Phenotypic profile of clones from early cultures of human metastatic melanomas and its modulation by recombinant interferon gamma

Fifty-six tumor clones isolated by cloning in soft agar from early cultures (before the 10th in vitro passage) of two different human metastatic melanomas (Me9229 and Me28) were characterized by FACS analysis for surface expression of class-I and class-II HLA antigens and of melanoma-associated antigens (MAA) with a panel of 15 monoclonal antibodies (MAbs). A marked phenotypic heterogeneity involving MAA and/or HLA markers was observed among the clones derived from both tumors. The differences among the tumor clones and between them and the uncloned melanoma were qualitative and quantitative for each antigen considered. Clones derived from Me9229 expressed the same HLA profile as the parental culture (class I+, class II-) while strong heterogeneity was observed for MAA expression. Clones from Me28 presented a marked heterogeneity for class-I and class-II HLA antigens but were more homogeneous for MAA. The phenotype of the clones was repeatedly checked over the first month in culture and found to remain generally unchanged and not linked to the cell cycle. However, major changes in antigenic expression of the clones could be observed upon treatment with recombinant interferon-gamma (rIFN-gamma): class-I and -II HLA antigens could be induced or augmented while a moderate inhibition was seen on MAA expression. Furthermore, an apparent hierarchy in expression and/or induction of class-II antigens by rIFN-gamma was observed among the tumor clones. DR antigens were more frequently expressed (Me28 clones) and upon treatment with rIFN-gamma reached higher levels than DP and DQ products. Taken together these results indicate that antigenic heterogeneity for MAA and HLA antigens can be detected in cells isolated from early cultures of human metastatic melanomas and suggest that the original uncloned tumor might be considered as a complex mixed population made up of a number of neoplastic cells each expressing a distinct phenotype which can be modulated by lymphokines such as IFN-gamma.     

Clonal heterogeneity in sporadic melanomas as revealed by loss-of-heterozygosity analysis

The major obstacle preventing effective treatment of melanoma is the biological heterogeneity of tumor cells. This study was performed to determine clonal genetic heterogeneity within primary melanoma and the evolution of these heterogeneous sub-clones during disease progression. DNA samples were obtained from 44 morphologically distinct areas identified within 10 primary tumors and from 15 metastases in the same patients. Loss of heterozygosity (LOH) analyses were performed using 17 microsatellite markers that mapped to chromosomes 6q, 9p, 10q and 18q, the most frequently deleted in melanoma. Of 10 primary tumors, 8 were revealed to have intratumoral genetic heterogeneity in terms of LOH of the 4 chromosome arms examined, 7 containing at least 2 different sub-clones harboring LOH of different chromosome areas, while the remaining one tumor showed prominent intratumoral genetic heterogeneity consisting of at least 6 genetically distinct sub-clones. LOH of 6q was detected only in a sub-set of multiple microdissected samples in most of the primary tumors, but was most frequently detected in metastases, suggesting that loss of this chromosome arm occurred late and played an important part in metastatic progression. Comparison of LOH between sub-clones within primary tumors and within metastases showed the divergence of metastatic clones from dominant populations within the primary tumor in 5 patients, whereas in the remaining three patients parent sub-clones were not identified, or constituted only a minor sub-population within the primary tumors. These results, showing considerable genetic heterogeneity in sporadic melanoma, have profound implications for the choice of future therapeutic strategies.     

Classes I and II HLA and melanoma-associated antigen expression and modulation on melanoma cells isolated from primary and metastatic lesions

