Melanoma: Grossniklaus HE

Folberg R, Salomao D, Grossniklaus HE, Proia AD, Rao NA, Cameron JD, Anonymous00355. · Department of Ophthalmology at the University of Illinois at Chicago, 60612, USA. · Hum Pathol. · Pubmed #12612878 No free full text.

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Akor C, Greenberg MF, Pollard ZF, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA. · J Pediatr Ophthalmol Strabismus. · Pubmed #14974837 No free full text.

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Dithmar S, Adams AW, Rusciano D, Grossniklaus HE. · Universitäts-Augenklinik Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg. · Ophthalmologe. · Pubmed #11552416 No free full text.

Abstract: 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 already 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.

Dithmar S, Albert DM, Grossniklaus HE. · L.F. Montgomery Ophthalmic Pathology, Laboratory, BT 428 Emory Eye Center, Atlanta, Georgia 30322, USA. · Melanoma Res. · Pubmed #10890373 No free full text.

Abstract: Many attempts have been made to develop a suitable animal model to study more effectively the aetiology, pathogenesis, diagnosis and therapy of intraocular (uveal) melanoma. Uveal melanoma may spontaneously occur in some animals, including dogs, cats, horses, rats, mice, birds and fish. The histological features, metastatic behaviour and unpredictable nature of occurrence of these uncommon spontaneous tumours detract from their suitability as a model. Several methods have been developed to induce intraocular melanoma chemically or by radiation in laboratory animals. Some of these induced tumours resemble human uveal melanoma, although the majority originate from the retinal pigment epithelium. Uveal proliferations have been biologically induced by feline leukaemia/sarcoma virus and simian virus 40, although the presence of virus in tumour cells and extraocular tumours resulting from shed virus detract from the utility of this model. Inoculation of tissue culture hamster, murine or human melanoma cells into animal eyes has the advantage that the inoculation site and size of inoculum can be controlled. Disadvantages include the immune suppression necessary for tumour growth in some models as well as the fact that many of the melanoma cell lines are of cutaneous origin. Transgenic murine models have been developed using the promoter region of the tyrosinase gene to target expression of oncogenes in melanin-producing cells. Spontaneous intraocular pigmented tumours and distant metastases may occur, although many, if not all, of the intraocular tumours arise in the retinal pigment epithelium.

Grossniklaus HE, Margo CE, Solomon AR. · Department of Ophthalmology, Emory University, Atlanta, Georgia, USA. · Trans Am Ophthalmol Soc. · Pubmed #10703122 links to  free full text

Abstract: PURPOSE: The purpose of this study is to test the hypothesis that a subset of conjunctival melanocytic proliferations exists that cannot be reproducibly classified as benign, malignant, or indeterminate. METHODS: Three groups of excisional biopsy specimens of conjunctival melanocytic proliferations were evaluated by 5 ophthalmic pathologists. These groups included lesions that were considered by the authors to represent benign (Group 1, n = 5), malignant (Group 2, n = 5) and indeterminate melanocytic proliferations (Group 3, n = 5). The panel classified the same sections in all 3 groups in a randomized, masked fashion, first without and then with a clinical history of patient age, sex and race. The kappa statistic (k) was used to quantify the degree of agreement among observers. RESULTS: There was strong concordance among the panel for both Group 1 (benign, k = 0.76) and Group 2 (malignant, k = 0.70) melanocytic proliferations. There was no concordance of the panel for Group 3 (indeterminate) lesions (k = -0.045). The concordance for Groups 1 and 2 and lack of concordance for Group 3 lesions were independent of knowledge of clinical history of age, sex, and race. CONCLUSIONS: A subset of melanocytic proliferations of the conjunctiva exists that cannot be reproducibly classified by pathologists as benign, malignant, or indeterminate.

Grossniklaus HE, Margo CE, Solomon AR. · Department of Ophthalmology, Emory University, Atlanta, GA, USA. · Arch Ophthalmol. · Pubmed #10496383 No free full text.

