Identification and quantification of subsets of mononuclear inflammatory cells in melanocytic and other human tumors

We used monoclonal antibodies and an indirect immunoperoxidase technique to identify mononuclear inflammatory cells associated with human tumors. The absolute number of the different types of inflammatory cells was assessed by using a point-counting technique. We studied tissues from six primary cutaneous melanomas, six metastatic melanomas, eight melanocytic nevi, 14 breast cancers, seven examples of fibrocystic disease of the breast, 11 lung cancers, and six colon cancers. Virtually all tumors were associated with substantial numbers of T lymphocytes (Leu3a-positive T helper-inducer cells predominating) and macrophages. Primary melanomas contained significantly more T lymphocytes (P less than .002), macrophages (P less than .005), and Langerhans/dendritic cells (P less than .002) than nevi or normal skin and had a higher proportion of T cells than metastatic melanomas (P less than .01). Breast cancers contained more T lymphocytes and macrophages than occur with fibrocystic disease (P less than .0001 and P less than .002, respectively) and more B lymphocytes. Cancers of the lung and colon contained moderate numbers of T lymphocytes and macrophages; however, colon cancers contained a higher proportion of B cells. Leu7-positive NK/K cells were noted in small numbers in all tumors examined.     

The Degree of Differentiation in Early- and Late-onset Forms of Platyfish-swordtail Hybrid Melanomas: A Morphological and Physiological Study in Primary Culture

The early- and late-onset forms of platyfish-swordtail hybrid melanomas (fry and adult melanomas) and the macromelanophores of platyfish and melanotic hybrids were cultured and characterized according to their cellular morphology and physiology. The fry melanomas contained many large and broad cells. The pigmentation of these cells was somewhat less than that of the macromelanophores of platyfish. Most of the fry melanoma cells responded rapidly to 10⁻⁶M epinephrine, exhibiting reversible melanosome aggregation. The adult melanomas consisted of small, dendritic, and sparsely pigmented cells. The physiological response of these adult melanoma cells varied widely from tumor to tumor. These findings are discussed in relation to the differentiation of fish melanophores.     

Heterogeneity of dendritic cells in rat apical periodontitis

Dendritic cells are important for the induction of T-lymphocyte-mediated immunity by acting as antigen-presenting cells. We have previously reported that dendritic cells are prevalent in the chronic non-expanding phase of rat apical periodontitis. To characterize these cells further, immunoelectron microscopy with three dendritic cell markers (CD11c, OX6, OX62) was conducted for samples from rat models of apical periodontitis. Dendritic cells were divided into two types (type I or type II). Most of the type I dendritic cells expressed CD11c, showed an irregular large profile, had typical cytoplasmic processes, and were recognized as the major dendritic cell population. Most of the type II dendritic cells expressed OX62, showed oval small profiles with a few thin short processes, and were sometimes observed infiltrating from blood vessels. Cell-to-cell contacts between type I dendritic cells and lymphocytes were the most frequently observed associations. These results suggest that dendritic cells are composed of heterogeneous populations that exhibit different phenotypes, morphologies, and maturation/differentiation/activation. This study was supported by Grants-in-Aid for Scientific Research (no. 11470402 to T.O., and nos. 15791091 and 18791393 to T.K.) from the Japan Society for the Promotion of Sciences.     

Histological types of polypoid cutaneous melanoma II

The aim of this study was to ascertain which histological types of melanoma can clinically and morphologically appear as polypoid melanomas. In 645 cases of primary cutaneous melanoma we have analyzed criteria for diagnosis of polypoid cutaneous melanoma and afterwards we have analyzed growth phase in each polypoid melanoma, histological type of atypical melanocytes, the number of epidermal ridges which are occupied by atypical melanocytes, and distribution according to age, sex and location, as well as the disease free survival. According to the criteria for polypoid melanomas we have found 147 (22.8%) polypoid cutaneous melanomas. Analyzing the growth phases, histological types of atypical melanocytes and the number of affected epidermal ridges in the group of polypoid melanomas we have ascertained 2 (1.4%) ALMs, 4 (2.8%) LMMs, 42 (28.6%) SSMs and 99 (67.2%) NMs. Our conclusion is that polypoid cutaneous melanomas are morphological forms of various histological melanoma types (ALM, LMM, SSM and NM) and they can all display polypoid morphological form. Polypoid cutaneous melanomas are most often of nodular histological type.     

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.     

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.     

Melanoma in people aged 65 and over in Scotland, 1979-89.