Human melanoma cells freshly isolated from 20 patients with primary and 73 patients with metastatic melanomas were analyzed by indirect immunofluorescence staining with monoclonal antibodies (MoAb) to class I (HLA-A, -B, and -C) and class II (HLA-DR and -DQ) antigens and to melanoma associated antigen (MAA). The latter included the GD3-MAA and the high molecular weight MAA. HLA class I antigens were present in 91 and 93% of primary and metastatic tumors, respectively. GD3-MAA was detected in 100% of primary and 80% of metastatic tumors. Whereas the high molecular weight MAA was expressed in 75% of tumors. Sixty % of primary and 50% of metastatic melanomas were stained by anti-HLA-DR MoAb, whereas 38 and 21% of cases, respectively, were stained by anti-HLA-DQ MoAb. Marked phenotypic heterogeneity was evident among primary and metastatic tumors, including different metastases from the same patient. Moreover, in vitro culture of melanoma cells isolated from metastases was associated with an increase from 50 to 75% of tumors stained by anti-HLA-DR MoAb but not of tumors positive for HLA class I antigens and MAA. In vitro incubation with partially purified or recombinant human gamma-interferon enhanced the expression of HLA-DR antigens on all short-term cultured melanoma cells tested but induced and/or augmented the expression of HLA-DQ antigens only in 5 of the 8 cases examined. The average increase in antigenic expression was higher for HLA-DQ than for HLA-DR antigens. Flow cytometric measurement of DNA content of melanoma cells treated with gamma-interferon revealed that the increase of HLA-DR and -DQ expression induced by gamma-interferon was independent from the cell cycle of the tumor cells.     

The differential reactivity of cells of the melanocytic lineage with four monoclonal antibodies against IFN-gamma inducible molecules.

The reactivity of four monoclonal antibodies (MAbs) directed against IFN-gamma inducible antigens with melanocytic cells was investigated in the course of local and systemic tumor progression of human malignant melanoma. Frozen sections of histologically defined melanocytic tissues at different stages of progression were stained with these MAbs using an indirect immunoperoxidase technique. The reactivity of MAbs Me15/B3 and Me15/F9, directed against two different epitopes of a 90-kDa molecule, was found to correlate with melanoma progression. Indeed, a significantly lower percentage of small than of advanced primary melanomas or metastases stained positively. A differential staining of nevocytic and dysplastic nevi was further observed for these two MAbs, which were also non-reactive with normal skin melanocytes. The reactivity of MAb Me14/D12, which identifies the intercellular adhesion molecule ICAM-1 and MAb Mel14/F12, directed against a 40-kDa molecule, was found to be independent of the Breslow thickness of primary melanomas. Both the latter MAbs stained a high proportion of nevocytic and dysplastic nevi. The co-expression of the surface molecules defined by MAbs Me14/D12, Me15/B3 and Me15/F9 in the course of melanoma progression was also analyzed. The frequency of this co-expression increased according to the Breslow thickness of primary melanomas. In addition, up to 100% of metastases, as opposed to 20% of dysplastic nevi, were found to be simultaneously stained by these three MAbs. It is therefore conceivable that high-risk melanocytic lesions might be identified by the use of a combination of MAbs directed against IFN-gamma regulated antigens.     

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.     

Antigenic profile of mucosal melanoma lesions

Ten primary and 6 metastatic mucosal melanoma lesions were tested, utilizing the immunoperoxidase reaction with a panel of 26 monoclonal antibodies (MAbs). The latter included MAbs recognizing membrane-bound and cytoplasmic-melanoma-associated antigens, adhesion molecules and their receptors, and HLA Class-I and Class-II antigens. The melanoma lesions displayed differential reactivity with the panel of MAbs. Mucosal melanoma lesions differ from other types of melanoma in their antigenic profile, since they are the only type of melanoma to display a higher expression of p97 MAA, CEA-MAA and NGF-R in primary lesions than in metastatic ones. Furthermore, the pattern of HMW-MAA expression in mucosal melanoma lesions is different from that in nodular and uveal melanoma lesions and only partly resembles that observed in acral lentiginous melanoma lesions. Like other types of melanoma, mucosal melanoma lesions display a heterogeneous expression of HLA antigens, a selective loss of reactivity with some of the anti-HLA Class-I MAbs and a higher frequency of abnormalities in HLA Class-I antigen expression in metastatic than in primary lesions. Expression of HMW-MAA, 110-kDa MAA, p97 MAA, CEA-MAA, NGF-R and GD₂ ganglioside in primary melanoma lesions was found to be associated with a poor prognosis. Although preliminary in nature, these results suggest that analysis of mucosal melanoma lesions with MAbs, in conjunction with information about the clinical course of the disease, will contribute to determine the biological and clinical significance of the distinct characteristics of the antigenic profile of mucosal melanoma lesions.     

