expansile nodules in the liver within 2 weeks and gave rise to pulmonary metastases within 4 weeks after injection. Vasculogenic mimicry patterns, composed of human laminin and identical with those in human primary and metastatic uveal melanomas, were detected in the animal model. The detection of human rather than mouse laminin in the vasculogenic mimicry patterns in this model demonstrates that these patterns were of tumor cell origin and were not co-opted from the mouse liver microenvironment. CONCLUSIONS: There are currently no effective treatments for metastatic uveal melanoma. This direct-injection model focuses on critical interactions between the tumor cell and the liver. It provides for translationally relevant approaches to the development of new modalities to detect small tumor burdens in patients, to study the biology of clinical dormancy of metastatic disease in uveal melanoma, to design and test novel treatments to prevent the emergence of clinically manifest liver metastases after dormancy, and to treat established uveal melanoma metastases.     

Role of Fas ligand in uveal melanoma-induced liver damage

BACKGROUND: Uveal melanoma, the most common adult intraocular malignancy, metastasizes preferentially to the liver. Areas of cell death surrounding uveal melanoma metastases were observed in the livers of mice. We hypothesized that uveal melanoma cells might express Fas ligand (FasL), facilitating FasL-mediated apoptosis of Fas-expressing hepatocytes. PURPOSE: To determine whether Fas ligand (FasL)-expressing human uveal melanoma cells induce apoptosis of human hepatocytes in vitro and in vivo. METHODS: Human uveal melanoma cell lines were assayed for FasL expression by flow cytometry and immunohistology. A human hepatocyte cell line was assayed for Fas expression by flow cytometry. Apoptosis of hepatocytes was detected by annexin V staining in vitro, and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in vivo. RESULTS: Human uveal melanoma cell lines expressed FasL, as determined by flow cytometry and immunohistology. Human hepatocytes were Fas-positive by flow cytometry. In vitro, annexin V staining revealed that human uveal melanoma cells induced apoptosis of human hepatocytes. TUNEL staining of liver metastases revealed apoptosis of murine hepatocytes in contact with metastatic human uveal melanoma cells. CONCLUSION: FasL-induced apoptosis of hepatocytes in contact with FasL-positive human uveal melanoma cells may contribute to hepatic failure during metastatic disease.     

Proteomic analysis of uveal melanoma reveals novel potential markers involved in tumor progression

PURPOSE: Patient survival in uveal melanoma may benefit from earlier recognition of potential metastases to the liver, but as yet, proper markers indicating metastases are not available. Identification of metastasis markers would therefore be of great value. The proteins that are expressed in two cell lines originating from two liver metastases were compared with the proteins expressed in a cell line obtained from the primary uveal melanoma of the same patient, to identify proteins that play a role in tumor progression as well as proteins that are expressed specifically in metastases. METHODS: Protein analysis was performed by using two-dimensional gel electrophoresis. A subset of proteins was subsequently identified with mass spectrometry. RESULTS: A set of 24 proteins was differentially expressed in both of the two metastatic cell lines compared with the cell line derived from the primary tumor. These proteins were subdivided into groups according to cellular function, with important roles in tumor development. CONCLUSIONS: Tumor progression and development of metastases is a multicomplex system. Comparing protein expression in two cell lines derived from metastases with a cell line derived from a primary uveal melanoma from the same patient identified proteins involved in tumor progression, and proteins specifically expressed in the metastases, which have the potential of becoming clinically useful biomarkers.     