OBJECTIVE--Detailed analysis of primary cutaneous melanoma first diagnosed in Scotland in patients aged 65 and over. DESIGN--Comparison of changing incidence, sex distribution, site, histogenetic type, tumour thickness, and prognosis of all primary cutaneous melanomas in patients aged 65 and over diagnosed in Scotland in the 11 years 1979-89 with similar data for patients aged under 65. SETTING--Data were obtained from the Scottish Melanoma Group''s database, established in 1979, which aims to record detailed clinical, pathological, and surgical follow up details of all primary cutaneous melanomas registered in Scotland. PATIENTS--1430 patients (954 women, 476 men) aged 65 and over; comprising over a third of the 3903 patients with primary melanoma recorded for all age groups in Scotland during this period. RESULTS--The overall incidence of melanoma in patients aged 65 and over increased from 12.2/100,000 in 1979 to 20.7/100,000 in 1989, with the greatest increase seen in older men, from 7.8/100,000 in 1979 to 18.0/100,000 in 1989. The site most commonly affected was the face in both men and women (33% of all tumours). The most common histogenetic type was superficial spreading melanoma. 526 patients (37%) had melanomas with a tumour thickness of 3.5 mm or greater in the older age group, compared with 453 patients (18%) in those aged under 65. The highest proportion of thick tumours was seen in older men. Five year survival figures for 616 patients diagnosed between 1979 and 1984 were 88%, 66%, and 47% for thin, intermediate, and thick tumours respectively. Overall five year survival for the older age group was 64% compared with 78% for the younger age group. CONCLUSION--The increase in melanoma in the elderly and the high proportion of thick tumours, especially in men, require a specific educational programme for both primary and secondary prevention directed towards the older population.     

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 study of the neuronal structure of the neostriatum in birds with different extrapolation capabilities

A comparative study of the neuronal structure of neostriatum in crows possessing a great capacity for extrapolation of the movement direction of an alimentary stimulus, and in pigeons deprived of it, did not reveal any fundamental differences in the size of cells, the number of dendritic endings and dendritic ramifications. Differences were observed in the neuronal morphology. In the crow neostriatum neurones, the dendrites are thinner, more sinous and provided with a denser cover of extremely fine protoplasmatic protrusions. In corresponding pigeon neurones the dendrites are thicker, more straight and with a smaller number of rather large protoplasmatic outgrowths. Such differences apparently set up morphological prerequisites for a finer analysis and processing of information, which may contribute to greater capacity of crows for extrapolation.     

Development of a cell with dendritic morphology from a precursor of B lymphocyte lineage

Cells developing dendritic morphology were detected in cultures of highly purified human B cells incubated with 4 beta-phorbol 12-myristate 13-acetate (PMA). After 72 hr of culture, 2 to 7% of the cells had assumed a dendritic shape provided that contact with a plastic or glass surface also occurred. Dendritic cells developed in cultures of B cells prepared by positively selecting cells that stained with the B cell-specific monoclonal antibody B1 with the fluorescence-activated cell sorter. By contrast, dendritic cells could not be detected in cultures of cells obtained from patients with Bruton's type agammaglobulinemia that lacked B cells. Cells with dendritic morphology were nonspecific esterase negative and not phagocytic. They expressed HLA-DR, DQ, and DP antigens, receptors for interleukin 2 and transferrin, and were stained by B1 and 60.3, an antibody that identifies the beta-chain common to lymphocyte function associated antigen-1, complement receptor 3, and the p150,95 antigen, but not by monoclonal antibodies to monocytes, complement receptors 2 or 3, NK cells, T cells, or Langerhans' cells. Formation of dendritic cells was inhibited by microtubule poisons (vinblastine, colchicine), a microfilament inhibitor (cytochalasin B), and the 60.3 monoclonal antibody, but not by inhibition of DNA synthesis. These data indicate that a subset of B cells is capable of assuming dendritic morphology after stimulation with phorbol esters and attachment to a surface. These dendritic cells exhibit characteristics that are quite similar to the interdigitating cells found in T cell-dependent areas of lymph nodes.     

Learning Reflectance Confocal Microscopy of Melanocytic Skin Lesions through Histopathologic Transversal Sections

Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM) features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane). The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods) with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features. Four melanomas and 2 nevi with different dermoscopic clues have been studied. Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections. We presented the possibility to perform transverse sections as a new approach to understand RCM features. Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections. Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion. Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.     