Abstract: OBJECTIVE: To test the hypothesis that a subset of conjunctival melanocytic proliferations exists that cannot be reproducibly classified as benign, malignant, or indeterminate. METHODS: Three groups of excisional biopsy specimens of conjunctival melanocytic proliferations were evaluated by a panel of 5 ophthalmic pathologists. These groups included lesions that we considered to represent benign (group 1 [n = 5]), malignant (group 2 [n = 5]), and indeterminate melanocytic proliferations (group 3 [n = 5]). The panel classified the same sections in all 3 groups in a randomized, masked fashion, first without and then with a clinical history of patient age, sex, and race. The kappa statistic was used to quantify the degree of agreement among observers. RESULTS: There was strong concordance among the panel members for both group 1 (benign [kappa = 0.76]) and group 2 (malignant [kappa = 0.70]) melanocytic proliferations. There was no concordance of the panel for group 3 (indeterminate) lesions (kappa = -0.045). The concordance for groups 1 and 2 and lack of concordance for group 3 lesions were independent of knowledge of clinical history of age, sex, and race. CONCLUSION: A subset of melanocytic proliferations of the conjunctiva exists that cannot be reproducibly classified by pathologists as benign, malignant, or indeterminate.

Eagle RC, Grossniklaus HE, Syed N, Hogan RN, Lloyd WC, Folberg R. · Department of Pathology, Wills Eye Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. · Arch Ophthalmol. · Pubmed #19204229 No free full text.

Abstract: OBJECTIVES: To report an important complication of ocular evisceration therapy for blind, painful eyes that has been unreported in the literature, and to stress the need for careful preoperative evaluation to exclude occult neoplasms prior to therapy. DESIGN: Multicenter, retrospective, nonrandomized clinicopathological case series of patients found to have previously unsuspected uveal malignant melanoma during histopathological examination of blind, painful eyes treated by evisceration. RESULTS: Histopathological examination of evisceration specimens disclosed previously unsuspected uveal melanoma in 7 patients who were treated for blind, painful eyes. Inflammation caused by necrosis of the tumor and other ocular tissues led to misdiagnosis as endophthalmitis, orbital cellulitis, or idiopathic orbital inflammation in several cases. Preoperative imaging was not performed in 3 cases and failed to detect tumors in the remaining 4 cases. Failure of necrotic tumors to enhance contributed to misdiagnosis. CONCLUSIONS: The presence of a malignant intraocular neoplasm should be excluded prior to evisceration of any blind eye or blind, painful eye, particularly with opaque media. Necrosis-related inflammation can confound the clinical diagnosis of occult lesions, as can failure of necrotic tumors to enhance on imaging studies.

Yang H, Fang G, Huang X, Yu J, Hsieh CL, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA. · Melanoma Res. · Pubmed #18337645 No free full text.

Abstract: The purpose of the study is to develop a mouse ocular melanoma model with human uveal melanoma cells that forms hepatic micrometastases. Human uveal melanoma Mel290 cells were transfected with a lentiviral-enhanced green fluorescent protein (EGFP) expression vector. Proliferation assays were performed by comparing Mel290-EGFP and Mel290 cells. After stable expression of EGFP and proliferation was ascertained, 1 x 10 Mel290-EGFP cells were introduced into NU/NU mice by posterior compartment (PC) inoculation or tail vein injection. Control groups were inoculated or injected with Mel290 cells. Ocular and hepatic frozen sections were examined by fluorescence microscopy, and the number of hepatic micrometastases was determined. EGFP expression was observed at 24 h after transfection. At 72 h after transfection, more than 70% of Mel290 cells expressed EGFP. At 45 days (six passages), 90% of Mel290 cells stably expressed EGFP. Histologic examination showed that Mel290-EGFP cells formed hepatic micrometastases after either PC inoculation or tail vein injection. A significant difference in the number of hepatic micrometastases between PC inoculation and tail vein injection (P<0.01) was observed. Mel290-EGFP cells stably expressed green fluorescent protein in vitro at 45 days (six passages). These cells formed hepatic micrometastases in NU/NU mice after PC inoculation or tail vein injection, with significantly more micrometastases developing in the PC inoculation model than after tail vein injection.