Inflammatory cell infiltrates in human melanoma at different stages of tumor progression

Progression of human melanoma is associated with changes in antigenic phenotypes of tumor cells. To establish whether inflammatory infiltrates in progressing melanoma also change, we studied 146 cutaneous melanomas at different stages of progression. Monoclonal antibodies (MAbs) against lymphocyte and macrophage subpopulations, interleukin-2 receptor (IL-2 R), immune interferon (IFN-gamma), and the IFN-gamma-inducible, progression-associated melanoma antigens HLA-DR and gp89 were applied in situ. During the course of melanoma progression, decreased amounts of peritumoral T cells, IL-2 R-expressing lymphocytes and dermal T6+ dendritic cells were found, while increased numbers of intratumoral T cells, inflammatory (27E10+) and mature (25F9+) macrophages were associated with local progression of primary melanomas. In metastases, most infiltrate components except 25F9+ macrophages were rare. Positive correlations were observed between: (1) dermal T6+ cells and IL-2 R+ lymphocytes, and (2) presence of IFN-gamma in the infiltrate and HLA-DR and gp89 antigens on tumor cells. In all stages, HLA-DR expression on tumor cells was correlated with: (1) a shift towards T8+ lymphocytes in the infiltrates and (2) a loss of IL-2 R expression. Our data suggest mutual influences between melanoma cells and mononuclear cell infiltrates in situ.     

Tumor progression in human malignant melanoma: five stages defined by their antigenic phenotypes

The antigenic profile of melanocytic cells in the course of local and systemic tumor progression of human malignant melanoma was investigated by the reactivity of a panel of monoclonal antibodies (MAbs) in frozen sections of histologically defined melanocytic lesions. Specific antigenic phenotypes made it possible to distinguish 5 groups of lesions which could be ranked in relation to each other due to the sequential acquisition or loss of progression markers. On this basis, a scheme of antigenic changes which accompany the stepwise transformation of normal skin melanocytes into highly malignant metastatic melanoma cells is proposed. The steps of tumor progression identified solely by phenotyping with MAbs were in complete concordance with the concept of melanoma progression derived from histological, statistical and clinical analyses. Furthermore, our finding that the expression of gp89 as well as HLA-DR antigens can be induced by interferon-gamma in vitro provides evidence that immune interferon may play a role in the regulation of genes leading to phenotypic changes in progressing melanoma cells.     

Two antigens detected on human ocular melanomas with the mouse monoclonal antibodies 2/10SN and 10/12SN.

Seven human ocular melanoma cell lines were established in vitro and 3 of these, GU-4, LLN-40 and its subline C17-11, were characterized. Mice were immunized with these ocular melanoma cell lines, and 2 hybridomas producing monoclonal IgG1 antibodies (MAb) were produced. MAb 2/10SN recognizes a 44-kDa monomeric protein, whereas MAb 10/12SN reacts with an 83/65-kDa heterodimeric protein. These melanoma-associated antigens (MAA) are detected at high concentrations in the cytoplasm of ocular melanoma cells. However, cell-surface labelling techniques suggest that these MAA are also associated with the cell-surface membrane. These 2 ocular MAA are also expressed by several skin melanoma cell lines. Immunohistochemical studies have localized these antigens to ocular and skin melanomas, to sweat ducts and basal squamous cells in normal skin, with limited expression in several other normal tissues and some carcinomas. Biodistribution studies in nude mice with human ocular melanomas have demonstrated good localization of 125I-labeled MAb 2/10SN at the tumor sites. Therefore, these 2 MAbs, 2/10SN and 10/12SN, recognize MAA which appear to be unique and may prove useful for imaging purposes.     

Differential expression of melanoma associated antigens in acral lentiginous melanoma and in nodular melanoma lesions.