Patients presenting with stage IV uveal melanoma: Lessons learned

Challenges persist in identifying patients with stage IV uveal melanoma. While clinical, histopathologic, and genetic features of the primary tumor have been shown to provide prognostic value for assessing metastatic risk, biopsy-related genetic analyses are expensive and not universally available. Therefore, this review will focus on clinical characteristics. Initial staging and follow-up screening protocols have evolved for patients with uveal melanoma. The Collaborative Ocular Melanoma Study (COMS) required a physical examination, chest X-ray, and hematologic survey (primarily liver function tests). Though these studies were found to have a high specificity, COMS investigators typically found late-stage metastases. More recently, protocols have concentrated on liver imaging (abdominal ultrasound, computed tomography, and magnetic resonance imaging). Though hepatic radiographic imaging has been found more likely to reveal earlier metastatic uveal melanoma, by definition it cannot detect most extrahepatic and multiorgan metastases. An international multicenter registry study recently focused on patients who were diagnosed with stage IV uveal melanoma simultaneously with their primary intraocular melanoma. Therein, utilizing center-specific diagnostic methods, stage IV was found to occur in about 2% of patients. However, subgroup analysis found that a disproportionate number of multi-organ metastases were discovered when whole-body positron emission tomography/computed tomography was used for staging. Herein, we review the literature on patients who present with stage IV uveal melanoma, how they were detected, and their outcomes.     

Rabbit model of uveal Greene melanoma: morphologic studies of metastatic lesions

We studied the morphology of metastatic tumors in three rabbits with implanted choroidal Greene melanoma. The tumor-bearing eyes were perforated and destroyed by the expanding tumors 10 to 12 weeks after implantation. The animals then lost body weight and became cachectic; they were suspected of having metastases and were sacrificed. The interval between the destruction of the eye and the discovery of metastases was 1 to 14 weeks. Widespread metastases involved the liver, lungs, kidneys, and subcutaneous tissue, with the greatest number of tumors in the liver and lungs. Histopathologic findings in the metastases and primary tumors were identical. This model may be considered appropriate for study of human uveal melanoma because (1) the biological behavior is similar to that of human uveal melanoma, (2) the primary and metastatic tumors are histologically similar, and (3) the large size of the rabbit's eye facilitates observation and manipulation.     

Using the direct-injection model of early uveal melanoma hepatic metastasis to identify TPS as a potentially useful serum biomarker

PURPOSE: To develop a method to screen for serum biomarkers of early hepatic metastasis from uveal melanoma. METHODS: Cytokeratin 18 (TPS) was identified from gene expression profiles as protein generated by highly invasive uveal melanoma cells. Sera were collected from two groups of 15 SCID mice 2 weeks after injection of either tissue culture medium or MUM2B human metastatic uveal melanoma cells into the mouse liver. Serum TPS levels were assayed in 53 healthy human controls, 64 uveal melanoma patients who were disease free for at least 10 years, and 37 patients with metastatic uveal melanoma. RESULTS: After 2 weeks, small hepatic nodules (0.1-2.8 mm; mean, 0.80 mm) developed in 11 of 15 mice injected with MUM2B cells. Serum TPS levels in media-injected mice (84.7 U/L) were substantially lower than levels in MUM2B-injected mice (601 mug/L). TPS levels were significantly higher (P < 0.0001) in patients with metastatic uveal melanoma (139.63 +/- 22.20) than in healthy controls (54.23 +/- 0.01) or in patients free of disease (69.29 +/- 9.76). Significant differences were found between TPS levels before and after the development of hepatic metastases (P < 0.01), and serum TPS levels became elevated in four patients at least 6 months before the detection of hepatic metastases by abdominal ultrasonography. CONCLUSIONS: The direct-injection model of uveal melanoma in the mouse liver may be used to screen for potential serum biomarkers of metastatic uveal melanoma.     

Whole-body bioluminescent imaging of human uveal melanoma in a new mouse model of local tumor growth and metastasis

