Expression Of Melanoma-associated Antigens Research Articles

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.

Feasibility of Immunotherapy for Lymphangioleiomyomatosis

This Commentary discusses the feasibility of vaccines against melanoma-associated differentiation antigens for treatment of lymphangioleiomyomatosis.

Expression of melanoma-associated antigens in human dendritic cells pulsed with an interleukin-2 gene encoded vaccinia melanoma oncolysate (rIL-2VMO)

Dendritic cells (DCs) possess the unique abilities to initiate a primary immune response and to present antigens to naïve T lymphocytes. Recently, there has been a rapidly growing interest in the use of DCs in active specific immunotherapy (ASI) for the treatment of patients with cancer. In the present study, we determined the ability of DCs to express Melanoma-Associated Antigens (MAAs) from a polyvalent Melanoma Vaccine (DC-MelVac; Patent# 11221/5) developed in our facility. The vaccine consists of a recombinant IL-2 gene-encoded vaccinia melanoma oncolysate (rIL-2VMO) derived from an established human melanoma cell line. Our results show that r-IL2VMO-pulsed DCs express MAAs presented by the Mel-2 melanoma cell line oncolysate used in this study. We believe that these promising results will prove useful as an active specific immunotherapeutic agent for patients with Stage III melanoma.

Expression of melanoma‐associated antigens in melanoma cell cultures

The efficiency of melanoma immunotherapy appears to depend on both melanoma- and immune system-specific factors. Melanoma-specific factors include melanoma-associated antigen (MAA) expression as well as HLA class I molecule expression. We investigated the expression of five MAA - Melan-A/MART-1, tyrosinase, gp100, MAGE-1 and MAGE-3 - by means of FACS analysis in 50 melanoma cell cultures and compared them to the cultures of human foreskin-derived melanocytes and melanoma cell line UKRV-Mel2. Melan-A, tyrosinase and gp100 expression was frequently reduced in melanoma cell cultures, compared to that in foreskin melanocytes, whereas MAGE-1 and MAGE-3 expression showed variable degree of upregulation, compared to that in foreskin melanocytes. The expression of all tested MAA demonstrated high interindividual variability. We further show that cell cultures derived from the same tissue sample are oligoclonal in nature, by demonstrating the presence of up to three cell populations bearing distinct MAA profile. Analysing samples derived from the same patient but each at a different time point, we show that MAA expression profile changes over time either in positive (increase) or in negative (decrease) direction. Finally, we demonstrate that brain metastasis-derived cell cultures significantly overexpress Melan-A and MAGE-3, compared to primary tumours and other metastatic sites (P-value range: 0.05-0.001). Elucidation of the MAA expression patterns and the kinetics within the same patient as well as during the course of the disease may help improve current and develop new immunotherapeutic strategies.

Differential expression of melanoma-associated antigens and molecules involved in antigen processing and presentation in three cell lines established from a single patient

Tumour cells are able to evade the immune system by using several 'escape mechanisms'. Downregulation of molecules involved in the processing and presentation of self-antigens has been reported. However, these adaptations have not been compared in metastases in different anatomical locations but derived from a single patient. We investigated three melanoma cell lines--MJT1 from the parietal lobe of the brain, MJT3 from the cerebellum and MJT5 from the left side of the neck--established from biopsies excised from a 45 year old female patient. Although human leukocyte antigen (HLA) class I was detected in all three cell lines by flow cytometry using an anti-HLA monomorphic antibody, further serological analysis demonstrated HLA B38 loss in all three cell lines, HLA B7 downregulation in MJT5 (skin metastases) and B7 loss in MJT3 and MJT1 (brain metastases) compared with the HLA type of the patient's normal autologous lymphocytes. Interferon-gamma (IFNgamma) treatment increased the expression of HLA class I and transporters associated with antigen processing 1 (TAP1) in all three cell lines. De novo HLA class II molecule expression was observed after IFNgamma treatment in MJT3 and MJT5. Western blot and reverse transcription-polymerase chain reaction results revealed heterogeneity of melanoma-associated antigen (MAA) expression in the cell lines: MJT3 cells expressed higher levels of MAAs than the other two cell lines. In conclusion, this study has demonstrated that three metastatic lesions from a single patient can have differential expression of molecules involved in antigen processing (TAP1) and presentation (HLA I and II), but that expression of these molecules is modulated by IFNgamma to a similar degree in all cell lines. In contrast, the downregulation of expression of specific MAAs between the three cell lines was unaffected by the addition of IFNgamma.

Expression of Melan-A/MART-1 in primary melanoma cell cultures has prognostic implication in metastatic melanoma patients.

