mart-1-antigen and Uveal-Neoplasms

To report the clinical and histopathologic features of two cases of eyelids metastases from uveal melanoma diagnosed in a metachronous and synchronous fashion.. Monocentric retrospective case series of histopathologically proven eyelids metastases from uveal melanoma at our institution.. Two patients were presented to our hospital for upper eyelids pigmented and firm lesions. Patient 1 had an history of left uveal melanoma treated conservatively with proton beam therapy 5 years earlier. Examination revealed bilateral upper eyelids lesions. Patient 2 had no malignancy history but was incidentally diagnosed with a cerebral nodule few months earlier. Examination revealed a right upper eyelid nodule and a previously unknown right uveal melanoma. Excisional biopsy was performed for both patients. Pathological assessment allowed the presence of melanoma cells. The lack of BAP1 nuclear expression on immunohistochemistry as well as the absence of cutaneous or mucosal melanoma were consistent with an uveal origin. Diffuse metastatic spread was noted for both patients. Systemic therapies were prescribed. Patient 1 died from metastatic spread (62 months and 4 months after uveal melanoma diagnosis and eyelids metastases removal, respectively) whereas patient 2 was still alive (14 months follow up).. Eyelids metastases from uveal melanoma is an exceptional finding. Excisional biopsy and pathological assessment are of main importance to confirm the diagnosis and to identify genetic mutations for further targeted therapies. Currently, prognosis remains poor.

Topics: Aged; Biomarkers, Tumor; Biopsy; Combined Modality Therapy; Eyelid Neoplasms; Fatal Outcome; Humans; Immunotherapy; Male; MART-1 Antigen; Melanoma; Middle Aged; Prognosis; Proton Therapy; Retrospective Studies; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Eye Enucleation; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Tumor Suppressor Protein p53; Uveal Neoplasms

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Conjunctival Neoplasms; Disease Models, Animal; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Neoplasm Proteins; Tomography, Optical Coherence; Ultrasonography; Uveal Neoplasms

To establish a mouse model with the aim of studying the tumour biology and metastasis formation of uveal melanoma.. Two human primary uveal melanoma cell lines (UMT2 and UMT42) were injected into the choroid of BALB/c nude mouse eyes. Intraocular tumour growth and metastasis formation in the liver and lungs were assessed after 13 to 22 weeks. Formalin-fixed, paraffin-embedded material was processed via haematoxylin and eosin staining for histological examination and periodic acid Schiff staining to search for extravascular matrix patterns. Immunohistochemistry for Melan A, CD34 and Ki67 was performed to assess the expression of a melanocytic lineage marker, angiogenesis and proliferative activity.. All eyes injected with UMT2 cells, but only 25% of eyes treated with UMT42, developed intraocular tumour growth. The morphology of intraocular melanomas resembled that of primary tumours and showed signs of malignancy, including retinal invasion, optic nerve invasion and scleral penetration with extraocular tumour growth. UMT2 tumours formed extravascular matrix patterns exclusively. Most of the tumour cells expressed Melan A. Intratumoural angiogenesis was detected in both tumour entities. Proliferative activity was verified in all but one tumour. However, no metastases appeared in the liver or lungs.. The mouse model presented with the UMT2 cell line allows for investigations of tumour biology of the primary UM because of the high degree of similarity between the tumours generated in the mouse eyes and the corresponding primary human UM. Unfortunately, the model is not suitable for investigations of metastasis formation.

Topics: Animals; Antigens, CD34; Biomarkers, Tumor; Cell Line, Tumor; Disease Progression; Follow-Up Studies; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasms, Experimental; Neovascularization, Pathologic; Pilot Projects; Retinal Neoplasms; Uveal Neoplasms

