Inflammation in uveal melanoma.

Leukocytic infiltration is a common feature of human cancers, including those that develop in immunoprivileged sites, such as the eye. The infiltration of myeloid and T cells into tumours is part of the host response against cancer. In uveal melanoma, high densities of immune cells seem to be involved in tumour progression, as they are associated with the loss of one chromosome 3. The nature of this tumour microenvironment might offer therapeutic opportunities.

Alterations of lymphocyte subpopulations in choroidal melanoma patients undergoing surgery.

PURPOSE: The alterations of lymphocyte subpopulations assessment after surgery in choroidal melanoma patients compared to cataract patients. MATERIAL AND METHODS: 12 patients with malignant melanoma of the choroid, 10 patients subjected to surgery due to cataract. Methods--flow cytometric measurement of absolute lymphocyte count, the number of all T cells (CD3+), T helper lymphocytes (CD3+ CD4+), T cytotoxic lymphocytes (CD3+CD8+), B lymphocytes (CD19+), NK cells (CD3-CD16+) and T cells (CD3+) cells with gammasigma TCR, on the day of surgery and two days after it. RESULTS: Comparable numbers of cells were observed in both groups prior to surgery, but the behavior of some populations differed: CD3+, CD3+CD4+ cells increased in melanoma patients whereas they decreased in reference group, the number of T lymphocytes with gammasigma TCR was significantly higher in melanoma patients before surgery and it did not differ after it. CONCLUSIONS: Though there were no significant differences in lymphocyte subpopulations between melanoma patients and the reference group, it seems that the presence of tumour influences the reactivity of the immune system to the trauma (surgery).

Pyrogenic cytokine interleukin-6 expression by a chordoid meningioma in an adult with a systemic inflammatory syndrome. Case report and review of the literature.

Chordoid meningioma is a rare meningothelial tumor characterized by chordoma-like histological features with lymphoplasmacellular infiltration. This tumor is often seen in children, but not in adults, with a systemic inflammatory syndrome (iron-resistant microcytic anemia and/or dysgammaglobulinemia) and very rarely with a persistent moderate hyperthermia. In the present report the authors describe a temporal chordoid meningioma in a 30-year-old woman who presented with fever, headache, and a serological inflammatory syndrome. The clinical symptomatology, chiefly the fever, disappeared immediately after removal of the tumor. To the authors' knowledge, only one similar patient with such clinical presentation and response to surgery has been mentioned in the literature. Interestingly, at immunohistochemical examination, the neoplasm showed focal positivity for the pyrogenic cytokine interleukin-6. The capacity of the tumor to produce this pyrogenic cytokine could explain both the patient's clinical presentation and her response to the surgical management.

[Serum antiretinal antibodies (ARA) in patients with choroidal melanoma--preliminary report]. / Przeciwciala przeciwsiatkówkowe (PPS) w surowicy chorych z czerniakiem naczyniówki--doniesienie wstepne.

PURPOSE: To estimate serum antiretinal antibodies (ARA) in patients with choroidal melanoma after therapy in 3 months follow-up period. MATERIAL AND METHODS: 24 patients at the age 37-72 years (mean: 57.8 years) with choroidal melanoma were examined. In all cases routine ophthalmic examination with A- and B-scan ultrasonography and fluorescein angiography were performed. In some subjects also indocyanine green angiography was performed. Ruthenium-106 plaques were used in 16 cases, in 5 cases treatment with Iodine-125 plaques was performed. Three patients underwent enucleation. In all cases ARA were determined in a serum dilution 1:10 by indirect immunofluorescence test on normal monkey retina as a substrate and FITC-labeled anti-human IgA, IgG, IgM serum (Euroimmun--Germany). ARA were scored before and within 3 months, after the therapy was performed. RESULTS: Before treatment ARA were present in 15 patients (62.5%) with choroidal melanoma in serum dilution 1:10. In a control group ARA were present in 3 cases (12.5%) in serum dilution 1:10. After therapy within 3 months follow-up period ARA occurred in serum of further 7 patients, all treated with Ruthenium-106 plaques. Fluorescence of outer retinal segments was present in 7 cases, while serum of remaining 15 patients demonstrated positive reaction within the whole tissue. Indirect immunofluorescence test on normal monkey retina revealed in 11 patients the presence of other autoantibodies; antinuclear and antinucleolar antibodies, reacting with nuclei of inner and outer nuclear layers of retina. CONCLUSIONS: The presence of ARA in serum of patients with choroidal melanoma before therapy may be a result of autoimmune reaction against the tumor tissue. The detection of ARA in serum after plaque therapy may be associated with reaction due to tumor irradiation. The appearance of other serum autoantibodies in patients with choroidal melanoma may indicate at more advanced and generalized process.