Avery RB, Diener-West M, Reynolds SM, Grossniklaus HE, Green WR, Albert DM. · Division of Ophthalmology, University of New Mexico, Albuquerque, USA. · Arch Ophthalmol. · Pubmed #18268211 links to  free full text

Abstract: OBJECTIVE: To describe the histopathologic findings in eyes with uveal melanoma that had secondary enucleation after failed brachytherapy plaque treatment. METHODS: Histopathologic findings in eyes that had secondary enucleation after plaque radiation therapy in the Collaborative Ocular Melanoma Study (COMS) were reported on a standardized data form. The findings were compared with eyes that had primary enucleation for uveal melanoma. RESULTS: Seventy-five eyes that had secondary enucleation were studied. Compared with primary enucleations, tumors in the irradiated eyes had lower mitotic activity, a smaller proportion of histologically intact tumor, more inflammation, more fibrosis, and more vascular damage within the tumor. In addition, compared with primary enucleations, eyes previously irradiated had a higher frequency of retinal invasion by the tumor and greater damage to the retinal vasculature, consistent with radiation retinopathy; neovascularization of the iris; and vitreous hemorrhage. Tumor growth or extrascleral extension was confirmed histopathologically in 25 of 42 eyes (60%) enucleated because of a reported failure of local control. CONCLUSIONS: Eyes with secondary enucleation after brachytherapy differ histopathologically from eyes with primary enucleation for uveal melanoma. These histopathologic differences may be due to the effects of radiation, tissue conditions related to plaque failure, and, in some cases, tumor growth. In 40% of eyes enucleated because of suspected failure of local control, increased tumor size could not be histologically confirmed.

Khalifa Y, Aaberg TM, Aaberg TM, Grossniklaus HE. · Department of Ophthalmology and Pathology, Emory University School of Medicine, Atlanta, Georgia, USA. · Indian J Ophthalmol. · Pubmed #17699951 links to  free full text

Abstract: We identified three eyes that had undergone enucleation after transscleral resection of uveal melanoma. Two enucleated eyes with microscopically positive margins of resection exhibited no evidence of residual melanoma and these patients were alive without metastasis with at least four years' follow-up. One eye with a transected melanoma contained residual melanoma and that patient died with metastatic melanoma to the liver three years after enucleation. There appear to be at least two general types of positive surgical margins of resection of uveal melanoma: microscopically positive margins and macroscopically positive (transected) margins of resection.

Yang H, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. · Curr Eye Res. · Pubmed #16769615 No free full text.

Abstract: PURPOSE: To evaluate the combined effect of neoadjuvant intracameral interferon alpha -2b and adjuvant low-dose angiostatin in reducing the number of hepatic micrometastases in a murine model of ocular melanoma. METHODS: The posterior compartments of the right eyes of C57BL6 mice were inoculated with 5 x 10(5) cells/2.5 microl of cells from the Queens, B16F10, or B16LS9 melanoma cell lines. The right eyes were enucleated at 7 days, and the mice were sacrificed at 28 days postinoculation, respectively. Hepatic micrometastases were counted. There were four treatment groups (n = 15 each) for each cell line as follows: group 1, intraperitoneal injections of 20 KIU interferon alpha -2b for 4 days prior to enucleation; group 2, intramuscular injections of 100 microl 0.1 microg/microl murine angiostatin every day for 14 days starting on day 1 after enucleation; group 3, treatment of group 1 and group 2 combined; group 4, intraperitoneal and intramuscular injections of equal volumes of phosphate-buffered saline (PBS) (control group). RESULTS: Results showed decreased micrometastases for groups 1 through 3 compared with group 4, with the greatest reduction in group 3 (p < 0.006). CONCLUSIONS: This study suggests that combined neoadjuvant interferon alpha -2b and adjuvant low-dose angiostatin therapy act synergistically to decrease hepatic micrometastases in a murine ocular melanoma model.

