Reiko F. Irie

S100 PROTEIN: A MARKER FOR HUMAN MALIGNANT MELANOMAS?

S100 protein is a nervous-system-specific cytoplasmic protein which is also present in human malignant melanoma cell lines. To test for its tumour specificity, biopsy specimens from normal tissue and a wide variety of tumours were tested for S100 by complement fixation and immunofluorescence tests. S100 was present in all of thirteen samples of malignant melanoma tissue and absent from nineteen samples of non-melanocytic tumours, and from normal skin and normal lymph nodes. S100 levels did not correlate with tyrosinase activity in the tissues examined. Detection and measurement of S100 protein may help distinguish poorly differentiated amelanotic malignant melanoma from tumours of obscure histological origin; they may also aid the detection of micrometastatic disease in the lymph nodes and other tissues of melanoma patients.

Ganglioside GM3:GD3 ratio as an index for the management of melanoma.

This report evaluates the relevance of the ratio of the melanoma‐associated ganglioside, GD3, and its precursor, GM3, to prognosis and management of Stage II disease. Tumor biopsy specimens from 42 melanoma patients were examined for the ratio and found that GD3 and GM3 constitute 80% of the total lipid bound sialic acids (LBSA) of melanoma. Although the ratio of GM3:GD3 in melanocytes, the progenitors of melanoma, is 19:1 (based on %LBSA), it ranged from 15:1 to 1:5 in tumor biopsy specimens. Patients are categorized into three groups based on their GM3:GD3 ratio (%LBSA) of tumor tissues as Group I (ratio ranged from 15:1 to 1.5:1) (10 of 42), Group II (1.4:1 to 1:1.4) (13 of 42), and Group III (1:1.5 to 1:5) (19 of 42). When the overall survival of patients from the onset of Stage II disease was evaluated among different groups, patients belonging to Group I survived significantly longer than patients of Group II (P = 0.02) and Group III (P = 0.01). Interestingly, Group III expressed immunogenic gangliosides (GD2, GM2, and O‐AcGD3) better than the other groups and may benefit more from therapies targeted against these gangliosides antigens. The results of this study indicate that GM3:GD3 ratio is an unusual but well‐defined biochemical criteria that may be useful for prognosis and therapeutic management of the disease. Therefore, we propose a routine analysis of the GM3:GD3 ratio of tumors excised after surgery. Screening the ratio is feasible since melanoma expresses a simple profile of gangliosides unlike other forms of cancer.

Activation of toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-κB and inflammatory response–related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression. [Mol Cancer Ther 2008;7(11):3642–53]

A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A

Fraction 4 (F4), a protein fraction isolated from aged garlic extract, enhanced cytotoxicity of human peripheral blood lymphocytes (PBL) against both naturalkiller (NK)-sensitive K562 and NK-resistant M14 cell lines. Although F4 treatment alone increased cytotoxicity, the effect was more remarkable when F4 was administered together with suboptimal doses of interleukin-2 (IL-2); combination treatment of 5 μg/ml F4 plus 10 U/ml IL-2 for 72 h generated lymphokine-activated killer activity equivalent to that produced by 100 U/ml IL-2 alone against M14. F4 enhanced IL-2-induced proliferation and IL-2 receptor (Tac) expression of PBL without significant increase of IL-2 production. The enhancement of cytotoxicity both by F4 alone and by F4 plus IL-2 was abolished by anti-IL-2 antibody. F4 also enhanced concanavalin-A(ConA)-induced proliferation of PBL. Radiolabeled-ConA binding assays revealed that F4 treatment greatly augmented the affinity and slightly increased the number of ConA binding sites in PBL. F4 also enhanced ConA-induced IL-2 receptor (Tac) expression and IL-2 production of PBL. Anti-IL-2 antibody inhibited the effect of F4 on ConA-induced proliferation. These data suggest that IL-2 is involved in augmentative effects of F4. Our results indicate that F4 is a very efficient immunopotentiator and may be used for immunotherapy.

Gangliosides from human melanoma immunomodulate response of T cells to interleukin-2

The gangliosides expressed by normal melanocytes are predominantly GM3 (greater than 90%) and GD3 (less than 5%). Malignant melanoma can express several other types of gangliosides in significant quantities, including GM2 and GD2. Melanoma patients can develop an immune response against some of these ganglioside antigens on autologous melanoma cells. The four major gangliosides expressed by human melanoma cells (GM3, GD3, GM2, and GD2) were examined for their immunomodulatory effect on lymph node lymphocytes from melanoma patients. Gangliosides were added exogenously to lymphocytes grown in the presence of IL-2. Preferential interactions of specific melanoma gangliosides on IL-2 stimulation were found. While GM2 and GD2 enhanced the lymphocyte response to IL-2, GM3 and GD3 significantly inhibited this response. GM2 and GD2 differ from GM3 and GD3 by the presence of a terminal N-acetylgalactosamine. Since different gangliosides can up-regulate and down-regulate lymphocyte responses to IL-2, the ganglioside phenotype of melanoma cells may play a major role in determining whether an individual tumor causes immune stimulation or suppression.