'Coronate' amacrine cells in the rabbit retina have the 'starburst' dendritic morphology

Acetylcholine-synthesizing cells in the rabbit retina are symmetrically distributed about the inner plexiform layer: one population of cholinergic amacrines has cell bodies in the inner nuclear layer and an equivalent population of displaced amacrines has cell bodies in the ganglion cell layer. It has been suggested that the morphological correlates of the acetylcholine-synthesizing cells are either coronate amacrine cells or starburst amacrine cells. Coronate cells have a characteristic nuclear morphology and can be selectively labelled by neurofibrillar methods or with the fluorescent dye4',6-diamidino-2-phenyl-indole (DAPI). Starburst cells have a characteristic dendritic morphology but have only been described from Golgi-stained retinae. This paper bridges the gap between the previous studies. DAPI-labelled coronate cells were impaled with a micropipette under microscopic control and filled with Lucifer yellow by iontophoresis. The results show that the coronate amacrines in the ganglion cell layer are type b starburst cells, and that those DAPI-labelled neurones in the inner nuclear layer with a coronate-like nuclear morphology are type a starburst cells. At a given eccentricity the dendritic field diameter of type a starburst cells is about 1.13 times larger than that of type b starburst cells. The dendritic field coverage of coronate (type b starburst) cells increases linearly with decreasing coronate cell density and ranges from 25 on the peak visual streak to 70+ in the superior periphery.     

A Technique for Predicting the Solvent-Producing Ability of Clostridium acetobutylicum

Changes in colony morphology were associated with the degeneration of solvent-producing strains of Clostridium acetobutylicum. The most efficient solvent-producing strains gave rise exclusively to colonies with dense centers containing large numbers of spores. Many outgrowths of various morphologies developed from the perimeter of such colonies after several days of incubation. The most degenerate cultures did not produce solvents and gave rise to large diffuse colonies that did not contain spores. These diffuse colonies did not produce outgrowths. Intermediate colony types were also observed. These could be derived from liquid cultures that were relatively poor solvent producers or from the outgrowths of colonies of efficient solvent-producing strains. Some of these intermediate types produced spores but did so less frequently than the high-solvent-producing strains. The spores of the intermediate types could not be distinguished from those of the most efficient solvent producers on the basis of heat sensitivity. The relationship observed between colony morphology and solvent production provides a method for predicting the solvent-producing potential of C. acetobutylicum cultures.     

The Study of Cutaneous Melanomas in Camargue‐Type Gray‐Skinned Horses (1): Clinical–Pathological Characterization

The clinical and pathological characteristics of cutaneous melanomas occurring in Camargue‐type gray‐skinned horses are reported. Examination of 83 tumor‐bearing horses revealed that the tumors occurred most frequently underneath the tail (93.9%) and at high rates in the peri‐anal region (43.0%), the lips (33.0%), and the eyelids (24.0%), but rarely in the vulva (3.8%). Tumorous lesions were characterized by the presence of either hemispheric nodules or large infiltrated plaques, or their combinations. Microscopic examination indicated that tumorous lesions were composed mostly of melanocytes and numerous melanophages and that component cells manifested a remarkable cellular atypia with anisocytosis and anisokarinosis. Pathological examination of lesions corresponding to earlier stages of the tumors disclosed that tumor formation and its related melanogenesis occurred in close topographical association with apocrine sweat glands, but not at the dermal–epidermal junction. Larger nodules of the tumors were often composed of a concentric deposition of cell layers, each being separated by dermal components from the epidermis. Because of the absence of their tropism toward the epidermis and their multiloculation, horse melanomas are considered to be different in their pathological characteristics from human melanomas.     

Immunohistochemical and histometrical studies of the human thymus with special emphasis on age-related changes in medullary epithelial and dendritic cells

Age-related changes in medullary epithelial and dendritic cells in the human thymus were examined quantitatively using immunohistochemistry and histometry. The material used was thymic biopsy specimens obtained from 105 patients during cardiac surgery, none of whom had immunological abnormalities. By using keratin and tissue polypeptide antigen markers to identify epithelial cells and S-100 protein and LN-2 markers to identify dendritic cells, the numbers of these cells in the medulla were counted morphometrically. The relative proportions of the cortex, medulla, Hassall’s bodies, perivascular space, connective tissue and fatty tissue were evaluated using a computer image-analysis system and the respective relative volumes were calculated. Our results indicate that the number of medullary dendritic cells/mm² and the relative volume of cortical thymocytes decrease steadily up to the age of 40 years, whereas no major change was found in the number of medullary epithelial cells/mm². Supported in part by a Grant-in-Aid (No. 61570664) for Scientific Research from the Japanese Ministry of Education     

Fine needle aspiration biopsy of metastatic melanoma. A morphologic analysis of 174 cases

