Immunohistochemical study of melanocytic nevus and malignant melanoma with monoclonal antibodies against S-100 subunits.

Immunohistochemical localization of S-100 protein alpha and beta subunits in the cells of melanocytic nevi and malignant melanomas was studied by using monoclonal antibodies directed against each subunit. Although polyclonal anti-S-100 reactivities have been demonstrated uniformly in all nevus cells and melanoma cells, monoclonal anti-S-100 alpha and anti-S-100 beta reactivities were either absent or rarely found in ordinary junctional nevi or junctional nests of ordinary compound nevi. However, in the junctional nests of dysplastic junctional nevi and junctional components of dysplastic compound nevi, monoclonal anti-S-100 alpha reactivity become more frequent, whereas monoclonal anti-S-100 beta reactivity remains negative. In the superficial variety of melanomas such as superficial spreading melanoma and lentigo maligna melanoma, monoclonal anti-S-100 beta is nonreactive until vertical growth or invasiveness begins. Most nodular melanomas are positively stained with both monoclonal anti-S-100 alpha and anti-S-100 beta. It is suggested that monoclonal anti-S-100 alpha can be an indicator of active junctional nevus of melanocytic nevi and the reactivity with monoclonal anti-S-100 beta may be related to vertical progression of superficial spreading melanomas and lentigo maligna melanomas.

A study of the so-called neurotization of nevi.

Fourteen nevi with neuroid zones were examined and compared with nine nevi without neuroid structures. At light microscopic level, nevus cells from the neuroid nevi and the control nevi show the same staining pattern with polyclonal antibodies against S-100 protein. Around the cells of the neuroid zones is a more intensive immunoreactivity with monoclonal antibodies against laminin and collagen type IV than around the nevus cells in the upper dermis and the nevus cells in the control nevi. Also, the Gordon-Sweet stain for reticulin shows a dense network around the cells of the neuroid zones. No immunoreactivity in the neuroid zones was found with monoclonal antibodies against myelin-basic protein, myelin-associated protein, and glial fibrillary acidic protein. At the electron microscopic level, nevus cells from the neuroid zones show stacks of elongated cytoplasmic processes surrounded by basal lamina material. This pattern explains the presence of the abundant cytoplasm seen at light microscopy. Because no features of neural or neurolemmal differentiation could be found, the exactitude of the term neurotization can be questioned.

S100 protein, neurone specific enolase, and nuclear DNA content in Spitz naevus.

The differentiation between Spitz naevus and melanoma is at times difficult. The present study was undertaken to define means to positively identify such melanocytic tumours of doubtful malignancy. Immunohistochemical staining intensity for S100 protein and neurone specific enolase (NSE) was measured in sections of 35 Spitz naevi using a microcomputer image analysis system. The data were compared with results previously obtained from 19 cases of malignant melanoma and 16 benign compound naevi. Disaggregated cells from paraffin-embedded material were stained by the Feulgen technique for DNA estimation. The nuclear DNA content distributions were measured using the same image analysis system. Compared with the malignant cases, the Spitz naevi showed significantly lower staining intensity for both S100 protein (P less than 0.0001) and NSE (P less than 0.0001). When compared with the benign compound naevi, the staining intensity was significantly lower for S100 protein (P = 0.003). The nuclear DNA distribution in Spitz naevi proved to be a normal diploid pattern in 31 cases. Four cases showed a small proportion of hyperdiploid nuclei. The results show that Spitz naevi can be significantly distinguished from malignant melanoma by staining intensity for S100 protein and NSE. A normal diploid DNA content distribution appears to be typical for Spitz naevi. Spitz and benign compound naevi show dissimilar expression of S100 protein which may indicate different patterns of differentiation in these two types of lesion. The image analysis equipment used in this study is accurate, simple to use, produces results rapidly, and is economic. Therefore, it is clinically practicable.

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma.

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.

Type IV collagen and laminin staining patterns in benign and malignant cutaneous lesions.