To establish a mouse model with histologic characteristics of uveal melanoma for investigation of intraocular tumor biology of melanoma.. After injection of 1 × 10(5) of HCmel12 melanoma cells, a cutaneous melanoma cell line, into the vitreous of CX3CR1(+/GFP) or C57Bl/6 mice (n = 12), tumor growth patterns, clinicopathological features, angiogenesis and metastatic behavior were analyzed by histology (hematoxylin and eosin, periodic acid-Schiff without hematoxylin) and immunohistochemistry (HMB45/MART-1-Ab, F4/80-Ab, green fluorescent protein (GFP)-Ab and VE-cadherin-Ab).. HCmel12 cells formed intraocularly growing tumor masses, which showed histologic features of intraocular melanoma such as angiotropism, intratumoral endothelial-lined vasculature, vasculogenic mimicry including prognostic significant extravascular matrix patterns, and invasion by inflammatory cells, in particular macrophages. There was no difference in tumor growth characteristics between CX3CR1(+/GFP) and C57Bl/6 mice. Five of 10 mice proceeded to extrascleral tumor growth and three of these developed metastases.. Intraocularly injected HCmel12 cells developed tumor masses with histologic characteristics of aggressive melanoma similar to human uveal melanoma. Since hematogenous dissemination to the liver was not observed, intravitreally injected HCmel12 cells do not qualify as a model for metastasizing intraocular melanoma. However, since the eye represents a semi-closed compartment with access to constant blood supply, these intraocular tumors represent a model for studies of isolated parameters in general tumor biology of intraocular melanoma.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Disease Models, Animal; Female; gp100 Melanoma Antigen; Green Fluorescent Proteins; Humans; Intravitreal Injections; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Skin Neoplasms; Transplantation, Heterologous; Uveal Neoplasms

The detection of circulating tumour cells in the bloodstream before and after surgical manipulation, and the qualitative detection of potential shedding of tumour cells during surgical manipulation of patients with uveal melanoma.. 202 patients treated for a newly diagnosed uveal melanoma were included in the study. Blood samples were acquired 24 h before and 30 min after the basic surgical steps. Detection of potential circulating melanoma cells was extrapolated from the presence of tyrosinase and MelanA/Mart1 transcripts by reverse transcription PCR.. Based on the measurement of tyrosinase transcripts, as a result of the first and second surgical manipulation there were three and zero transitions from negative to positive respectively, while there were two and one transitions from positive to negative, respectively. According to MelanA/Mart1 transcripts, there were 19 and 5 transitions from negative to positive respectively, and 15 and 2 transitions from positive to negative, respectively. No statistically significant differences were documented, concerning the presence of circulating tumour cells in the blood samples acquired before and after the first surgical manipulation or the second one.. The change in the percentage of patients with detected tumour cells in their bloodstream was not statistically significant. The frequent shifts from negative to positive samples as well as from positive to negative samples comparing preoperative to postoperative samples indicates discontinuous shedding or variation due to measurements close to the threshold of detection. As a conclusion, the surgical manipulation does not seem to have a measurable contribution to the spread of melanoma cells in the bloodstream.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Humans; Iatrogenic Disease; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplastic Cells, Circulating; Ophthalmologic Surgical Procedures; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Uveal Neoplasms

Topics: Aged; Histocytochemistry; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Magnetic Resonance Imaging; Male; MART-1 Antigen; Melanoma; Microscopy; Radiography; Uveal Neoplasms

Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors.. Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice.. Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice.. Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.

Topics: Animals; Blotting, Western; Cell Count; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Mice; Mice, Nude; Microscopy, Phase-Contrast; Neoplasms, Experimental; Polymerase Chain Reaction; RNA, Neoplasm; Uveal Neoplasms

Topics: Biomarkers, Tumor; Ciliary Body; Eye Banks; Eye Enucleation; Humans; Ki-67 Antigen; Limbus Corneae; Male; MART-1 Antigen; Middle Aged; Neoplasm Invasiveness; Nevus, Pigmented; S100 Proteins; Tissue Donors; Uveal Neoplasms

Topics: Aged; Biomarkers, Tumor; DNA Mutational Analysis; DNA, Neoplasm; Eye Enucleation; Fatal Outcome; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; HSP27 Heat-Shock Proteins; Humans; Male; MART-1 Antigen; Melanoma; Multiplex Polymerase Chain Reaction; Neoplasms, Multiple Primary; Polymorphism, Single Nucleotide; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms; Visual Acuity