The lack of melanoma-associated antigen (MAA) expression has been associated with the reduced overall survival in melanoma patients. In order to investigate whether the MAA expression detected on cell cultures established from melanoma patients might relate to the overall survival in these patients, we screened primary cell cultures derived from 37 melanoma metastases for the expression of five known MAA: Melan-A, tyrosinase, gp-100, MAGE-1 and MAGE-3 by polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS). MAA expression detected by PCR was found at a high percentage in evaluated melanoma cell lines: 25 of 28 (89%) were positive for Melan-A, 22 of 28 (79%) were positive for tyrosinase, 26 of 28 (93%) were positive for gp-100, and 18 of 28 (64%) were positive for MAGE-3 expression. Using the FACS method the percentage of MAA-positive cell lines was much lower: 14 of 31 (45%) cell lines were positive for Melan-A, eight of 31 (26%) were positive for tyrosinase, 13 of 31 (42%) were positive for gp-100, six of 31 (19%) were positive for MAGE-1, and 14 of 31 (45%) were positive for MAGE-3 expression. Kaplan-Meier survival analysis demonstrated that the patients whose cell lines were positive for Melan-A expression by PCR had significantly longer overall survival time as Melan-A PCR-negative cases (P=0.0038). This could not be shown for any of the markers tested by FACS. Our results suggest that the expression of Melan-A/MART-1 in patient-derived cell cultures may help to identify a group of melanoma patients with prolonged survival.

Phase I-II trial of a novel mixed haptenized autologous melanoma vaccine

2563 Background: We have reported positive immunological and clinical results in patients treated with an autologous tumor cell vaccine modified with the hapten, dinitrophenyl (DNP). Now we have developed a second generation technology consisting of irradiated, autologous tumor cells, half of which are modified with DNP and half with a second hapten, sulfanilic acid (SA). Methods: Freshly-dissociated, cryopreserved tumor cells (TC) are irradiated (2500 cGy); then half of the cells are modified with DNP and half with SA. After combining them, the “mixed haptenized” cells are fixed in a low concentration of ethanol, aliquotted, and frozen. Each vaccine batch is tested for sterility, endotoxin, and flow cytometric expression of melanoma-associated antigens. In an ongoing phase I–II trial, patients with stage IV melanoma are administered the vaccine at one of three dosage levels (0.05x106, 0.50 x106, or 5.0 x106 cells/dose) using a previously determined optimal schedule of administration. Before and after a 1...

Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells.

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction

The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed cDNA in the different samples after RNA isolation and reverse transcription, a beta-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.

Impact of interferons on the expression of melanoma-associated antigens in melanoma short-term cell cultures.

Some immunotherapeutic approaches based on melanoma-associated antigens rely on in vitro cultivation of melanoma cells. A beneficial effect of interferons has been shown in melanoma. This study aimed to determine whether stimulation of patient-derived melanoma short-term cell cultures using interferon-alpha and -gamma changes the expression pattern of melanoma-associated antigens. Lymph node, skin and brain metastases were cultivated for up to 3 weeks and treated with interferon-alpha, interferon-gamma or mock stimulation. Expression of the melanoma-associated antigens MAGE-3, MelanA/MART-1 and tyrosinase was determined by flow cytometry and compared with the expression pattern of HLA class I molecules. We found consistently enhanced expression of HLA class I molecules, whereas the melanoma-associated antigens showed mixed responses, with moderate induction, suppression or no visible effect. The reaction to interferon stimulation was similar for all the antigens examined within a single melanoma cell culture. In contrast to the HLA class I molecules, which showed induced expression with interferon, the melanoma-associated antigens showed a varied response to interferon stimulation. Differential reaction to interferon stimulation is of importance to immunotherapeutic modalities and might influence progression of the disease. We therefore suggest that evaluation of variation in melanoma-associated antigen expression in the clinical setting may help to identify patients who would profit from adjuvant interferon therapy.

Endogenous Tumor necrosis factor alpha unmasks the binding sites for N-acetylglucosaminyl-(beta1-4)-N-acetylmuramyl-alanyl-D-isoglutamine on the surface of melanoma cells

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 +/- 200 per cell) and affinity (Kd = 10 +/- +/- 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitro with GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 +/- 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor alpha (TNF-alpha). The mechanism of the TNF-alpha action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.