The presence of both laminin and type IV collagen was sought at the dermo-epidermal junction and in the dermis adjacent to benign melanocytic naevi of the junctional, compound, and intradermal types; dysplastic naevi; and both primary and secondary melanoma. In all, 154 lesions were studied, using antibodies to laminin and type IV collagen and an indirect immunoperoxidase technique. The staining patterns seen with the two antibodies were virtually identical, although that of laminin was generally fainter. Breaks in and thinning of the normally continuous line of type IV collagen and laminin at the dermo-epidermal junction were seen in association with the junctional activity of benign naevi, and in malignant melanomas in association with invasive tumour cells. Both benign and malignant cells of the melanocyte series showed relatively light pericellular staining around individual cells and clusters of cells in the papillary dermis. This staining pattern was much stronger in the deeper reticular dermis. It is concluded that the pattern of staining of these two antibodies and in particular the presence of breaks in type IV collagen and laminin at the dermo-epidermal junction are not specific for either benign or malignant melanocytic lesions and cannot be used as a diagnostic marker of invasive malignancy.

A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue.

The expression of human nerve growth factor (NGF) receptor in tumors and normal tissue was investigated with the use of a monoclonal antibody recently developed against that protein. This antibody, NGFR5, reacted strongly with 100% of 25 nerve sheath tumors. Eight of nine pheochromocytomas and three of three paragangliomas also had positive results, but the immunoreactivity was restricted to the sustentacular cell population. Within cells of melanocytic lineage, there was no immunostaining of melanocytes in normal epidermis, whereas 13 of 14 benign nevi had positive results, primarily involving spindled nevocytic structures within the dermis. NGF receptor was scarcely expressed in human melanoma; 9 of 19 melanomas had positive results, but immunoreactivity was generally restricted to rare cells within the larger tumor cell population. Among nonneurogenic mesenchymal tumors, results were generally negative: 0 of 5 chondrosarcomas, 0 of 6 malignant fibrous histiocytomas, 0 of 3 meningiomas, and 1 of 8 leiomyosarcomas were immunoreactive. Carcinomas were variable in immunoreactivity: 12 of 16 squamous cell carcinomas had positive results, whereas adenocarcinomas demonstrated focal, basal epithelial immunoreactivity and neuroendocrine tumors generally had negative results. Among normal tissues, in addition to expected neural immunostaining, NGFR 5 reacted positively with several nonneural cell types, including lymphoidal follicular dendritic cells, myoepithelial cells, vascular adventitia, and basal epithelium of oral mucosa and hair follicles. Antibodies to NGF receptor may play a role in the identification of benign and malignant soft tissue lesions.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

[Primary tissue culture of nevus cell nevus--in comparison with the findings from neurofibroma culture].

Forty-two nevus cell nevi excised surgically from 40 patients were processed for primary culture. The characteristic features of cultured nevi were compared with those of neurofibroma reported previously. 1) Based on phase-microscopic findings, S-100 protein staining, and slow motion picture, most of the cultured macrophage-like cells or cells with dendrites were considered to be nevocytes. 2) The emigration of nevocytes was observed in 31 of the 42 nevus cultures (73%). Nevocytes classified histologically as the intradermal type with fatty degeneration and nevi obtained from the face and aged patients exhibited higher tendencies to ward emigration when they were cultured. 3) The cultured Schwann cells in neurofibroma and the nevus cell nevus, mainly c-type nevocytes, are both of neural crest origin. They were observed to have closely similar morphological and S-100 protein staining features.

[Immunohistochemical study of nevocellular nevi].