Human uveal melanoma expresses NG2 immunoreactivity.

BACKGROUND/AIMS: NG2 is the rat homologue of the human melanoma proteoglycan (HMP), also known as the high molecular weight melanoma associated antigen. Most cutaneous melanomas, as well as glioblastomas, chondrosarcomas, and some leukaemias express NG2 immunoreactivity, recognised using monoclonal antibody (mAb) 9.2.27. This antibody has also been used for molecular targeting in targeted alpha therapy for melanoma. The purpose of this study was to evaluate the expression of NG2 immunoreactivity in human uveal melanoma and normal ocular tissue using mAb 9.2.27. METHODS: Enucleated eyes from 26 patients with choroidal or ciliary body melanoma (n=26) were available as paraffin sections, and stained with haematoxylin and eosin to assess for tumour cell type and histopathology. Additional slides were investigated for NG2 immunoreactivity using mAb 9.2.27 and alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining. Two independent observers graded immunostaining using a semiquantitative scale from 0 (negative) to 3 (strong). RESULTS: Immunostaining for mAb 9.2.27 could not be graded in 7/26 cases with dense pigmentation of the tumour. For the remaining cases, grade 2 (moderate) or more immunostaining was seen in 18/19 tumours (95%). The retina, retinal pigment epithelium (RPE), and choroid displayed weak immunostaining (grade 0.5-1.5) in the majority of melanoma affected eyes. Normal retina and choroid (n=5) appeared negative for mAb 9.2.27. Optic nerve axon bundles in both control and melanoma affected eyes displayed moderate immunostaining. CONCLUSION: In the present study, the majority of human uveal melanomas expressed NG2 immunoreactivity, as detected using mAb 9.2.27. This antibody may be a suitable candidate for radioimmunotherapy to target ocular melanoma.

Frequent loss of heterozygosity on chromosome 6p in uveal melanoma.

Lack of expression of HLA class I antigens is frequently observed on primary uveal melanoma, and is correlated with improved patient survival. Several mechanisms may contribute to the observed loss of HLA class I expression, including changes at the DNA level. In this study, we used microsatellite analysis as a molecular genetic approach to examine loci on chromosome 6p for loss of heterozygosity (LOH). Three pairs of microsatellite markers were used to screen 20 formalin-fixed, paraffin-embedded uveal melanomas for LOH on the short arm of chromosome 6. In all cases, normal adjacent scleral tissue was used as a control. We identified LOH in eleven cases from microsatellite locus D6S105 to the telomere, in eight cases from microsatellite locus D6STNFa to the telomere (area includes D6S105), and in seven cases from microsatellite locus D6S291 to the end of chromosome 6p (includes D6STNFa and D6S105). In seven cases, retention of heterozygosity was found at all three loci using these primers. Our results suggest that loss of heterozygosity on chromosome 6p is a common feature in uveal melanoma. We did not find a correlation between the presence of LOH and locus-specific HLA-A and -B expression.

Effects of transpupillary thermotherapy on immunological parameters and apoptosis in a case of primary uveal melanoma.

Transpupillary thermotherapy (TTT) is a new treatment modality for uveal melanoma. We studied whether application of TTT influences the immunogenicity of the tumour cells in vivo or the expression of molecules related to apoptosis. Immunohistochemistry using monoclonal antibodies directed against HLA molecules, HMB45, P53, Fas ligand (FasL), Fas, Bcl-2 and tumour-infiltrating cells was applied to sections of an enucleated eye containing a uveal melanoma that received TTT 1 week before enucleation. The innermost part of the tumour which had been exposed directly to the laser treatment showed no staining for HLA antigens, nor for Fas or FasL epitopes. The intermediate part of the tumour showed a wet necrosis and HLA expression similar to the expression in the peripheral tumour. A large number of macrophages were observed in the necrotic as well as the intact tumour tissue, especially bordering the wet necrotic area. FasL and Bcl-2 were only expressed in the viable, outer part of the tumour. This immunological evaluation of one case of uveal melanoma treated with TTT revealed that TTT may not only have a direct destructive effect on the primary tumour, but may also influence the immunogenicity of uveal melanoma cells, induce infiltration of macrophages into the tumour, and induce apoptosis. The presence of many macrophages suggests that they play a role in the removal of the TTT-treated tumour tissue by phagocytosis.

Alpha/beta- and gamma/delta TCR(+) lymphocyte infiltration in necrotising choroidal melanomas.