Intraocular melanocytoma is a rare naevus variant that can be located at the optic disc or within the uvea, and belongs to the group of non-epithelial-associated melanocytic lesions. We wanted to gain an understanding of the role of GNAQ, GNA11 and BRAF V600E in the pathogenesis of uveal melanocytoma and in cases of transformation to uveal melanoma and also to perform a differential immunohistochemical study comparing melanocytoma with uveal melanoma.. Two patients were identified with melanocytoma, one of which had transformed to melanoma. In the latter case, the melanocytoma exhibited an immunophenotype that featured nuclear p27 and no HMB45 staining, with very low Cyclin D1 expression compared with the melanoma that featured little nuclear but more cytoplasmic p27 positivity, much higher Cyclin D1 expression and HMB45 positivity. The melanocytomas were negative for CD68 allowing distinction from melanophages. Both melanocytomas and the melanoma harboured mutations in GNAQ, with no mutations of GNA11 or BRAF V600E.. GNAQ mutations are present in uveal melanocytomas and in a case of transformation to melanoma, implicating GNAQ-dependent mitogen activation signals, in the pathogenesis of uveal melanocytoma. This assists in explaining why a proportion of uveal melanocytoma can transform to uveal melanoma, known to harbour high-frequency GNAQ mutations at exon 5, codon 209.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; gp100 Melanoma Antigen; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mutation; Nevus, Pigmented; Polymerase Chain Reaction; Proto-Oncogene Proteins B-raf; Retrospective Studies; Uveal Neoplasms

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; MART-1 Antigen; Melanoma; NM23 Nucleoside Diphosphate Kinases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms

Uveal melanoma primarily metastasizes hematogenously with metastases often confined to the liver. The aim of this study was to investigate the presence of circulating tumor cells (CTC) in patients with metastatic disease as a marker for systemic disease and to determine their prognostic relevance.. Blood samples from 68 patients were collected at the time of initial treatment of metastases. mRNA expression of tyrosinase and MelanA/MART1 as a surrogate marker for the presence of CTC was analyzed by real-time RT-PCR and compared with patient characteristics.. CTC were detected in 63% of all patients and in 67% of the 48 patients with only liver metastases. Univariate and multivariate analyses revealed PCR results and serum lactate dehydrogenase as independent prognostic factors for progression-free (hazard ratios 2.2/3.5) and overall survival (hazard ratios 4.0/6.5). Combination of PCR and lactate dehydrogenase divided the patient cohort into 3 groups with distinct prognosis.. CTC as evidence for systemic disease can be found in the majority of patients with metastatic uveal melanoma, including patients with visible disease confined to the liver. Detection of CTC-specific mRNA transcripts for tyrosinase and MelanA/MART1 by PCR is a poor prognostic factor for progression-free and overall survival. Characterization of CTC could improve the understanding of their biology.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Karnofsky Performance Status; L-Lactate Dehydrogenase; Liver Neoplasms; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplastic Cells, Circulating; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Uveal Neoplasms

To assess the functional significance of intraocular tumor-associated lymphatic vessels in ciliary body melanomas with extraocular extension.. Twelve consecutive patients enucleated for a malignant melanoma of the ciliary body with extraocular extension and immunohistochemical presence of intraocular LYVE-1-positive and podoplanin-positive lymphatic vessels were examined for proliferation status and tumor invasion into tumor-associated lymphatics. Proliferating lymphatic vessels were identified using LYVE-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as the proliferation marker. Tumor invasion into lymphatic vessels was assessed using Melan-A as the melanoma marker. Kaplan-Meier analyses of survival and metastasis were performed.. Intraocular proliferating lymphatic vessels were detected in all 12 ciliary body melanomas with extraocular extension. The ratio of proliferating lymphatics was significantly higher in the intraocular vs extraocular tumor compartment (P < .001). Extraocular lymphatic invasion by tumor cells was observed in 5 patients (42%), intraocular lymphatic invasion in 4 (33%), and synchronous intraocular and extraocular lymphatic invasion in 3 (25%). Detection of melanoma cells in intraocular and extraocular lymphatic vessels was significantly associated with higher risks of lymphatic spread (P < .001) and lower metastasis-free survival rates (P = .03).. Intraocular tumor-associated lymphatic vessels contain proliferating endothelial cells and can be invaded by cancer cells in ciliary body melanomas with extraocular extension. Lymphatic invasion by tumor cells seems to be associated with an increased risk of lymphatic spread and mortality in these affected patients.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Ciliary Body; Eye Enucleation; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lymphangiogenesis; Lymphatic Metastasis; Lymphatic Vessels; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Uveal Neoplasms; Vesicular Transport Proteins