PURPOSE: Human uveal melanoma develops in one of the most capillary-rich tissues of the body and has a pure hematogenous dissemination. Radiodiagnostic examinations, such as ultrasonic diagnostic resonance imaging and chest radiographs plus liver enzyme studies in blood, are methods used to detect liver and other distant metastases in patients. Nevertheless, the mortality rate is high, because of the frequent occurrence of metastases and the lack of systemic therapy. Therefore, the development of novel anticancer strategies is urgent, and more sensitive and less invasive methods of detecting and monitoring in vivo tumor growth and metastatic disease in cancer models are needed. METHODS: A luciferase (Luc)-positive human uveal melanoma cell line (OCM-1 FRT/luc) was established. Tumor cells were inoculated into the anterior chamber of murine eyes for induction of orthotopic growth or into the left heart ventricle to mimic hematogenous micrometastatic spread. Development of metastases and tumor growth was monitored weekly by whole-body bioluminescent reporter imaging (BLI). RESULTS: Injection of cancer cells into the anterior chamber of the eye of mice closely mimicked orthotopic tumor growth of uveal melanoma. Tumor progression could be quantitatively monitored 3 weeks after inoculation of 10(5) OCM-1 FRT/luc cells. Of the mice injected, 83% exhibited a detectable tumor within 5 weeks. Intracardiac injection of tumor cells resulted in metastatic growth, especially in bone. Mice had bone (maxillofacial region and femora) and visceral (lung and mediastinum) metastases after 4 to 6 weeks. OCM-1 FRT/luc cells may also have a propensity to colonize the eye after intracardiac inoculation. CONCLUSIONS: BLI enables continuous quantitative monitoring in the same animal of growth kinetics for each tumor and its metastases. This model will accelerate the understanding of the pathogenesis and treatment of uveal melanoma and metastasis.     

Uveal melanoma expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and susceptibility to TRAIL-induced apoptosis

PURPOSE: The study had two purposes: to examine the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors on uveal melanoma cells and metastases arising from uveal melanoma and to determine the susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. METHODS: Nine human uveal melanoma cell lines and three cell lines derived from uveal melanoma metastases were examined for TRAIL receptor expression by flow cytometry. In vitro apoptosis assays were performed to determine the relative susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. Annexin V staining was also used to determine the capacity of either cycloheximide or interferon-beta to enhance TRAIL-induced apoptosis. RESULTS: Five of the nine uveal melanoma cell lines expressed TRAIL-R2 on more than 60% of the cells. All three of the cell lines derived from uveal melanoma metastases expressed TRAIL-R2 on more than 50% of the cells. Cycloheximide exerted a profound effect in enhancing TRAIL-induced apoptosis in all but two of the uveal melanoma cell lines and in all three of the metastases cell lines. Interferon-beta produced a similar enhancement of TRAIL-induced apoptosis, even in cell lines that were previously shown to be resistant. CONCLUSIONS: TRAIL is a potentially useful therapeutic modality for the management of uveal melanomas and their metastases. Moreover, pharmacological agents and biological response modifiers that independently display antineoplastic properties can enhance TRAIL-induced apoptosis in resistant uveal melanoma cells.     

Metastatic uveal melanoma. Development of neoadjuvant treatment in animal experiments

Background. Up to 50% of patients with uveal melanoma develop metastases but none of the existing treatments of the primary tumor has been able to reduce the metastatic rate. Probably, micrometatases have aleady developed before treatment of the uveal melanoma and dormant micrometastases can persist for years before they start growing. This long time-span provides the possibility to treat micrometastases. Methods. In order to develop an animal model for metastatic uveal melanoma, B16 melanoma cells were injected into the posterior ocular compartment of C57BL6 mice. These cells grew and metastasised to the lungs and liver. Immunological factors for the metastatic process and possible neoadjuvant treatments were investigated. Results. Natural killer cells (NK) are of significance in the rejection of metastases and HLA-I expression of uveal melanomas correlates with the melanoma cell type. Interferon-alpha-2b increases the activity of NK cells and reduces the metastatic rate in the animal model. Conclusion. Treatment with interferon-alpha-2b results in decreased metastases from intraocular melanoma in a murine model.     