Immunohistochemical analyses were performed on 64 nevocellular nevi (12 compound nevi and 52 intradermal nevi). S-100 protein and its alpha- and beta-subunits were almost always demonstrated in type A, B and C cells, and the staining intensity tended to increase in the type C cells. Neuron-specific enolase was detected in each type of cell; however, the population of positive cells was smaller among type C cells. Beta 2-microglobulin was occasionally demonstrated, but only in type A cells. Vimentin was frequently revealed in every type of cell. Neither myelin basic protein nor glial fibrillary acidic protein was observed in any type of cell. In contrast, normal epidermal melanocytes were positive for vimentin, but negative for S-100 protein and its subunits and neuron-specific enolase. Schwann cells were positive for S-100 protein and its beta-subunit, but negative for the alpha-subunit. Thus, the nevus cells shared a common nature with epidermal melanocytes and Schwann cells which originate from the neural crest; however, the former cells were somewhat different from the latter two kinds and from benign and malignant tumors derived from these cells in the expression of these antigenic substances. Such differences in the expression of antigenic substances may be due to dysontogenic manifestations in nevus cells.

[Prostacyclin (PGI2) biosynthetic activity in variously differentiated neoplasms and the surrounding tissue]. / Zur Prostazyklin- (PGI2-) Biosyntheseaktivität in unterschiedlich differenzierten Neoplasien und umgebendem Gewebe.

For further elucidation of the connection between tumor differentiation, intensity of stroma reaction and the capacity of prostacyclin biosynthesis comparable investigations were performed on the PGI2 formation and the microscopic pattern of 10 tissue samples from naevus cell tumors, skin basalioma, oral pavement epithelium carcinoma and their direct adjoining region. The effects were compared with 10 tissues from healthy oral mucosa and facial skin. The results indicate that the PGI2 activity depends on the quantity of cells within the tissue. The various cells were stimulated in a different manner. The highest values of the PGI2 biosynthesis were found in cells of an inflammatory proliferation direct adjoining the oral pavement epithelium carcinoma. This shows a connection between tumor differentiation, intensity of stroma reaction and the activity of prostacyclin biosynthesis, possibly indicating a criterion of malignity for the prognosis of the tumor disease.

Fibrous papule: a tumor of fibrohistiocytic cells that contain factor XIIIa.

Factor XIIIa, a blood coagulation factor, has been found in a variety of cell types, including dendritic reticulum cells and fibroblast-like mesenchymal cells. We hypothesized that fibrous papule, a lesion of uncertain histogenesis, was composed of dermal stellate cells and in this report demonstrate that this neoplasm consists of cells that contain this factor. Nevus cells do not contain factor XIIIa.

Melanoma cell heterogeneity. A study of two monoclonal antibodies compared with S-100 protein in paraffin sections.

Fifty-six formalin, Bouin's, and Carnoy's fixed, paraffin-embedded malignant melanomas (21 primary, 35 secondary), were studied by avidin-biotin complex immunohistochemistry using monoclonal antibodies (MoAb) HMB-45 and B1.1, comparing reactivity with polyclonal anti-S-100 protein. B1.1 (anti-CEA MoAb) was expressed in a minor percentage of cells of the invasive component of some primary melanomas, and weak to moderately in scattered metastic melanoma cells. MoAb HMB-45 prepared against melanocytic tumors reacted with over 90% of all tumors studied, being weakly reactive in one, and nonreactive in four metastases. This antibody stained some primary melanomas and their dysplastic nevus components in a heterogeneous manner, but was largely nonreactive in deep dermal nevus cells that were in association with invasive melanoma, enabling recognition of the deepest penetration of melanoma cells in the dermal nevus component. MoAb HMB-45 appears specific for melanoma cells, with no cross-reactivity with nonnevomelanocytic malignant tumors (unlike polyclonal anti-S-100 protein). MoAb HMB-45 is more sensitive in detecting malignant melanoma cell heterogeneity than anti-S-100 protein.

Immunohistochemical demonstration of S-100 protein and melanoma-associated antigens in melanocytic nevi.

