Cytologic detection of penile malignant melanoma of soft parts in pleural effusion using monoclonal antibody HMB-45

A case of penile malignant melanoma of soft parts ("clear cell sarcoma") with pulmonary metastases and malignant effusions is reported. The tumor cells in the pleural effusion were scattered singly and admixed with reactive mesothelial cells. They had abundant granular cytoplasm and round nuclei with prominent nucleoli. Although staining for S-100 protein was positive in sections from the penile lesion, it was negative in sections of a cell block prepared from the effusion; however, the effusion tumor cells demonstrated immunoreactivity with HMB-45, an antimelanoma monoclonal antibody.     

Metastatic balloon cell melanoma: a case report

BACKGROUND: The protean morphology of malignant melanoma is diagnostically challenging. Balloon cell melanoma is a histologic variant composed predominantly or entirely of large cells with abundant, vacuolated cytoplasm. It shares the cytologic features of the other subcategories of malignant melanoma, such as discohesion, nuclear pleomorphism and intranuclear cytoplasmic pseudoinclusions, but generally lacks melanin pigment and, as the name would suggest, is characterized by the presence of numerous cytoplasmic vacuoles. CASE: A 55-year-old man presented with an enlarged right cervical lymph node. Clinically and radiographically this mass was considered to be metastatic; however, the patient had no known primary neoplasm. Fine needle aspiration biopsy (FNAB) and cytologic examination showed numerous discohesive, variably sized, malignant cells with abundant, vacuolated cytoplasm and pleomorphic nuclei with irregular nuclear contours, macronucleoli and frequent intranuclear cytoplasmic pseudoinclusions. Pigment was not identified. These features, along with strong immunohistochemical positivity for S-100, HMB-45 and Melan-A, suggested the diagnosis of balloon cell melanoma. A right parotidectomy and lymph node dissection were performed, and histologic tissue evaluation confirmed the diagnosis. CONCLUSION: This case of lymph node balloon cell melanoma metastasis was diagnosed by FNAB.     

Effect of sodium butyrate on S-100 protein levels and the cAMP response

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression of two glioma cell-associated proteins, glial fibrillary acidic protein (GFAP) and S-100 protein in human glioma-derived cell line (RF), and of S-100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S-100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S-100 levels by two-fold over basal levels. NaB was found to inhibit the induction of S-100 protein but exhibited no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutyrate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta-adrenergic and the forskolin-mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S-100 protein by attenuating cAMP levels.     

Latent transforming growth factor-beta-binding protein 2 is an adhesion protein for melanoma cells

Of the four latent transforming growth factor (TGF)-beta-binding proteins (LTBPs), LTBP-2 is different in the respect that it does not bind small latent forms of TGF-beta. LTBP-2 is therefore likely to have other roles in the extracellular matrix. LTBP-2 contains an RGD putative integrin recognition site, suggesting a role in cell adhesion. We carried out a study on cell attachment to LTBP-2. Purified recombinant LTBP-2 was used as substratum in cell adhesion and migration studies. We found that, unlike most adherent cell lines, all of the melanoma cell lines tested adhered to LTBP-2 very efficiently and in a concentration-dependent manner. Bowes melanoma cells bound most efficiently to LTBP-2 and were used for further characterization. Cell adhesion assays with recombinant LTBP-2 fragments indicated that the adhesive site is located in an N-terminal region of LTBP-2. The attachment of melanoma cells to LTBP-2 was prevented with monoclonal antibody against beta1 integrin in a concentration-dependent manner, suggesting an important role for beta1 integrin in the process. Antibodies against integrin subunits alpha3 and alpha6 decreased melanoma cell adhesion as well. The beta1 and alpha3 integrins were localized on the cell surface, especially in lamellipodia, as observed by immunofluorescence. In addition to integrin antagonists, heparin also markedly decreased melanoma cell adhesion. LTBP-2 also supported Bowes cell migration in modified Boyden chamber assays in a manner similar to the migration on fibronectin. Current data indicate that LTBP-2 can play a role in melanoma cell adhesion.     