The aim of this study was to determine in patients with high-risk primary uveal melanoma whether the detection of circulating tumor cells by quantitative reverse transcription-PCR (RT-PCR) is of prognostic relevance.. Blood samples from 110 patients with high-risk nonmetastatic uveal melanoma were collected on the occasion of primary treatment or follow-up visit. mRNA expression of tyrosinase and MelanA/MART1 were analyzed by real-time RT-PCR and compared with clinical data at presentation and follow-up by univariate and multivariate analyses.. The RT-PCR assay yielded a positive result in 11 of 110 patients, with five positive findings for tyrosinase and five for MelanA/MART1, and one sample positive for both markers. At a median follow-up of 22 months, 25% of patients had developed metastases and 15% had died. Univariate statistical analysis revealed RT-PCR and the largest tumor diameter as important prognostic factors for the development of metastases and for survival. In a Cox proportional hazard model, RT-PCR result and largest tumor diameter predicted metastases (hazard ratios 7.3 and 2.6, respectively), whereas PCR result, largest tumor diameter, and Karnofsky performance status were significant variables for disease-specific survival (hazard ratios 22.6, 4.7, and 6.0, respectively). Analysis of individual RT-PCR results revealed both tyrosinase and MelanA/MART1 transcripts as independent prognostic factors.. The presence of tyrosinase or MelanA/MART1 transcripts is an independent prognostic factor in patients with high-risk primary uveal melanoma for subsequent development of metastases and for survival and can be used to select patients for adjuvant treatment studies.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cohort Studies; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Uveal Neoplasms

Uveal melanoma is the most common primary malignant ocular cancer in adults. This tumor has a distinct expression pattern of markers compared with cutaneous melanoma. MC1R is under study as a potential target for antitumor immunity. Because of the potential immunogenicity of MC1R, it is important to evaluate its expression on uveal melanomas.. Two novel monoclonal antibodies (MP1.1C11 and MP1.1B7) were used to examine the expression of MC1R in uveal melanomas. Tissue samples obtained from 17 patients were analyzed for expression of MC1R by immunohistochemistry. Additionally, uveal melanoma cell lines were treated with proinflammatory cytokines, after which MC1R cell surface expression was analyzed by flow cytometry.. Results demonstrated that MC1R is expressed by uveal melanoma to a significantly greater extent than other melanoma markers. With the use of MP1.1C11 or MP1.1B7, MC1R was detected in 95% of the tested melanoma tissues, including one liver metastasis. In contrast, MART-1, S100-specific protein, and gp-100 were only expressed by 66%, 33%, and 67% of the analyzed samples, respectively. Results also demonstrated that even though MC1R is mainly located intracellularly, its cell surface expression can be promoted by cytokines such as IFN-gamma, TNF-alpha, IL-4, and IL-10.. These observations support the inclusion of MC1R in the panel of markers for the diagnosis of uveal melanoma. Therapeutic use of MC1R-specific antibodies targeting cytokine-induced MC1R potentially requires expression of the target molecule on the surfaces of tumor cells. Data presented here support MC1R as a new marker and a putative therapeutic target for uveal melanoma.

Topics: alpha-MSH; Antigens, Neoplasm; Biomarkers, Tumor; Blotting, Western; Cytokines; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunoenzyme Techniques; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Receptor, Melanocortin, Type 1; S100 Proteins; Tumor Cells, Cultured; Uveal Neoplasms