Yang H, Xu Z, Iuvone PM, Grossniklaus HE. · Departments of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA. · Mol Vis. · Pubmed #16735992 links to  free full text

Abstract: PURPOSE: Our previous experiments have shown that low dose angiostatin results in decreased hepatic micrometastasis in a mouse model of uveal melanoma. The purpose of these experiments is to evaluate the effect of angiostatin on in vitro migration of melanoma cells and to explore the in vivo mechanism of angiostatin in our model. METHODS: For in vitro studies, quantitative RT-PCR was used to detect VEGF and PEDF mRNA in mouse B16LS9 melanoma cells and Mel290 human uveal melanoma cells with or without supplemental 0.1 mug/ml murine or human recombinant angiostatin. A wound healing assay was used to measure cellular migration in these two groups of cells. For the in vivo mechanism, aliquots of tissue culture B16LS9 cells treated with or without 0.1 mug/ml murine angiostatin were heterotopically inoculated into the posterior compartments of the right eyes of C57BL/6 mice. Frozen hepatic tissue was prepared and stained with hematoxylin using an RNase-free technique. Hepatic micrometastatic uveal melanoma cells were obtained by laser capture microdissection (LCM). Levels of VEGF and PEDF mRNA were detected by real time RT-PCR in the hepatic micrometastases. RESULTS: After in vitro treatment of the cell lines with angiostatin, the ratio of VEGF/PEDF mRNA significantly decreased in the B16LS9 (0.88+/-0.11 [mean+/-standard deviation] versus 2.70+/-0.15 in the control group; p=0.00006) and Mel290 (0.12+/-0.02 versus 0.68+/-0.04 in the control group; p=0.00346). However, the absolute VEGF mRNA and PEDF mRNA did not significantly change (p>0.08 for both cell lines). The migration assay showed significantly decreased migration at 24 h and 48 h after angiostatin treatment for both B16LS9 (p<0.01) and Mel290 (p<0.01) cell lines. For the in vivo experiments, pretreatment with angiostatin resulted in a decreased VEGF/PEDF mRNA ratio in B16LS9 cells compared to controls (0.0274+/-0.0070 versus 0.1726+/-0.0313; p=0.0014). Additionally, there was significantly increased PEDF mRNA (2.14+/-0.12 versus 0.30+/-0.05 in the control group; p=0.00002) in the liver metastases after pretreatment with angiostatin. CONCLUSIONS: Angiostatin inhibits the migration of melanoma cells in vitro. Angiostatin significantly decreases the ratio of VEGF/PEDF mRNA level in vitro and in hepatic micrometastatic melanoma cells. Angiostatin increases PEDF mRNA in melanoma metastases.

Gallardo MJ, Randleman JB, Price KM, Johnson DA, Acosta S, Grossniklaus HE, Stulting RD. · Department of Ophthalmology, University of Texas Health Sciences Center, San Antonio, TX, USA. · Am J Ophthalmol. · Pubmed #16387002 No free full text.

Abstract: PURPOSE: To report cases of ocular argyrosis that developed after long-term self-application of commercially available eyelash tint. DESIGN: Observational case series. METHODS: Case review, clinicopathologic analysis, and literature review. RESULTS: Three patients developed ocular argyrosis after the long-term self-application of Revlon Professional Roux Lash and Brow Tint (Colomer USA Corp, New York, New York, USA). Clinical evaluation revealed various degrees of silver deposition on the upper eyelid, lid margin, caruncle and conjunctiva, and diffuse Descemet's membrane deposits. In one case, histologic examination demonstrated silver deposition in the basement membrane and superficial substantia propria of the conjunctiva. CONCLUSIONS: Argyrosis can occur after long-term application of readily available eyelash tints, and the deposition of silver may be permanent. In certain circumstances, conjunctival argyrosis may simulate benign and malignant lesions, including conjunctival melanoma. These products should only be applied by trained cosmetologists.

Yang H, Akor C, Dithmar S, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA. · Mol Vis. · Pubmed #15623988 links to  free full text