Immunoreactivity with the Anti-MAGE Antibody 57B in Malignant Melanoma: Frequency of Expression and Correlation with Prognostic Parameters

The melanoma-associated antigen (MAGE) family consists of a number of antigens initially recognized by cytotoxic T lymphocytes, which are currently being investigated for immunotherapy of patients with metastatic melanoma and other tumor types. Expression of MAGE mRNA in melanocytic tumors is said to be restricted to invasive malignant tumors and absent in nevi. Recently, a monoclonal antibody (57B) has become available to examine MAGE protein expression in archival material. In this study, we performed immunohistochemical analysis on 132 melanocytic nevi and 205 melanomas (85 primary cutaneous melanomas and 120 metastatic tumors) to determine the frequency of MAGE expression and to explore a potential correlation with various prognostic parameters. None of the melanocytic nevi and none of the 20 in situ melanomas was immunopositive with the antibody 57B. Immunoreactivity was present in 17 of 65 (26%) primary invasive melanomas of the skin and in 30 of 120 (25%) metastatic tumors. Positive immunostaining did not correlate with tumor stage (P =.66), Breslow thickness (P =.39), Clark level (P =.5), or the histologic type of melanoma (P =.23) but was associated with a brisk infiltrate of lymphocytes involving the vertical growth phase of melanomas (P =.01). Because tumor-infiltrating lymphocytes in melanoma are associated with longer survival, our findings suggest a potential prognostic role for MAGE. Furthermore, the seeming restriction of immunopositivity to invasive malignant tumors suggests a potential diagnostic role for the antibody 57B in confirming the malignant potential of a melanocytic tumor.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in anIn vitro melanoma model

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

First Comparative Delineation of the T Cell Receptor Repertoire in Primary and Multiple Subsequent/Coexisting Metastatic Melanoma Sites

At present, very little is known about the types and heterogeneity of T cell responses and immunodominant epitopes of melanoma-associated antigens at coexisting sites of primary melanoma and metastatic lesions. To address this issue, we compared the T cell receptor (TCR) gene usage, complementary-determining region 3 diversity, and melanoma-associated antigens expression patterns of primary and metastatic melanoma specimens from three patients with partially homologous HLA class-1 types. Results obtained showed an overall predominance of a very limited number of TCRV regions with AV13 and BV14 being most frequently overexpressed. Sequencing of the dominating TCR transcripts confirmed the restricted usage of certain TCR specificities and, in two of the three patients, identified several identical TCR clonotypes at more than one metastatic site. Nevertheless, we failed to detect TCR transcripts that were common to all tumor deposits in a given patient and, within the majority of coexisting metastases, tumor-infiltrating lymphocytes preferentially used individual site-specifically expanded TCR beta-chain VJ segment combinations. This occurrence of individual responses simultaneously executed at and influenced in their specificity by the different sites of tumor growth, has important implications for the type of strategies chosen in the development of efficacious vaccines for patients with metastatic melanoma.

Expression of melanoma-associated antigens in short-term melanoma cultures detected by RT-PCR and subsequent ELISA.

Expression of melanoma-associated antigens in short-term melanoma cultures detected by RT-PCR and subsequent ELISA.

Endogenous tumour necrosis factor-alpha sensitise melanoma cells to glucosaminylmuramyl dipeptide

Flow cytometry was used to demonstrate that cultured human melanoma BRO cells expressed membrane-bound tumour necrosis factor-alpha (TNF-α) and were able to release TNF-α upon treatment with glucosaminylmuramyl dipeptide (GMDP). The released TNF-α was shown to prime melanoma cells, previously unable to respond to GMDP by increasing expression of melanoma-associated antigens, making them sensitive to GMDP treatment.

Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo.

MART-1/MelanA and Pmel17/gp100 are melanoma-associated antigens (MAAs) that can be recognized by tumor-infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co-factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART-1/MelanA (monoclonal antibody [MAb] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno-histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART-1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART-1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART-1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)-negative lesions. Of the 217 specimens, 155 were obtained from HLA-A*0201 patients, of which 6% were negative for HLA-A2, 8% were negative for MART-1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions.

Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro.

Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful in enhancing the immunogenicity of tumor antigens and mediating tumor regression. Endogenous expression of antigen by DC offers the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. In this report, we show that human DC are (a) capable of infection by recombinant poxviruses encoding melanoma-associated antigen (MAA) genes and (b) capable of efficiently processing and presenting these MAA to cytotoxic T cells. In 6/6 HLA A*0201-expressing melanoma patients tested, the virally driven expression of MART-1/Melan A MAA by DC was sufficient to generate CD8+ T lymphocytes that could recognize naturally processed epitopes on tumor cells. In most cases, specific anti-MART-1 reactivity could be detected after a single stimulation. Analysis of epitope dominance revealed that the amino acid sequence recognized by these cytotoxic T lymphocytes (CTL) corresponded to the MART-1(27-35) residues previously shown to be most commonly recognized by cytotoxic T lymphocytes expanded from metastatic melanoma lesions. These data show that the virally driven expression of MAA by DC can be exploited for the efficient induction of clinically relevant cytotoxic T-cell responses. This has clinical implications for active immunization therapy, and currently vaccine trials have been proposed for patients with metastatic melanoma.

Melanoma associated antigen expression in advanced primary cutaneous melanoma

Melanoma associated antigen expression in advanced primary cutaneous melanoma

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions.

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA-A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2-7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in < 50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.