The reactivity in an avidin-biotin complex immunoperoxidase reaction with a large panel of anti-human melanoma associated antigen (MAA) and anti-HLA monoclonal antibodies of 24 primary and 11 metastatic acral lentiginous melanoma (ALM) lesions was compared to that of 12 primary and 12 metastatic nodular melanoma (NM) lesions. The expression of the membrane bound vitronectin receptor, Mr 110,000 MAA, Mr 97,000 MAA, and intercellular adhesion molecule-1 was significantly lower in both primary and metastatic ALM lesions than in their NM counterparts. Furthermore, primary ALM lesions displayed a significantly lower expression than primary NM lesions of the membrane bound high molecular weight melanoma associated antigen (HMW-MAA), Mr 110,000 MAA, Mr 100,000 MAA, 9-O-acetyl-GD3, GD2-GD3, and GD2, of the cytoplasmic monoclonal antibody 465.12 defined MAA and of transferrin receptor and of HLA-DQ and DP antigens; ALM metastases expressed a significantly lower level of carcinoembryonic antigen-MAA than NM metastases. These antigenic differences do not reflect an antigenic paucity of ALM cells, since ALM lesions express a higher level of T4-tyrosinase than NM lesions and a level of HLA Class I antigens similar to that of NM lesions. In view of the use of HMW-MAA, Mr 97,000 MAA, and GD3 in immunoscintigraphy and/or in immunotherapy, it is noteworthy that the three antigens are expressed in a similar high percentage of ALM metastases and of primary and metastatic NM lesions, while the HMW-MAA is expressed in a markedly lower percentage of primary ALM lesions than Mr 97,000 MAA and GD3. However, the degree of heterogeneity of HMW-MAA within a positive primary ALM lesion, as measured by the percentage of stained melanoma cells, is lower than that of Mr 97,000 MAA and GD3. The expression of the antigens investigated in ALM and NM lesions was not correlated with the presence of lymphocyte infiltrates, melanin content of melanoma cells, and epithelioid and spindle type of melanoma cells in the lesions. On the other hand, the survival of patients with ALM was inversely correlated with the expression of intercellular adhesion molecule 1 or HMW-MAA in their primary lesions. A potential role of HMW-MAA in the course of the disease is suggested by its significantly higher expression in metastatic than in primary ALM lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

An immunocytochemical investigation of antigen expression by skin melanoma cells cultured for vaccine production

There is a great variety of histological patterns of skin melanoma and, in particular, that of its metastatic patterns. Malignant melanocytes are capable of influencing tumor-associated antigen expression. As of now, several varieties of melanoma-associated antigens (MAA) have been identified: MART1/melan A, tyrosinase, MITF, gp100, members of MAGE family, S100, CD63 and CD146. Peptides isolated from such molecules can induce MHC-restricted response of cytotoxic T-lymphocytes. It has been shown that level and nature of specific antigen expression caused by melanocytes correlate with tumor stage and a relationship between survival and MAA expression on tumor cells identified. Morphological features, growth pattern and proliferation rate varied in melanoma cell cultures used in our study. Our experiments involved evaluation of changes in the properties of antigens HLA (class 1 and 2), tumor tissue samples and cells isolated from them, which were capable of stable proliferative activity during passages 5, 10, 15, 20, 25, 30 and 35, and assay for MAA content. Levels of the antigens were significantly lower following long-term culturing melanoma cell melanoma cells in vitro. At initial passages (1-5), antigen profile in most cultures was similar to that in tumor tissue samples. Later on each cell population showed greater antigen expression heterogeneity matched by increased number of cells going through mitotic cycle; their nuclei were stained with antibodies to Ki-67. No HLF A/B/C molecule expression took place during tumor cell culturing: stained cells--in 68.9% of cultures (passages 1-5) and 36.3% (passage 35). However, HLA DQ/DP/DR molecule identification showed an inverse relationship: 44.1% (passage 5) while virtually all the cell lines did synthesize those molecules after passage 35. Hence, MAA and MHC (class 1 and 2) antigens expression in tumor cell should be monitored when they are used for preparation of autologuos and allogenic vaccines. In case of allogenic vaccine production, cell lines capable of stable production of MAA should be selected.     

Progression markers in metastasizing human melanoma cells xenografted to nude mice (review)

The past decade transplants of human tumors in nude mice have been increasingly used as an experimental model for local tumor growth and dissemination. A few human melanoma cell lines have been described that give rise to metastases in nude mice after subcutaneous inoculation. First we give an overview of some relevant literature with respect to the pathogenesis of tumor metastasis, models to study human cancer metastasis, neoplastic progression and the detection of antigens involved in metastasis. Finally we describe our results concerning the morphological and immunohistochemical profile of six different human melanoma cell lines and their xenograft lesions in nude mice using a set of monoclonal antibodies recognizing different categories of human melanoma-associated antigens. From the data we conclude that the nude mouse mouse model appears suitable to study the role of melanoma-associated progression markers in the pathogenesis of metastasis.     