Expression of histocompatibility (HLA) antigens on tumor cells and normal cells from patients with melanoma

The expression of HLA antigens and β2‐microglobulin (β2‐μ) on cultured melanoma cells originated from 11 patients has been quantitated and compared with that on fibroblasts and cultured human lymphoid cells originated from the same patients. No qualitative or quantitative difference was detected with the exception of one melanoma line. HLA antigens were also quantitated in sera from melanoma patients: two sera reacted with anti‐HLA‐B7 antibodies although this specificity was not expressed on lymphocytes from whom the sera were obtained. A technique to quantitate HLA antigens on cells developed in the course of this study is described. Cancer 40:36–41, 1977.

Chapter 19 Tumor gangliosides as targets for active specific immunotherapy of melanoma in man

Publisher Summary This chapter discusses various important aspects of gangliosides as vaccines for active specific immunotherapy of melanoma. The chapter presents new data on the antibody response to gangliosides induced in patients receiving Merkel cell polyomavirus. Several lines of evidence indicate that gangliosides modulate immune functions. Among the documented immunoregulatory properties of gangliosides are (1) inhibition of normal human lymphoproliferative responses to mitogens and antigens, (2) inhibition of interleukin-2 dependent cell proliferation, (3) inhibition of cytotoxic effector functions, and (4) binding to helper cells and modulated expression of CD4. Gangliosides have also been shown to modulate the humoral immune response. Melanoma cells in the radial growth phase have a GM3 and GD3 profile similar to that of normal melanocytes. GM3 and GD3 inhibits the proliferation of human lymphocytes induced by phytohemagglutinin (PHA) and interleuhn-2. Further, studies show that gangliosides—particularly GM3—significantly inhibits the concanavalin A-induced proliferation of peripheral blood lymphocytes. The lymphocytes of melanoma patients seemed to be more sensitive to the influence of exogenous gangliosides than did those of healthy controls. The same was true for mixed lymphocyte reactions.

Aberrant Expression of Gangliosides in Human Renal Cell Carcinomas

Aberrant and elevated ganglioside expression has been observed in neoplasms, and has been shown to be an important marker of tumor progression. We therefore studied the gangliosides of renal cell carcinoma (RCC) by analyzing gangliosides from 18 RCC biopsies, 10 RCC lines and 5 normal kidney biopsies. A comparison of tumor with normal tissue revealed a significant difference in individual ganglioside expression in which the former consistently expressed eight major gangliosides, GM3, GM2, GM1, GD3, GD1A, GD2, GD1B and GT1B, according to the nomenclature of Svennerholm. There was a notable significant mean increase in the expression of GM2, GM1 and GD1A and a significant decrease in the expression of GD3 in tumor tissue compared with normal kidney tissue. Compared with tumor biopsy tissue, RCC cell lines showed a significant decrease in the expression of GM3, but a significant increase in GM2, GM1 and GD2. There was a marked increase in a pathway gangliosides (GM2, GM1, and GD1a) in RCC biopsies and cell lines compared with normal kidney. These studies indicating that RCC have markedly aberrant ganglioside expression similar to neural origin tumors may relate to the activation of a ganglioside pathway enzymes. Gangliosides expressed on RCC tumors may be important markers of tumor progression and target antigens for immunotherapy.

Establishment of an ascitic human melanoma cell line that metastasizes to lung and liver in nude mice

SummaryUCLA-SO-M14, a human melanoma cell line, was cultured for ten passages in vitro. The line was converted to an ascitic form, M14-A, by transplanting M14 into CD-1 nude mice and back into tissue culture. The minimum number of M14-A cells that formed ascites in all mice (within 10–21 days) was 5×105, and over 80% of such mice developed macroscopic liver metastases. M14-A inoculated subcutaneously formed tumor at the site of injection, but rarely led to the development of metastases. However, when M14-A was inoculated subcutaneously in young mice (1–14 days old), more than 50% developed lung (but not liver) metastases. The reproducibility of the formation of ascites and metastases was confirmed by testing M14-A at various passages. M14-A may be useful as a model for the metastatic process in human melanoma.

Redirected lysis of human melanoma cells by a MCSP/CD3-bispecific BiTE antibody that engages patient-derived T cells.

Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8+ T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.