AIM: To detect specific tumour infiltrating T cells (TIL) carrying antigen specific MHC-I restricted receptor genes on necrotising and non-necrotising malignant melanomas and to correlate the findings with clinical data. METHODS: alpha/beta- and gamma/delta- TIL were determined by immunohistochemical staining in melanomas of patients with known follow up of more than 10 years. An antigen retrieval method was used to determine variable genes delta1 and gamma1 on TCR(+) cells by an anti-TCR Vdelta1 and anti-CrgammaM1, and of Valpha and Vbeta TCR(+) by an anti-pan-TCR(+) alpha/beta antibody. RESULTS: Intratumoral TIL were present in 86 of 113 (76.1%) necrotising melanomas (NMM) v 21 of 100 (21%) in non-necrotising melanomas (MM); of these, Valpha/beta- TCR(+) cells were present in 52 of 74 (70.3%) TIL harbouring NMM v four of 21 (19%) MM; Vgamma1 in 29 of 74 (39.2%) NMM v two of 21 (10%) MM; and Vdelta1 in 39 of 74 (52.7%) NMM v three of 21 (14%) MM. Extratumoral lymphocytic infiltration was seen in 86 (76.1%) NMM including Valpha/beta TCR(+) cells in 10 (11.6%) cases, v five (5%) MM cases with no Valpha/beta TCR(+) cells detected. Vgamma1 and Vdelta1 TCR(+) cells were not found in extratumoral infiltrates. CONCLUSIONS: In NMM, the median survival was 69.3 (range 6-237) months, 19 of 74 patients (25.7%) survived 5 years, and mortality was associated with advanced stage (p<0.001), patient age (p<0.023), and extent of necrosis (p<0.048). Survival was increased with evidence of Vgamma1 and Vdelta1 TCR(+) cells (p<0.026).

Uveal melanomas contain antigenically specific and non-specific infiltrating lymphocytes.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) were recovered from a series of human choroidal melanomas and expanded in cultures containing interleukin-2 (IL-2) to determine whether TIL contained cytotoxic cells that could be activated in vitro. METHODS: TIL were recovered from six ocular melanoma patients and expanded in vitro with IL-2. Cytotoxic activity was tested in a standard 4-hr 51Cr release assay. The HLA class I phenotype of patients was determined, using peripheral blood lymphocytes and the Amos modified-cytotoxicity test. HLA class I expression on tumor cells was determined by flow cytometry. RESULTS: TIL from four patients lysed autologous ocular melanoma cells. Two of these patients possessed TIL that displayed specific cytotoxic activity and failed to lyse tumor cells from other patients (HLA-mismatched, or -matched). TIL from the remaining two patients possessed non-specific cytotoxic cells that lysed ocular melanoma cells from a variety of other patients (HLA-mismatched). TIL from patients that failed to lyse autologous tumor cells possessed cytotoxic activity for ocular melanoma cells from other HLA-mismatched patients. CONCLUSIONS: Ocular melanomas accumulate lymphocytes with the potential to kill tumor cells. Our results imply that elimination of tumor cells may be possible by activation of cytotoxic cells present within progressively growing ocular tumors.

[Clinical and histopathological aspects of 113 necrotizing malignant melanomas of the choroid. 2. Immunogenetic characterization of T-cell receptor-positive tumor-infiltrating lymphocytes and survival of patients with necrotizing melanomas of the choroid]. / Klinische und histologische Aspekte von 113 nekrotisierenden malignen Melanomen der Aderhaut. 2. Immungenetische Charakterisierung T-Zell-Rezeptor-positiver Tumor-infiltrierender Lymphozyten und Uberlebensrate von Patienten mit nekrotisierenden Melanomen der Aderhaut.

BACKGROUND: T-cell receptor (TCR) gene analysis of tumor infiltrating lymphocytes (TIL) has been recognized to play a pivotal role in the immunosurveillance of solid neoplasms. PATIENTS AND METHODS: Therefore, 113 necrotizing choroidal melanomas (NMM) were compared to 100 non-necrotizing melanomas (MM) histologically and immunohistochemically. RESULTS: NMM showed TIL more frequently (76.11% vs. 21%, p < 0.001). V alpha/V beta- (70.3% of NMM) and V gamma 1- (39.2% of NMM) and V delta 1- (52.7% of NMM) TCR+ cells were distributed focal or diffuse, and correlated with tumor volume, diameter, and scleral invasion. 74.33% of NMM patients had died after 240 months (5 year survival: 58.1%). Mortality was not significantly associated with TIL (p < 0.33) or V alpha/V beta-TCR+ cells (p < 0.2), however, survival was significantly increased with evidence of V gamma 1- and V delta 1-TCR+ cells (p < 0.026). CONCLUSIONS: V alpha/V beta-, V gamma 1- and V delta 1-TCR+ tumor infiltrating lymphocytes can be demonstrated in choroidal melanomas. This is a basis for functional studies providing a rationale for the evaluation of immunotherapeutic modalities.