Abstract: PURPOSE: To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma. METHODS: Angiostatin and plasminogen expression was detected in three murine melanoma cell lines (Queens, B16F10, and B16LS9). The three cell lines were heterotopically inoculated into the posterior compartment (PC) of the right eyes of C57BL/6 mice. After enucleation, the mice were given injections of 100 microl PBS and low dose (0.1 microg/microl) or high dose (0.3 microg/microl) murine recombinant angiostatin every day for 14 days after enucleation. The mice were sacrificed at 21 days post-enucleation and hepatic micrometastases were counted. In vitro migration/invasion assays were performed with low (0.1 microg/microl) and high (50 microg/microl) concentration angiostatin supplementation. Quantitative RT-PCR detected mRNA and Western analysis determined protein expression of VEGF for all cell lines. Evaluation of TdT mediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of the micrometastases determined apoptosis and proliferation ratios. RESULTS: There was a decrease in micrometastasis in the low dose group for Queens (p<0.05), B16F10 (p<0.05), and B16LS9 melanoma (p<0.01) cell lines. Two of the cell lines (B16F10 and B16LS9) elaborated plasminogen and were able to cleave plasminogen into K1-K4 (angiostatin). There was a decrease in the in vitro migration and invasion after supplementation with low concentration compared to high concentration angiostatin (p<0.01). VEGF mRNA and protein expression decreased in all cells lines in low concentration angiostatin, with the greatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios were increased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low dose angiostatin. CONCLUSIONS: There were significantly fewer micrometastases in treated compared to controls with low dose compared to high dose angiostatin. This treatment results in apoptosis in the micrometastases. The mechanism appears to be related an anti-migratory effect and altered VEGF expression by melanoma cells.

Yang H, Dithmar S, Grossniklaus HE. · Department of Ophthalmology, Emory University, Atlanta, Georgia 30322, USA. · Invest Ophthalmol Vis Sci. · Pubmed #15223777 links to  free full text

Abstract: PURPOSE: To investigate in a murine model the mechanism by which micrometastatic melanoma, which spreads from the eye to the liver, is controlled by interferon (IFN)-alpha 2b. METHODS: Major histocompatibility (MHC) class I antigen (H-2, all haplotypes) expression in three murine melanoma cell lines (Queens, B16LS9, B16F10) was determined by flow cytometric immunophenotyping. The cell lines were heterotopically inoculated into the posterior compartments (PCs) of C57Bl/6 mice, and the mice were given intraperitoneal (IP) injections of IFN-alpha 2b or PBS for 1 or 4 days before enucleation at 7 days after inoculation. Groups of mice were made NK deficient or depleted with subcutaneous (s.c.) injection of anti-asialo GM1. The mice were killed at 28 days or 56 days (survival experiment) after inoculation, and the number of hepatic micrometastases was histologically determined. NK cells were isolated from the spleen and liver at necropsy, and propidium iodide labeled target-specific cytolysis was determined by flow cytometry. The micrometastases were evaluated for apoptosis and proliferation with TUNEL and MIB1 immunostaining, respectively, and TUNEL-to-MIB1 ratios were determined. Hepatic NK cells were immunostained with CD49b. RESULTS: MHC class I antigen was expressed in the three cell lines in the order of Queens < B16LS9 < B16F10. All cell lines grew, were confined to the PC, and formed hepatic micrometastases. A decrease in micrometastases, an increase in target-specific cytolysis, and an increase in survival correlated with decreased HLA class I expression by the melanoma cells. The IFN-alpha 2b treatment resulted in a boost of intrinsic hepatic NK cells, demonstrated in NK-deficient but not NK-depleted mice. The treatment effect corresponded to increased apoptosis (TUNEL)-proliferation (MIB1) ratios in the micrometastases. Immunostaining demonstrated an increased number of intrahepatic NK cells associated with the micrometastases in treated groups. CONCLUSIONS: Neoadjuvant IFN-alpha 2b results in decreased hepatic micrometastasis and increased survival time through increased intrinsic hepatic NK cell-mediated tumor apoptosis in a murine model of metastatic ocular melanoma.

Zaldivar RA, Aaberg TM, Sternberg P, Waldron R, Grossniklaus HE. · Department of Ophthalmology, Emory University, Atlanta, Georgia, USA. · Am J Ophthalmol. · Pubmed #12719073 No free full text.

Abstract: PURPOSE: To review the clinicopathologic features of eyes enucleated after failed transpupillary thermotherapy. DESIGN: Retrospective review. METHODS: Pathology reports in the L.F. Montgomery Laboratory between 1998 and 2002 were searched for enucleated eyes with choroidal melanoma that had been treated only by transpupillary thermotherapy (TTT) prior to enucleation. The clinical features of the patients, including ultrasonography examination, were reviewed and compared with the pathologic findings. RESULTS: Seven eyes from seven patients had been enucleated, representing 8% of eyes treated with TTT at our institute during the period studied. The primary indication for enucleation was tumor growth. The melanomas tended to grow laterally, with minimal if any increase in thickness. Five of the seven eyes histologically demonstrated extrascleral extension, which was detected by ultrasonography prior to enucleation in only one of those eyes. CONCLUSIONS: Choroidal melanoma may continue to grow along a path of least resistance after TTT. The melanoma may extend laterally in the choroid and through emissary canals. Early extrascleral extension may be difficult to detect by ultrasonography.