Reduction of liver metastasis of intraocular melanoma by interferon-beta gene transfer

PURPOSE: This study determined whether adenovirus-mediated transfer of the murine interferon-beta (AdCMVIFN-beta) gene protects against liver metastases arising from intraocular melanomas in mice. METHODS: A replication-deficient adenovirus vector (AdCMVIFN-beta) was used for the in vivo transfer of the murine IFN-beta gene into intraocular melanoma-bearing mice. AdCMVIFN-beta was injected either intravenously or directly into the intraocular melanomas. The effect of gene transfer on liver metastases was ascertained by histopathologic analysis of the livers and by measuring serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are two enzymes associated with liver metastases in patients with uveal melanoma. RESULTS: Mice treated with two intratumoral injections of AdCMVIFN-beta had a 68% reduction in metastatic liver lesions (P = 0.016) and a 51% reduction in liver enzyme levels compared with control mice (P = 0.02). However, the antimetastatic effect of AdCMVIFN-beta was not directly attributable to the adenovirus vector or virus-mediated cytolysis of tumor cells. Intravenous treatment with AdCMVIFN-beta resulted in an 86% reduction in the number of metastatic foci in the liver (P = 0.014) and a 61% reduction of serum AST levels compared with mice treated with AdCMVLacZ (P = 0.015). AdCMVIFN-beta treatment produced a sharp increase in the NK cell activity that was demonstrable in vivo and in vivo. In vivo depletion of NK cells by anti-asialo GM1 antibody abrogated the antimetastatic effects of AdCMVIFN-beta. CONCLUSIONS: The results support the feasibility of activation of NK cell function through gene transfer as one possible therapeutic strategy for reducing hepatic metastases of uveal melanomas.     

HLA expression in choroidal melanomas: correlation with clinicopathological features

PURPOSE: In this retrospective study, we analyzed the relevance of human leukocyte antigen (HLA) expression to clinical behavior of uveal melanoma and correlated it with conventional light microscopic parameters. METHODS: HLA class I antigen and Beta 2 microglobulin expression were analyzed in 45 primary choroidal melanoma lesions by immunoperoxidase staining with monoclonal antibodies to HLA class I antigen and beta 2 microglobulin and correlated with the known clinicopathological parameters. Immunoanalysis was done by a semi quantitative method. The tumors were divided into 3 groups. Group A: Tumors with no extrascleral extension/no liver metastases, group B: tumors with only extrascleral extension and group C: tumors with liver metastases. For statistical analysis we analyzed the negative, weak (heterogeneous) and the positive expression of HLA and beta 2 microglobulin with known clinicopathological parameters. RESULTS: In-group A (n = 35) tumors with no extrascleral extension and no liver metastases HLA class I antigen and beta 2 microglobulin are negative (absent staining) in 30 choroidal melanomas. In-group B (n = 4) they are weak (heterogeneous) in 3 tumors. In-group C (n = 6) all the 6 tumors have a positive (bright staining) immunoexpression. No statistical significance was obtained when HLA and beta 2 microglobulin immunoreactivity was compared against largest tumor diameter (LTD), tumor infiltrating lymphocytes (TIL), mitosis and nuclear grade. The difference of HLA class I and beta 2-microglobulin imunoreactivity among the various cell types spindle, mixed and epithelioid was statistically significant (p = 0.003), (p = 0.004). The difference in immunoreactivity between tumors with no liver metastases and tumors with liver metastases was statistically significant (p < 0.001). CONCLUSIONS: HLA class I antigen and beta 2 microglobulin are negative in melanomas with no extrascleral extension and liver metastases and weak in melanomas with extrascleral extension and are positive in melanomas with liver metastases. HLA expression is independent of the conventional markers in uveal melanoma.     

Regional chemotherapy for uveal melanoma liver metastases

Chemotherapy remains an important approach for the treatment of liver metastases from uveal melanoma (UM). Compared with systemic chemotherapy, regional chemotherapy has similar efficacy and fewer systemic adverse effects. Regional chemotherapy for UM liver metastases includes hepatic artery infusion (HAI), transarterial chemoembolization (TACE), and isolated hepatic perfusion (IHP). In this review, we aim to examine the efficacy of regional chemotherapy and compare HAI, TACE, and IHP in terms of overall survival (OS). The three approaches showed no obvious difference in OS results.     