Immunohistochemical expression of S-100 beta in choroidal melanomas.

OBJECTIVE: To investigate the expressivity of S-100 beta antibodies in choroidal melanomas and to compare it with that of S-100 protein and HMB-45. DESIGN: Twenty-seven choroidal melanoma specimens obtained from the McGill University Ophthalmic Pathology Registry were classified as spindle cell, epithelioid cell or mixed-cell type. Immunohistochemistry was performed using the standard peroxidase-antiperoxidase technique with monoclonal HMB-45, polyclonal S-100, polyclonal S-100 beta and monoclonal S-100 alpha beta antibodies in formalin-fixed, paraffin-embedded sections. OUTCOME MEASURE: Intensity of immunoreaction. The result was considered positive when at least five focal areas of stained cells were observed within the tumour. RESULTS: All 27 tumours were positive for HMB-45, 19 (70%) for S-100, 23 (85%) for S-100 beta, and 21 (78%) for S-100 alpha beta. No correlation was found between the intensity of the immunoreaction and cell classification. CONCLUSIONS: HMB-45 is the most reliable marker for choroidal melanomas. S-100 beta is a more sensitive marker than S-100 for choroidal melanomas regardless of cell type. Contrary to previous reports, S-100 beta should not be considered a useful immunomarker to differentiate between primary choroidal melanoma and cutaneous melanoma metastatic to the choroid.

Effect of hyperthermia on expression of histocompatibility antigens and heat-shock protein molecules on three human ocular melanoma cell lines.

Hyperthermia is used as a new treatment modality for ocular melanoma. We wondered whether this treatment would affect the antigenicity of melanoma cells and studied the effect of hyperthermia on the expression of histocompatibility antigens (HLA), beta 2-microglobulin, as well as heat-shock proteins (HSP-60 and HSP-70) on choroidal melanoma cells. Uveal melanoma cell lines were exposed to different temperatures (39-45 degrees C) in a waterbath. Antigen expression was determined with fluorescence-activated cell sorting analysis, using monoclonal antibodies against HLA and HSP. In a 51Cr-release cytotoxicity assay we studied the effect of heat on natural killer (NK) cell susceptibility. Exposure to 45 degrees C for 30 min reduced expression of HLA class I antigens and beta 2-microglobulin. A greater reduction was observed after longer exposure times. Expression of HSP-70 was increased after exposure to 45 degrees C at all time intervals, while expression of HSP-60 was not induced by heat treatment. We did not find a significant difference in the NK cell susceptibility between heated and unheated cells. Hyperthermia has a time- and temperature-dependent effect on expression of HLA class I and HSP-70 molecules on the cell surface of uveal melanoma cells. Hyperthermia did not alter the susceptibility to NK cell lysis.

Flow cytometry analysis of peripheral blood lymphocytes in patients with choroidal melanoma.

PURPOSE: To evaluate peripheral blood lymphocyte subpopulations in patients with choroidal melanoma. METHODS: In this prospective study, peripheral blood lymphocytes of 226 patients afflicted with choroidal melanoma were analyzed by flow cytometry and compared with those of 49 age-matched and gender-matched control subjects. Subpopulations of peripheral blood lymphocytes were further identified by monoclonal antibodies specific to cell-surface markers. Statistical analysis was performed by a Student t test. RESULTS: There was no overall difference between the patients with choroidal melanoma and the control subjects with regard to peripheral blood lymphocyte subpopulations. However, when the patients were divided into subgroups based on their clinical characteristics, differences in natural killer (NK) cell population and activated T cells were noted in two subgroups. Patients with ciliary body involvement showed a statistically significant reduction in NK cells (194 +/- 101 vs 260 +/- 178 per mm3; P = .01). The number of activated T cells in this subgroup of patients was increased but not statistically significantly (7.32 +/- 4.79 vs 6.09 +/- 4.34 per mm3; P = .08). In patients with extrascleral extension, a statistically significant increase in activated T cells was noted (9.84 +/- 7.41 vs 6.25 +/- 4.3 per mm3; P = .02). The NK cells in this subgroup of patients were also reduced, but the reduction did not achieve statistical significance (178 +/- 123 vs 248 +/- 167 per mm3; P = .24). CONCLUSIONS: We noted statistically significant differences in peripheral blood lymphocytes in two subgroups of patients with clinically less favorable choroidal melanoma.