Usefulness of silver intensification of immunostaining for cytologic diagnosis of primary melanoma of the female genital organs

OBJECTIVE: To improve the procedure for diagnosing vaginal melanoma with cytopathologic analysis of HMB-45. STUDY DESIGN: The study examined silver intensification of immunostaining of HMB-45 in nine cases of primary melanoma of the vagina and vulva using archival Papanicolaou-stained smears. RESULTS: All nine samples showed positive staining for HMB-45. Five cases showed intensive staining, two moderate and two weak. The positive staining was black in the cytoplasm of melanoma cells but was detected in neither the background nor normal squamous cells. Though destaining of Papanicolaou stain was not performed before immunostaining, the positivity of immunostaining was easily judged. CONCLUSION: After morphologic observation, immunocytochemical study of HMB-45 is possible even though time has passed since the cytologic specimen was obtained. When there is a suspicion of amelanotic melanoma or scantily pigmented melanoma of the vagina and vulva, cytogenesis with HMB-45 is helpful, especially because it involves little invasion.     

Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization.

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.     

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Protein delivery by the cell-penetrating peptide YTA2

In most cases, the transport of cell-penetrating peptide (CPP) with a cargo molecule over the plasma membrane requires a cross-linking of the cargo molecule to the peptide. Lately, a method of cargo delivery, coincubation with CPP, has been applied. We have studied uptake and toxicity of the CPP, YTA2, in the Bowes human melanoma cell line and human MDA-MB-231 breast cancer cell line and compared the results with known cell-penetrating peptides. The results show that fluoresceinyl YTA2 is taken up by the Bowes cells with 3.23 nmol/mg protein and shows low membrane toxicity to the cells with an EC50 of 60 microM. Furthermore, we show that YTA2 is capable of delivering cargo proteins, such as beta-galactosidase and tetramethyl rhodamine iso-thiocyanate (TRITC) labeled streptavidin into cells by coincubation. The delivery of TRITC-labeled streptavidin was quantified to 42.4 pmol streptavidin/mg protein. The delivery of proteins into the cells by mere coincubation is an advantage, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted. In addition, the flexibility in CPP cargo delivery is increased.     

Amelanotic malignant melanoma arising in an ovarian cystic teratoma: a case report

BACKGROUND: Malignant melanoma arising in a cystic teratoma is extremely rare. We report the clinicopathologic and cytopathologic features of an amelanotic malignant melanoma arising in an ovarian cystic teratoma. CASE: A 55-year-old woman presented with an asymptomatic right ovarian mass, showing features of cystic teratoma according to preoperative computed tomography and magnetic resonance imaging. The resected teratoma was suspected to include a nonepithelial malignancy in a touch preparation from a solid component. The tumor showed immunoreactivity for Melan-A, S-100 and HMB-45 in the absence of melanin granules, which established the diagnosis of amelanotic malignant melanoma arising in an ovarian cystic teratoma. CONCLUSION: Cytopathologic findings from touch preparations and immunohistochemical staining are useful for the diagnosis of amelanotic malignant melanoma arising in an ovarian cystic teratoma.     

S-100 protein and neuron-specific enolase in parathyroid glands and C-cells of the thyroid

Normal parathyroid glands and parafollicular cells (C-cells) of man, rat and rabbit, and also human parathyroid adenomas and medullary carcinomas were investigated for the presence of S-100 protein and neuron-specific enolase (NSE). For determination of the proteins immunoperoxidase methods were applied, i.e., the PAP method and the avidin-biotin system. The antisera, of polyclonal origin, were specifically directed against cow S-100 protein and rat or bovine NSE. The respective antisera are known to crossreact with S-100 protein from man, rat, and rabbit, as well as with NSE from man and rat. Surprisingly, the test for S-100 protein was found to be strongly positive in the parathyroid glands of rat and rabbit and was focally positive in normal and adenomatous human parathyroid glands, but completely negative in C-cells and medullary carcinoma cells. NSE was present in C-cells of rat and man, and in medullary carcinoma cells, but was absent in normal and adenomatous parathyroid cells. The results support data that indicate that both parathyroid cells and C-cells are derived from elements of the neural crest, but undergo different maturation processes during embryological development.     