The authors investigated the expression of S100A1, S100A6, S100B, MelanA, and CEA in conjunctival naevi, primary acquired melanosis (PAM), conjunctival melanoma, and uveal melanoma in order to assess their potential usefulness in the pathological differential diagnosis of these entities.. Paraffin embedded sections of 18 conjunctival naevi, 14 PAM, 16 conjunctival melanomas, and 20 uveal melanomas were immunostained for S100A1, S100A6, S100B, MelanA, and CEA, and expression was scored semiquantitatively.. Expression of S100A1 differed significantly between conjunctival naevi and conjunctival melanoma, with percentages of positive cells of 30.6% and 71.4%, respectively. Conjunctival melanomas had high average scores for S100A1 and S100B (71.4%, 62.9%, respectively), while uveal melanomas also had high S100A1 but low S100B scores (88.5%, 18.5%, respectively). MelanA was highly variable; naevi and uveal melanoma had higher average scores than conjunctival melanoma. CEA was hardly detectable in all four groups.. S100A1 seems to be a possible candidate to differentiate conjunctival naevi from conjunctival melanoma. S100B seems to differentiate between uveal melanoma and conjunctival melanoma. However, the study size was small and therefore the data have to be confirmed by others.

Topics: Antigens, Neoplasm; Biomarkers; Carcinoembryonic Antigen; Cell Cycle Proteins; Conjunctival Diseases; Conjunctival Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanosis; Neoplasm Proteins; Nerve Growth Factors; Nevus; S100 Calcium Binding Protein A6; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Uveal Neoplasms

Downregulation of melanoma-associated antigens (MAAs), against which natural cytolytic T lymphocytes (CTLs) exist in humans, is one of the mechanisms that aids in evasion of immune surveillance. In view of putative re-expression strategies for MAAs during immunotherapy, this study was conducted to investigate MAA silencing in malignant melanoma.. The expression of the MAA Melan-A/MART-1 was analyzed in 10 uveal and 10 cutaneous patient-derived melanoma cell lines by Western blot analysis and RT-PCR. Expression characteristics of four other MAAs-Tyr, Tyrp1, Dct, and gp100/Pmel17-were analyzed by RT-PCR. DNA methylation patterns at the Melan-A/MART-1 promoter region were investigated by methylation-sensitive restriction enzyme digestion and subsequent Southern blot analysis. Exogenous promoter activity was assessed in all 20 melanoma cell lines to correlate the DNA methylation patterns with Melan-A/MART-1 expression.. MAA expression was observed in 15 of the 20 melanoma cell lines. Furthermore, there is a direct correlation between DNA methylation patterns at the Melan-A/MART-1 promoter region, exogenous Melan-A/MART-1 promoter activity, and Melan-A/MART-1 protein expression. These data reveal the division of patient-derived melanoma cell lines into two distinct subsets, which are identical for both uveal and cutaneous tumor types.. The authors propose a categorization of melanoma cell lines into two different panels based on shared MAA-expression characteristics: panel I, MAA-expressing cell lines, and panel II, MAA-deficient cell lines. This categorization can be used to obtain knowledge about the regulation of MAA-expression and for further research concerning MAA-based immunotherapy.

Topics: Antigens, Neoplasm; Blotting, Southern; Blotting, Western; DNA Methylation; DNA, Neoplasm; Gene Expression; Genes, Reporter; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured; Uveal Neoplasms

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytotoxicity Tests, Immunologic; Drug Administration Schedule; Epitopes, T-Lymphocyte; Freund's Adjuvant; HLA-A2 Antigen; Humans; Injections, Subcutaneous; Lipids; Longitudinal Studies; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Uveal Neoplasms; Vaccines, Subunit

Aggressive melanoma cells may express endothelial markers that can be used to calculate microvascular density (MVD). High MVD has been associated with adverse outcome in uveal melanoma. If tumor cells label with endothelial cell markers, then MVD may not accurately reflect a tumor's vascularity. This study was designed to study the influence of melanoma cell labeling by endothelial cell markers on MVD.. Tissue sections of 200 ciliary body or choroidal melanomas were stained with CD34 alone, and the MVD was calculated by counting discrete foci of CD34 labeling in hot spots, as described previously. From adjacent sections double labeled by fluorescent immunohistochemical stains for S100 protein and CD34, tumor cells labeling with both markers were identified. The relationship between marker coexpression and MVD was tested. Tissue sections were also double labeled for Melan-A and CD34.. MVD was found to be associated with death from metastatic melanoma as reported previously. However, colocalization of both Melan-A and S100 protein with CD34 was demonstrated. The labeling of tumor cells by CD34 was associated with an elevated calculation of MVD (P < 0.0001) but not with cell type, mitotic figures, tumor-infiltrating lymphocytes, or PAS-positive patterns.. CD34 may label uveal melanoma cells and may contribute to computation of MVD. Although MVD is prognostically significant in uveal melanoma, this feature is not an exclusive measure of tumor vascularity.