Membrane-bound regulators of complement activation in uveal melanomas. CD46, CD55, and CD59 in uveal melanomas.

PURPOSE: To identify the presence of membrane-bound regulators of complement activation (m-RCA) on uveal melanomas and uveal melanoma cell lines and to examine their role in the inhibition of complement-mediated lysis in vitro. METHODS: Immunohistochemistry and flow cytometric analysis with monoclonal antibodies directed against m-RCA CD46, CD55, and CD59 were applied to tissue sections of 10 uveal melanomas, three primary uveal melanoma cell lines, and one uveal melanoma metastatic cell line. A microcytotoxicity test was used for measuring antibody-dependent complement-mediated lysis. RESULTS: The tissue sections and all four uveal melanoma cell lines expressed CD46, CD55, and CD59. Complement-mediated lysis in the presence of human complement was increased after partial removal of the m-RCA CD55 and CD59 with phosphatidylinositol-specific phospholipase C from the uveal melanoma cell line 92-1. CONCLUSIONS: These results demonstrate that CD46, CD55, and CD59 are expressed in uveal melanomas and that CD55 or CD59, or both, plays a role in resistance to complement-mediated cytotoxicity. The finding that m-RCA are expressed in uveal melanomas may have implications for the effectiveness of the anti-tumor response and in the therapeutic application of monoclonal antibodies directed against tumor-associated antigens.

The immunoalkaline phosphatase technique in immunohistochemistry: the effect of permanganate-oxalate melanin bleaching upon four final reaction products.

Melanin bleaching prior to the application of primary antibody can alter the immunoreactivity of a number of antigens. The effect of melanin bleaching subsequent to antigen visualisation by four different chromogens used with an immunoalkaline phosphatase technique was investigated. Vector black was the only final reaction product that withstood exposure to the permanganate-oxalate sequence for a time sufficient to bleach melanin.

[Analysis of infiltrating lymphocytes in choroidal melanoma].

We performed immunohistochemistry on tumor infiltrating lymphocytes (TILs) in a 78-year-old man with choroidal malignant melanoma. Cell suspensions of TILs from fresh specimens and peripheral blood lymphocytes (PBLs) were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD29, anti-CD45RA, and anti-human leukocyte antigen (HLA)-DR monoclonal antibodies and analyzed using three-color flow cytometry. In light microscopy, the number of infiltrating lymphocytes around the tumor was very small. Immunohistochemically, T lymphocytes were more numerous than B lymphocytes. Flow cytometric analysis showed that CD8+ cells were more numerous than CD4+ cells in CD3+ cells in TILs, and most of these cells also expressed HLA-DR antigen. CD29+ (memory) cells were increased and CD45RA+ (naive) cells were decreased in CD4+ cells in TILs as compared with PBLs. We concluded that the increase in the percentage of activated memory T lymphocytes and the decrease of naive T lymphocytes may reflect a localized antigen-specific immunological response in choroidal malignant melanoma.

[Antigen pattern in choroid melanoma in correlation with immunoscintigraphy]. / Antigenmuster beim Aderhautmelanom in Korrelation zur Immunszintigraphie.

We have reported that the sensitivity of immunoscintigraphy in ocular melanoma is fairly low in comparison with (metastasizing) cutaneous melanoma. No significant correlation was found between the histological data for ocular melanoma and the immunoscintigraphic results. We therefore wanted to see whether we could demonstrate an antigen pattern that was different from that of cutaneous melanoma, which might explain our previous results. Our study comprised tumor tissue from 20 patients with ocular melanoma who had undergone previous immunoscintigraphic examination. Using immunohistochemical techniques, tumor immunoreactivity was investigated against 225.28S, the antibody used for immunoscintigraphy, on cryosections in 12 cases, and against anti-HMB-45, and anti-S-100 and anti-vimentin on paraffin sections in all 20 patients. In summary, there was marked immunohistochemical heterogeneity, and none of the antibodies examined showed a significant correlation with immunoscintigraphy. Even 225.28S that was used for the immunoscintigraphic examination did not retrospectively allow a predictable immunoscintigraphic outcome. When comparing our results with the literature on cutaneous melanoma we were also able to confirm differences in immunoreactivity with regard to the other antibodies. We conclude that the comparatively poor results in ocular immunoscintigraphy obtained with 225.28S are due to antigenic differences between ocular and cutaneous melanoma.