Iwamoto S, Burrows RC, Grossniklaus HE, Orcutt J, Kalina RE, Boehm M, Bothwell MA, Schmidt R. · Division of Dermatology, Department of Medicine, University of Washington Medical Center, Box 356524, Seattle, WA 98195-6524, USA. · Arch Ophthalmol. · Pubmed #12470134 No free full text.

Abstract: OBJECTIVE: To characterize the immunophenotypic expression pattern of conjunctival melanomas, with the use of standard melanoma markers as well as microphthalmia transcription factor and p75 neurotrophin receptor. DESIGN: Eleven conjunctival melanomas, including 1 caruncular melanoma, were immunolabeled with a panel of antibodies that included S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, microphthalmia transcription factor, and p75 neurotrophin receptor. The results were tabulated on the basis of intensity and pervasiveness of labeling and compared with a previous study of uveal melanomas. RESULTS: Immunolabeling with S100 was at significantly higher levels in conjunctival melanomas than in uveal melanomas. Tyrosinase, HMB-45 and HMB-50 combination, melan-A, and microphthalmia transcription factor were expressed at high levels in conjunctival melanomas, whereas p75 neurotrophin receptor was not expressed. CONCLUSIONS: Melanomas of the conjunctiva, including the caruncle, expressed S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, and microphthalmia transcription factor at high levels, suggesting that these are good markers for this melanoma subtype. Expression of S100 was significantly higher in conjunctival melanomas than in uveal melanomas. The immunophenotypic pattern of conjunctival melanomas is most similar to the epithelioid subtype of cutaneous melanomas.

Dithmar S, Crowder J, Jager MJ, Vigniswaran N, Grossniklaus HE. · Universitäts-Augenklinik Heidelberg, lm Neuenheimer Feld 400, 69120 Heidelberg. · Ophthalmologe. · Pubmed #12227276 No free full text.

Abstract: PURPOSE: The prognosis of uveal melanoma is correlated with its histologic cell type. The epithelioid cell type is associated with a higher metastatic rate than the spindle cell type. The Human Leucocyte Antigen Class I (HLA-I) expression of the melanoma also correlates with the prognosis. In this study, we analyzed HLA-I antigen expression of uveal melanomas to determine whether a relationship exist between antigenic expression and melanoma cell type. METHODS: Formalin-fixed, paraffin-embedded spindle cell type (n = 11) and epithelioid cell type (n = 11) uveal melanomas were immunostained with the HC10 antibody (1:80) for HLA-I antigen expression with appropriate positive and negative controls. Sections were assessed semiquantitatively according to the percentage of stained cells. RESULTS: Among the spindle cell type melanomas, 2 out of 11 (18%) stained with HC10 antibodies. The staining intensity was less than 25% of the cells in these two melanomas. Among the epithelioid cell type melanomas, 9 out of 11 (82%) stained with HC10. The staining intensity was more than 25% of the cells in 5 of these 9 melanomas. CONCLUSIONS: It is unknown why spindle and epithelioid cell type uveal melanomas have different prognoses. Human uveal melanoma cell lines with low HLA-I expression are susceptible to NK cell-mediated lysis in vitro and in murine studies. The prognostically more favorable spindle cell type melanoma expresses less HLA-I than the epithelioid cell type melanoma. These results stress the role of NK cells in the rejection of uveal melanoma.

O'Reilly FM, Brat DJ, McAlpine BE, Grossniklaus HE, Folpe AL, Arbiser JL. · Department of Dermatology, Emory University School of Medicine, 5309 Woodruff Memorial Bldg., Atlanta, GA 30322, USA. · J Am Acad Dermatol. · Pubmed #11511840 No free full text.