S-100 protein and neuron-specific enolase in parathyroid glands and C-cells of the thyroid

Summary Normal parathyroid glands and parafollicular cells (C-cells) of man, rat and rabbit, and also human parathyroid adenomas and medullary carcinomas were investigated for the presence of S-100 protein and neuron-specific enolase (NSE). For determination of the proteins immuno-peroxidase methods were applied, i.e., the PAP method and the avidin-biotin system. The antisera, of polyclonal origin, were specifically directed against cow S-100 protein and rat or bovine NSE. The respective antisera are known to crossreact with S-100 protein from man, rat, and rabbit, as well as with NSE from man and rat. Surprisingly, the test for S-100 protein was found to be strongly positive in the parathyroid glands of rat and rabbit and was focally positive in normal and adenomatous human parathyroid glands, but completely negative in C-cells and medullary carcinoma cells. NSE was present in C-cells of rat and man, and in medullary carcinoma cells, but was absent in normal and adenomatous parathyroid cells. The results support data that indicate that both parathyroid cells and C-cells are derived from elements of the neural crest, but undergo different maturation processes during embryological development.     

Granular cell tumors: evidence for heterogeneous tumor cell differentiation

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.     

An immunoperoxidase study of S-100 protein in neoplastic cells in serous effusions. Use as a marker for melanoma

S-100 protein has been demonstrated on histologic sections in a number of neural and nonneural tissues, including a variety of neoplasms. Since pleural or peritoneal effusions are frequently the initial presentation of cancer, a study was undertaken to determine if S-100 protein in exfoliated cancer cells could be used as a marker for melanoma. Cells in 36 serous fluids obtained from 32 patients were retrospectively examined for S-100 protein by the peroxidase-antiperoxidase technique. All samples had been previously studied as Papanicolaou-stained cytology specimens, and 25 samples had been studied by transmission electron microscopy. All benign effusions were negative for S-100 protein. Malignant effusions were negative except for some that contained malignant melanoma cells: two of five pigmented melanomas and both cases of amelanotic melanoma. This study indicates that S-100 protein in malignant cells is a useful marker for malignant melanomas, especially the amelanotic type.     

Comparative histopathology of grey-horse-melanoma and human malignant melanoma

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.     

S-100 protein stimulates cellular proliferation

Summary S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [³H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95–31.25 g/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 g/ml). Maximum stimulation of PBL was at 31.25 g/ml. PBL were not inhibited even at high concentrations of S-100p (125 g/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an autocrine tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

HMB-45 staining for cytology of primary melanoma of the vagina. A case report

BACKGROUND: Malignant melanoma in the vagina is very rare, but its diagnosis is usually easy if a melanin pigment is present. With cytodiagnosis, however, it is difficult to differentiate amelanotic melanoma or scantily pigmented melanoma from other conditions. In the present case, monoclonal antibody HMB-45, the efficacy of which has been established in histologic studies, was used in the cytodiagnosis of amelanotic melanoma in the vagina. CASE: A woman, aged 78 years, presented with a brownish, nodular tumor, diameter 3 cm, in the vagina. Scraping smears with Papanicolaou staining showed nonepithelial malignant cells without granules suggesting melanin. Smears stained with HMB-45 showed positive immunoreactivity. The diagnosis underwent histologic confirmation of amelanotic melanoma on the initial biopsy. CONCLUSION: Cytodiagnosis was made with HMB-45, which proved very effective in the differential cytodiagnosis of amelanotic melanoma and scantily pigmented melanoma, particularly because it obviated the need for tissue invasion.