Cripto-1 expression in uveal melanoma: an immunohistochemical study

Human Cripto, the founder member of the epidermal growth factor-Cripto-FRL1-Cryptic (EGF-CFC) family, plays an important role during early embryonic development and in particular in carcinogenesis and the development of cancer metastases. Cripto-1 is over-expressed in most cancers, but is absent or only weakly expressed in normal cells. For this reason, Cripto-1 could be of potential value in the targeted treatment. There is no information on the expression of Cripto-1 in human uveal melanoma. Cripto-1 reactivity was evaluated by immunohistochemistry on 36 archival uveal melanomas using the polyclonal antibody to Cripto-1. The tumors were divided in to 2 groups. There were 18 uveal melanomas with no intrascleral or extrascleral extension and 18 uveal melanomas with intrascleral/extrascleral extension/liver metastasis. Cripto-1 reactivity was correlated with tumor aggressiveness and cell type. Furthermore, we studied the immunolocalization of Cripto-1 in 4 uveal melanoma cell lines OCM-1, OCM-8, and 92-1, and OMM-1 and in 2 primary uveal melanocyte cultures. Cripto-1 was expressed in both the non-invasive and aggressive uveal melanomas. Cripto-1 was positive in the 4 uveal melanoma cell lines and absent in the primary uveal melanocyte cultures. Retinal tissue did not express Cripto-1. The results suggest that Cripto-1 is expressed in uveal melanoma, negative in the non-neoplastic ocular tissue and point to its use as a target for therapy.     

Osteopontin expression and serum levels in metastatic uveal melanoma: a pilot study

PURPOSE: This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma. METHODS: Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed. RESULTS: By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%. CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.     

Uveal melanoma: Ocular and systemic disease

Although rare, uveal melanoma is the most common intraocular tumor in adults. Most cases arise from the choroidal layer of the uvea, displaying a discoid, collar-button, or mushroom shaped growth. Histopathologically, neoplasms are classified by the dominant cell type: spindle, epithelioid or mixed spindle cell type. The most important prognostic factors are cell type, nucleolar size, largest tumor dimension, and mitotic figures. Patient prognosis is poor when metastases occur in the liver, one of the main reasons that despite advances in the diagnosis and treatment of uveal melanoma, the mortality rate has not change significantly since 1973.     

Microcirculation and tumor-infiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases

PURPOSE: To investigate the relationship between progression to hepatic metastasis and tumor-infiltrating macrophages and microcirculation attributes in uveal melanoma, a cancer that almost invariably disseminates hematogenously to the liver. METHODS: A cross-sectional histopathologic analysis of 48 hepatic metastases and corresponding primary choroidal and ciliary body melanomas was conducted, by using statistical tests appropriate for paired data. Main outcome measures were the number and type of CD68-immunopositive, tumor-infiltrating macrophages, extravascular matrix loops and networks identified with periodic acid-Schiff stain, and microvascular density (MVD) counted as the number of discrete structures labeled by monoclonal antibody QBEND/10 to the CD34 epitope. RESULTS: Hepatic metastases had a significantly lower grade of pigmentation (P < 0.0001), more frequent epithelioid cells (P = 0.0027), more intermediate and dendritic types of CD68-immunopositive macrophages than round ones (P = 0.0031), and a higher MVD (median difference, 15 counts more/0.313 mm2, P = 0.0003) than the primary uveal melanomas that spawned the metastases. The frequency of tumors with extravascular loops and networks did not increase on metastasizing. The survival of the patient after diagnosis of disseminated disease tended to be shorter if hepatic metastases had a high MVD (P = 0.098), adjusting for the size of the specimen. CONCLUSIONS: Of the markers studied, the presence of epithelioid cells and MVD most closely parallel progression of uveal melanoma from primary tumor to metastasis. These two tumor characteristics may be interrelated, and high MVD may help to predict survival after detection of hepatic metastases. The results also suggest that the grade of pigmentation and morphologic type of tumor-infiltrating macrophages are interrelated.