Abstract: BACKGROUND: Melanoma is the most lethal form of skin cancer. Diagnosis of amelanotic melanoma and detection of micrometastases in sentinel lymph nodes pose diagnostic and therapeutic dilemmas for the dermatopathologist and clinician. OBJECTIVE: The purpose of this article is to determine the utility of immunohistochemistry using antibodies specific for microphthalmia in the identification of melanocytic lesions in the skin, eye, central nervous system, and sentinel lymph nodes. METHODS: Paraffin-embedded, formalin-fixed specimens of cutaneous melanoma, including amelanotic melanoma and lentigo maligna melanoma, were stained with antibodies specific for microphthalmia. In addition, paraffin sections of extracutaneous lesions, including sentinel lymph nodes, uveal melanoma, and central nervous system melanocytomas, were stained with the specific microphthalmia antibody. RESULTS: All cutaneous melanomas stained positively with microphthalmia, as did uveal melanomas and central nervous system melanocytomas. These findings confirm the melanocytic origin of melanocytomas and uveal melanomas and demonstrate that microphthalmia staining can be used to establish melanocytic origin of neoplasms. In addition, micrometastases were easily detected in sentinel lymph nodes. CONCLUSION: Microphthalmia transcription factor immunohistochemistry is a valuable tool in the identification of melanocytic lesions in numerous sites. Use of this stain may facilitate detection of micrometastases in sentinel lymph nodes.

Dithmar S, Rusciano D, Lynn MJ, Lawson DH, Armstrong CA, Grossniklaus HE. · No affiliation provided · Arch Ophthalmol. · Pubmed #10922203 No free full text.

Abstract: OBJECTIVES: To investigate the treatment of metastasis from uveal melanoma and to test the effect of interferon (IFN) alfa-2b in a murine model. METHODS: The B16-LS9 tissue culture melanoma cells were inoculated into the posterior intraocular compartment of 3 groups of C57BL/6 mice. The inoculated eyes were enucleated at 9 days and the mice were euthanized at 26 days after inoculation; the site and number of metastases were determined using standard histologic techniques. Group 1 was the control group; group 2 was given 20,000 international units (IU) of IFN alfa-2b intramuscularly 12 hours before enucleation, and group 3 received daily injections of 20,000 IU of IFN alfa-2b intramuscularly starting 4 days before enucleation. RESULTS: Pulmonary metastases were detected in 57%, 33%, and 0% of groups 1, 2, and 3, respectively; hepatic micrometastases were detected only in group 1. These results showed a significant decrease in hepatic metastases in mice receiving IFN alfa-2b vs controls (P =.005). CONCLUSION: Treatment with IFN alfa-2b results in decreased hepatic metastases from intraocular melanoma in a murine model. Arch Ophthalmol. 2000;118:1085-1089

Dithmar S, Rusciano D, Grossniklaus HE. · Department of Ophthalmology, Emory University, School of Medicine, Atlanta, Georgia, USA. · Melanoma Res. · Pubmed #10711634 No free full text.

Abstract: The aim of this study was to compare the transcorneal and transconjunctival techniques for the implantation of intraocular melanoma cells and development of metastasis in a murine model. Groups of C57BL/6 mice were given either transconjunctival or transcorneal inoculations of 2.5 x 10(5)/2.5 microl tissue culture B16-LS9 melanoma cells into the intraocular posterior compartment (PC). The eyes were enucleated at 4-11 days post-inoculation and histologically examined. The mice were sacrificed 14 days after enucleation and necropsies were performed with histological evaluation for visceral metastases. Intraocular and extraocular tumour growth was present in all of the eyes inoculated via the transconjunctival route. Pulmonary metastases were found in this group if the eye was enucleated 7 or more days post-inoculation. The melanoma remained confined to the inside of the eye in the transcorneal group until day 7. Haematogenous metastases to the lung and liver developed from the intraocular melanoma in this group. Transcorneal inoculation of tissue culture melanoma cells into the murine PC provides a useful animal model for visceral metastasis of ocular melanoma.

Dithmar S, Diaz CE, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA. · Retina. · Pubmed #10696752 No free full text.