Flow cytometric screening of monoclonal antibodies for drug or toxin targeting to human cancer

Monoclonal antibodies (MAbs) that are candidates for antibody-directed therapy were evaluated by a flow cytometric method. This method accurately quantitates the intensity of staining and the percentage of cells from freshly derived primary tumors expressing the relevant cell surface antigens. This method was applied to human colorectal, gastric, and ovarian carcinomas. It allowed calculations of the number of drug molecules that potentially could be delivered by each MAb as well as selection of the optimal combinations of antibodies for treatment of each type of cancer. The binding of all the MAbs varied among the tumors, although combinations of antibodies reduced this problem. A combination of MAbs C14 and NCRC-23 recognized 97% of colorectal tumors. A combination of C14, NCRC-23, and 791T/36 recognized 95% of gastric tumors. Combinations of either 791T/36 and C14 or 791T/36 and NCRC-11 recognized 80% of ovarian tumors. The number of cells binding with a single MAb varied within the tumor. The optimal anti-colorectal tumor antibody was NCRC-23 (anti-carcinoembryonic antigen), which recognized a mean of 65% of the large cells within a tumor at a mean antigen density of 4.9 X 10(5) sites/cell. The optimal anti-gastric tumor antibody was C14 (anti-Y hapten), which recognized a mean of 66% of the large cells within a tumor at a mean antigen density of 4.4 X 10(5) sites/cell. The optimal anti-ovarian antibody was 115/D8, which recognized 54% of the large cells at a mean antigen density of 4.2 X 10(5) sites/cell. These antigen densities were similar to those calculated for HLA/ABC antigens in colorectal and ovarian cancers. However, the gastric tumors expressed elevated levels of major histocompatibility complex class I antigens, with a mean density of 7.3 X 10(5) sites/cell. Combinations of antibodies that recognize a high proportion of tumor cells are likely to be necessary for MAb-drug targeting to prevent tumor recurrence and/or metastases.     

Autologous cellular immune response to primary and metastatic human melanomas and its regulation by DR antigens expressed on tumor cells

Summary Evidence for heterogeneity of several biological features of human malignant melanoma (Me) like morphology, cytogenetics, oncogenes activation, antigenic expression, metastatizing capacity and procoagulant activity are briefly reviewed in an attempt to distinguish findings related to primary vs. metastatic lesions. In our own studies monoclonal antibodies were used to study expression of MHC class I, class II products and of Me-associated antigens (MAA) on primary and metastatic Me cells. High expression of class I antigens was found in a high percentage of both primary and metastatic tumors, whereas DR and MAA showed a significant variation (from 3 to 90% of cells) in expression both in primary and in metastatic Me. When autologous cell-mediated immune responses were evaluated, it was found that Me cells from primary tumors but not those from lymph node metastases were able to stimulate autologous lymphocytes to proliferate and become cytotoxic for autologous Me.Clonal analysis of cytotoxic lymphocytes was then carried out in order to see whether the lack of lymphocytes reactivity to metastatic cells was due to the absence or to a low frequency of cytotoxic cells in the unstimulated PBL. CTL clones cytotoxic for autologous Me (Auto-Me) cells were indeed isolated. Three classes of CTL clones were identified: 1) one which is cytotoxic for Auto-Me; 2) a second one which lyse Auto-Me and allogeneic Me; and 3) a third one which is cytotoxic for Auto-Me and allogeneic normal and neoplastic cells. Metastatic Me cells, however, had the ability to suppress the stimulation of autologous PBL by alloantigens or IL-2. This effect was dose-dependent and was not due to absorption of IL-2 by Me cells. Since it has been reported that Me cells express class II MHC antigens, we investigated whether there was any correlation between autologous immune responses and DR expression on Me cells. Autologous lymphocytes stimulation was found to occur only with DR+ Me cells from primary lesions, whereas metastatic cells, either DR+ or DR-, did not stimulate autologous PBL. Moreover, the suppressive effect of metastatic Me cells was associated with their expression of DR antigens. The modulation of DR antigens on Me cells by Interferon-gamma correlated positively with their suppressive capacity.Thus, it appears that primary Me can behave differently from the metastatic one in their interactions with the immune system of autologous host. These findings suggest that DR antigens on Me cells may have an important role in the regulation of autologous immune responses.     