Topics: Antigens, CD34; Antigens, Neoplasm; Biomarkers, Tumor; Cell Count; Endothelium, Vascular; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neovascularization, Pathologic; Prognosis; S100 Proteins; Staining and Labeling; Survival Rate; Uveal Neoplasms

A novel antibody A103, which recognizes melan-A/MART-1, has been found to be more sensitive than the antibody HMB-45, which recognizes gp100, in melanocytic lesions of the skin and might therefore also be useful in the diagnosis of uveal and conjunctival melanocytic lesions. In this study we compared the staining characteristics of anti-melan-A, anti-S100 protein and HMB-45 in 13 conjunctival, 11 iris and 37 ciliary and choroidal malignant melanomas. The ciliary and choroidal melanomas comprised 13 spindle cell (10 spindle B and three spindle A), 14 mixed cell and 10 epithelioid cell tumours. In the conjunctival melanomas the diagnostic sensitivity was 100% for anti-S100 and anti-melan-A and 85% for HMB-45. In the iris melanomas the sensitivity was 100% for anti-S100 and anti-melan-A and 55% for HMB-45. A high staining intensity of anti-melan-A was particularly noticed in iris melanomas. In the choroidal malignant melanomas, the spindle cell and mixed cell types showed a sensitivity of only 69-79% with all three antibodies. In the epithelioid cell type the sensitivity was 80% for anti-S100 and 100% for HMB-45 and anti-melan-A. In conclusion, anti-melan-A was found to be a useful addition to antibody panels for ocular melanocytic lesions. Anti-melan-A has a higher sensitivity than HMB-45 in conjunctival and iris melanomas, but the sensitivity is similar to HMB-45 in choroidal melanomas. Anti-melan-A stains in a very similar pattern to anti-S100, but the staining intensity of anti-melan-A is higher than that of anti-S100 in iris melanoma.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Conjunctival Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Uveal Neoplasms

In the treatment of cutaneous melanoma, provisional therapeutic strategies have been designed to combat tumour load using T cells that are sensitized with peptides derived from melanoma autoantigens, such as glycoprotein 100 (gp100), melanoma antigen recognized by T cells 1 (MART-1 or MelanA), tyrosinase and tyrosinase-related protein 1 (TRP-1). We recently found that gp100, MART-1 and tyrosinase are heterogeneously expressed in human cutaneous melanoma (De Vries et al (1997) Cancer Res 57: 3223-3229). Here, we extended our investigations on expression of these immunotherapy candidate proteins to uveal melanoma lesions. Cryostat sections from 11 spindle-type, 21 mixed and epithelioid tumours and four metastasis lesions were stained with antibodies specifically recognizing gp100, MART-1, tyrosinase and TRP-1. In addition, we used the DOPA reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results. High expression of gp100, MART-1 and tyrosinase was found in the uveal melanoma lesions: 80% of the lesions displayed 75-100% positive tumour cells. TRP-1 positivity was slightly less: approximately 65% of the lesions stained in the 75-100% positive tumour cell category. All uveal melanoma lesions were positive for the four markers studied, this being in contrast to cutaneous melanoma where 17% of the advanced primary lesions and metastases were negative. The presence of these antigens was a little lower in metastases. We conclude that uveal melanomas and their metastases express melanocyte-lineage immunotherapy candidate proteins very abundantly. Uveal melanomas differ in this respect from cutaneous melanoma, in which the expression of these immunotherapy antigens was much more heterogeneous. This makes uveal melanoma a suitable candidate tumour for immunotherapeutic approaches.

Topics: Antigens, Neoplasm; Autoantigens; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Oxidoreductases; Protein Biosynthesis; Uveal Neoplasms