Abstract: PURPOSE: To report two cases of regional lymphatic spread of primary uveal melanoma. METHODS: The clinical records of two patients who underwent enucleation for uveal melanoma and later developed regional lymph node metastases were reviewed. One of the two eyes was initially treated with proton beam irradiation. Histologic sections of the enucleated eyes and excised lymph nodes were examined. RESULTS: The melanomas arose in the choroid and ciliary body of the two patients and spread to regional lymph nodes 2 years after enucleation. The choroidal melanoma recurred after irradiation, diffusely infiltrated the uveal tract, and extended into the conjunctiva via an emissary canal. The ciliary body melanoma spread through the trabecular meshwork to the conjunctiva. CONCLUSIONS: Choroidal and ciliary body melanoma may rarely exhibit regional lymph node metastasis. This mode of metastasis may occur after extraocular spread and invasion of conjunctival lymphatics.

Dithmar SA, Rusciano DA, Armstrong CA, Lynn MJ, Grossniklaus HE. · Emory University School of Medicine, Department of Ophthalmology, Atlanta, GA, USA. · Curr Eye Res. · Pubmed #10520219 No free full text.

Abstract: PURPOSE: To study the role of natural killer (NK) cells in growth of spontaneous hepatic metastasis in a murine intraocular melanoma model. METHODS: Tissue culture B16-LS9 melanoma cells were analyzed by flow cytometry for MHC class I expression of all haplotypes and inoculated into the posterior compartment (PC) of one eye of C57BL6 mice. The eyes were enucleated at 12 days post-inoculation and histologically examined for tumor growth. One group of mice (n = 10) were given intraperitoneal injections of anti-asialo GM1 for NK cell depletion post-enucleation and a second group of mice (n = 9) served as controls. The mice were sacrificed at 24 days post-inoculation and necropsies were performed to determine the number and size of metastasis. RESULTS: The B16-LS9 cells failed to express MHC class I antigen. Tumor grew in the PC of all eyes and metastasized to the lungs and livers of all mice, with the average number of hepatic micrometastases greater in the NK depleted group versus the control group (p =.009). There was no significant difference in the average number of pulmonary metastases in the treated versus the control group (p =.072). Hepatic metastases grew to an average diameter of 600 microm in diameter in two NK depleted mice. CONCLUSIONS: NK depletion in this model of metastatic ocular melanoma results in increased number and growth of hepatic micrometastases.

Dithmar S, Völcker HE, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA. · Ophthalmology. · Pubmed #10406619 No free full text.

Abstract: PURPOSE: To describe two eyes from two patients with multifocal primary intraocular melanoma. DESIGN: Two case reports. METHODS: The history and histologic findings in the enucleated eyes of two patients with multifocal intraocular melanoma are described in comparison to previously reported cases. MAIN OUTCOME MEASURES: Pathologic examination of enucleated eyes. RESULTS: One of the two eyes contained mixed cell type melanomas, and one eye contained spindle cell type melanomas. Examination of serial sections showed no continuity between the intraocular melanomas. There were no associated ocular or systemic conditions with the multifocal intraocular melanomas. CONCLUSIONS: Multifocal primary intraocular melanoma is rare. There is no known predisposing factor to this condition.

Diaz CE, Rusciano D, Dithmar S, Grossniklaus HE. · Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA. · Curr Eye Res. · Pubmed #10223656 No free full text.

Abstract: PURPOSE: To create a murine ocular melanoma model that consistently metastasizes to the liver. METHODS: Twelve-week-old C57BL6 mice (n=10) were inoculated in the posterior compartment (PC) of one eye with 5x10(5) tissue culture B16LS9 melanoma cells. The inoculated eyes were enucleated at two weeks and the mice were sacrificed with necropsies performed at four weeks post-inoculation. RESULTS: Melanoma grew and was confined to the eyes of all 10 mice. The melanoma hematogenously spread to the lungs in 9 of 10 mice and the liver in 8 of 10 mice. There was a positive correlation between pulmonary and liver metastasis (r=0.94). CONCLUSIONS: B16LS9 melanoma cells consistently hematogenously spread to the liver when implanted into C57BL6 mice eyes. The value of this model is unknown at this time since it is not known if the intrahepatic melanoma is capable of growing and killing the host.