Spatial distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

The time-dependent (1-72-h) spatial distribution of three biotinylated anti-melanoma monoclonal antibodies (MAbs), a control MAb, and several macromolecular tracers was studied in two small (4-12-mg), well-characterized human melanoma xenografts (SK-MEL-2, M21) growing in the s.c. space of athymic nude mice. The specific MAbs (436, IND1, and 9.2.27) recognize two different melanoma cell surface antigens (Mr 125,000 glycoprotein melanoma-associated antigen and high molecular weight melanoma-associated antigen) and have equilibrium association constants differing by two orders of magnitude (10(8)-10(10) M-1). SK-MEL-2 tumors were poorly vascularized and were composed of one or several collections of tumor cells with few intratumor blood vessels. In contrast, M21 tumors induced a strong angiogenic response and were organized into multiple small tumor cell nests separated from each other by fine blood vessels. Neither tumor developed extensive connective tissue stroma. In both tumors, hyperpermeable blood vessels were concentrated at the tumor-host interface but some intratumor vessels in M21 tumors were also leaky. Macromolecular tracers extravasated extensively from leaky vessels into tumor stroma but penetrated poorly into tumor parenchyma. All three tumor-specific MAbs stained tumor cell surfaces in a time-dependent fashion such that one-half or more of all tumor cells were stained by 24-48 h. Tumor cell staining was favored by increased density of tumor cell antigens but, at the doses studied, was little affected by differences in affinity among tumor-specific antibodies. The distribution of MAb staining was nonuniform in two respects: (a) peripherally situated tumor cells were more likely to be stained than centrally placed cells, and only in the smallest tumors did MAb reach centrally placed tumor cells; and (b) staining was nonuniform in different parts of the same tumor. The inhomogeneity of tumor cell staining by tumor-specific MAb was attributable to several factors, including: tumor blood vessel number, distribution, perfusion and permeability; distribution of tumor connective tissue stroma; small volume of the parenchymal interstitial space and relatively impaired diffusion of macromolecules in that space (low effective diffusivity of MAb); and interactions between specific MAbs and tumor cells. Of these factors, those associated with the parenchymal compartment apparently were rate limiting, and strategies that enhance parenchymal penetration are likely to improve solid tumor therapy with MAbs.     

The initial steps of tumor progression in melanocytic lineage: a histochemical approach.

Antigen expression was studied by immunohistochemistry in 133 human melanocytic skin lesions to gain insight into the initial steps of tumor development, i.e. in particular the change from melanocytes to benign nevi. We refer to the proposed progression model of Clark and co-workers. The following types of antigens were investigated: (i) intermediate filament antigens (vimentin), (ii) melanoma-associated antigens (HMB-45, NKI/C3, MA-930, LS59), (iii) proliferation-associated antigens (S-100, Ki67, Ro/SSA, calmodulin), (iv) progression-associated antigens (HLA-DR, ICAM-1), and (v) basal membrane antigens (bullous pemphigoid antigen, laminin, fibronectin, collagen type IV). The intensity of expression and the topography of immunoreactive pigment cells were compared with the stage of tumor progression. Special attention was paid to the early steps of this process, i.e. the disturbance of the epidermal melanin unit and the development of melanocytic ("nevocellular")nevi. A dramatic shift of antigen expression (antigen types [i] to [v]) was noted in benign nevi compared with melanocytes. Nevi with cellular atypia disclosed a tendency towards an increased percentage of tumor cells reactive for melanoma- and progression-related antigens (types [ii] and [iv]). However, there was no clear cut level of distinction of antigen expression (types [i] to [v]) between benign and primary malignant melanocytic tumors. So-called dysplastic nevi resembled benign tumors or melanocytes rather than malignant melanoma. Metastatic melanoma of skin showed a relatively high number of Ki67-positive, cycling melanoma cells. The results have a bearing on the concepts of melanocytic nevus ontogenesis and "maturation". It appears that melanocytes lose maturity on their way down to the dermis in contrast to traditional concepts (Abtropfung); this might be of importance for our understanding of melanoma development in association with melanocytic nevi. Our findings are discussed with regard to Clark's model of tumor progression.     