Thirty-nine melanocytic nevi of varying histologic structure and stages of development were examined for the presence of S-100 protein and melanoma-associated antigens (MAA) by the indirect immunoperoxidase technique, using a polyclonal anti-serum to S-100 protein and monoclonal antibodies to MAA. S-100 protein and MAA were found in the cells of all types of nevi, including dysplastic and congenital nevi. While S-100 protein was present in nevus cells at the epidermodermal junction and at all levels of the dermis, the melanoma-directed monoclonal antibodies BM 24-2, NKI/C-3, Mel-1, and Mel-2 reacted most prominently with the superficial (A-type) nevus cells; the B- and C-type cells being negative in these cells. An exception to this rule was melanoma-monoclonal antibody 34.1 which reacted with nevus cells at all levels; deep C-cells often being more intensely stained than the subepidermal A-cells. The cells of the different nevus cell layers, known to differ morphologically and enzymatically, are, thus, also different in their expression of MAA. Our data suggest that for most antibodies these variations may be due to differences in the metabolic activities of nevus cells at various distances from the epidermis.

MACG1, a mouse monoclonal antibody detecting a monosialoganglioside expressed in tumor-infiltrating macrophages.

MacG1 is a mouse monoclonal antibody (mAb) directed against a ganglioside, which is differentially expressed by macrophages infiltrating malignant melanomas and benign melanocytic lesions. mAb MacG1 was obtained by immunization with liposomes containing a mixture of gangliosides extracted from malignant melanoma. The antibody was selected for binding to melanoma gangliosides and for reactivity with frozen tissue sections of malignant melanoma. mAb MacG1 showed reactivity in 25 of 46 melanomas examined but in only 1 of 51 nevi tested. The mAb did not react with melanoma cells but did with cytoplasmic granules and deposits associated with large dispersed cells, which were also found in some nonmelanomatous tumors and in some lymphoid tissues. Using mAbs directed against differentiation antigens these cells were identified as macrophages. In nearly all reactive tissues MacG1-positive macrophages accounted for a minority of the total macrophages. The difference in reactivity between malignant melanomas and nevi could not be explained by the variable numbers of total macrophages in these lesions. It is suggested that mAb MacG1 may define a functionally distinct subpopulation of tumor-infiltrating macrophages. Staining of cells other than macrophages was observed in some normal and malignant neural tissues. MacG1 bound to a monosialoganglioside extracted from melanoma and reacted only with NeuAc alpha 2-3Gal beta 1-4Glc-Cer when tested with a panel of ganglioside standards.

Human nevocellular nevus cells are surrounded by basement membrane components. Immunohistologic studies of human nevus cells and melanocytes in vivo and in vitro.

Dermal nevus cells in the skin are surrounded by electron-dense deposits resembling a basement membrane (BM), and epidermal melanocytes rest on the epidermal BM. Using antibodies directed against various BM components, we have determined that the BM-like structure surrounding nevus cells in vivo contains type IV collagen, laminin, and BM-1 proteoglycan, analogous to BM throughout the body, but not bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen that are keratinocyte-associated proteins present in the epidermal BM. Moreover, in vitro, both nevus cells and melanocytes derived from adult donors display intracellular and extracellular fibronectin, BM-1 proteoglycan, type IV collagen, and laminin. In contrast, newborn melanocytes maintained under identical culture conditions display none of these BM components, emphasizing the influence of donor age on cell behavior. The data suggest that dermal nevus cells manufacture a BM in vivo, as do certain other neural crest-derived cells. The apparent shared ability of cultured nevus cells and melanocytes to synthesize BM components, coupled with other previously noted behavioral and morphologic similarities in vitro, suggests that these cell types are very closely related; and that morphologic or histochemical differences present in vivo are the result of environmental influences rather than intrinsic differences.

Melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution.

Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, auto-stimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio-Gel P30, reverse phase-high performance liquid chromatography and heparin-sepharose steps. This modified procedure provides a 10-fold improved yield of MGSA over previously published procedures. Purified MGSA-stimulated melanoma cell growth in both 3H-thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13-14 Kd based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF beta), and epidermal growth factor (EGF) in combination with TGF beta did not stimulate 3H-thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three-fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.