Lack of comparability between binding of monoclonal antibodies to melanoma cells in vitro and localization in vivo

The affinity and specificity of monoclonal antibodies (MAbs) to tumor-associated antigens are often determined by in vitro assays. The specific binding of two anti-human melanoma antibodies (96.5 and ZME-018) and a control antibody (ZCE-025) to three human melanoma cell lines (DX3, A375-M, and Hs294t) was examined under in vitro conditions and compared to in vivo localization of 111In-labeled antibodies to the same cells growing as solid tumors in the subcutis of nude mice. The in vitro binding of the specific MAbs to the tumor cells did not predict in vivo localization. Under in vitro conditions, MAb ZME-018 bound to all three cell lines at levels exceeding that of 96.5, yet ZME-018 did not show superior localization to subcutaneous tumors. MAb 96.5 bound to cultured DX3 cells at levels exceeding those observed with A375-M cells. Yet, 96.5 localized better to A375-M xenografts in nude mice than to DX3 or Hs294t xenografts. Antigen expression differed between in vitro and in vivo growing cells, as evidenced by alteration in binding of 96.5 to tumor cells dissociated from solid subcutaneous tumors. Collectively, the data suggest that in vitro parameters do not predict the clinically relevant localization of MAbs to tumors.     

Biological Study of R24 Mouse Monoclonal Antibody in Patients Undergoing Thoracotomy for Pulmonary Metastases from Soft Tissue Sarcoma

The disialoganglioside GD3 is expressed on the surface of soft tissue sarcoma, malignant melanoma, and other malignant cells and is, therefore, a potential target for therapeutic monoclonal antibodies. Intravenously administered R24, a murine IgG₃ monoclonal antibody to GD3, induces inflammation and tumor regression at sites of metastatic malignant melanoma. R24 5 mg/m² was given intravenously every other day for six doses to 10 patients with pulmonary metastases from a primary soft tissue sarcoma of the extremity for whom thoracotomy was planned. Resected tissue was available from 7 patients. All metastases expressed GD3; however, expression was heterogeneous within tumors, and in no tumor were more than 80% of the cells GD3 positive. A mild to moderate infiltrate consisting of mononuclear cells with T-cell markers was identified around or within pulmonary metastases in 6 patients. Tolerable acute allergic reactions occurred in all patients, but 3 patients had severe chest tightness and bronchospasm that limited the planned therapy. The setting of thoracotomy for metastatic disease provides an ideal system for studies on the pharmacology and biological effects of monoclonal antibodies that target soft tissue sarcoma antigens.     

Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens

Surgically removed benign and malignant human skin lesions of nonmelanocyte origin have been tested with monoclonal antibodies to la antigens, to the HLA-A,B antigenic molecular complex, and to melanoma-associated antigen(s) (MAA). MAA include a high-molecular-weight (HMW) MAA, a 115,000-molecular-weight MAA, a 94,000-molecular-weight MAA, and a cytoplasmic MAA. Indirect immunofluorescence was used as the assay system because of the limited amount of tissue available. When the amount of tissue available was sufficient, double determinant immunoassays (DDIA) were used to quantitate the level of the HMW MAA and of the cytoplasmic MAA. The results of the DDIA were in agreement with those of indirect immunofluorescence in more than 75% of the cases. Malignant skin tumors of various histiotypes displayed three types of changes: 1) appearance of la antigens and cytoplasmic MAA, 2) increase in the level of the HMW MAA, of a 115,000- and a 100,000-molecular-weight MAA, and 3) reduction in the level of HLA-A,B antigens and beta 2-microglobulin. A significant heterogeneity was found in the antigenic profile among various lesions of a given histiotype as well as among tumor cells within a given lesion.