The prognostic and therapeutic decisions in cases of metastatic melanoma depend upon the morphologic documentation of metastatic disease, which may rapidly and accurately be done by fine needle aspiration (FNA) biopsy of clinically suspicious lesions. The tumor cells derived from malignant melanomas demonstrate a wide range of appearances, however, and other neoplasms may be mimicked. Furthermore, additional neoplasms of other types are more frequent in melanoma patients: the possibility of a new primary tumor must be considered if the morphology of the tumor cells is uncharacteristic. Therefore, a study was undertaken to analyze the morphologic changes seen in FNA biopsy specimens from metastatic malignant melanoma and to determine which features could be the most useful in establishing a definitive diagnosis. A total of 174 consecutive cases, comprising 151 malignant aspirates and 23 inconclusive aspirates, were reviewed. The most significant features for identification of melanoma over other tumor types were the cell shape and nuclear position, the presence of numerous isolated neoplastic cells and occasional binucleated or multinucleated cells. Intracellular melanin in neoplastic cells was diagnostic when present, but it was absent in 60% of the cases. Macronucleoli and/or intranuclear cytoplasmic invaginations were characteristic but variable features. Morphology was also found to vary by site and cell type. Lung aspirates were less cellular and more likely to contain melanin. Aspirates of subcutaneous nodules were more often composed of spindle-shaped cells or of other variant cell types. Lymph node aspirates more often yielded epithelioid cells with macronucleoli and/or intranuclear invaginations. Spindle-cell melanomas usually demonstrated inconspicuous nuclei and rarely showed enlarged nucleoli. Epithelioid-cell tumors contained multinucleated cells and areas of cell wrapping more frequently than did spindle-cell tumors. The findings in this study emphasize that a full awareness of the spectrum of morphologic presentations of metastatic melanoma as well as of the clinical history are needed for greater precision in its diagnosis and for avoidance of the pitfall of misdiagnosing nonmelanomas with similar appearances.     

Establishment of an outgrowth culture system to study growth regulation of normal human epithelium

Summary To study the growth regulation of epithelial cells as a sheet, I developed an outgrowth culture system for normal human ectocervical epithelial (NHCE) cells, whereby outgrowths from tissue explants increase their radius in a constant rate over time. Cinematographic observation revealed that throughout the outgrowths the cells coordinately migrate and proliferate. To date, all 59 specimens examined have shown similar growth characteristics, with explant size not causing any difference in the growth rate; 10⁸ cells/specimen can easily be obtained in 3 wk. Cell densities of outgrowths also remain constant. Moreover, there is no fibroblast contamination, and removal of explants does not affect growth rate. Therefore, pure epithelial outgrowth in uniform growth condition can be prepared for further experiments. The results demonstrate that the outgrowth culture system is an attractive model for analysis of growth control mechanisms in normal human epithelium in vitro.     

The locus coeruleus of cat

Summary The locus coeruleus of cat is populated by two types of neurons: medium sized ones, with plump cell bodies and relatively short dendrites; and small ones, with triangular bodies and relatively long dendrites. The former type is regarded here as typical of the centre, whereas the second type could simply represent displaced neurons from the adjacent griseum centrale. Electron microscopy failed to reveal any outstanding richness in pigment granules in kittens up to five weeks old. Very characteristic somatic appendages were found, mostly in the medium sized neurons. These somatic spines communicate with the perikaryon by means of a narrow neck region. A complex, multilayered, glial sheath surrounds the cells. This glial sheath is pierced by the somatic appendages, which are not surrounded by glia and make contact with axonal knobs. Typical dendritic spines appear to be absent. Axodendritic synapses are made on medium sized dendritic trunks. By and large, most of the synaptic vesicles present in the centre are of the small, clear-centered type. However, dense core vesicles extremely variegated in size and appearance were found, both in presynaptic and postsynaptic profiles. The possibility that dense core vesicles should be regarded as atypical lysosomes rich in by-products of the metabolism of catecholamines (melanine) has been considered.Supported by grant MA 4183 from the Medical Research Council of Canada.     

Sex differences in the motor nucleus of cranial nerve IX-X in Xenopus laevis: a quantitative Golgi study

In the clawed frog (Xenopus laevis), motor neurons in cranial nerve nucleus IX-X control contraction of laryngeal muscles responsible for sexually dimorphic vocal behaviors. We examined sex differences in dendritic arbors of n.IX-X cells using the Golgi-Cox method. Three morphological classes of somal types (ovoid, triangular, and elongate) are present in similar frequencies in n.IX-X of both males and females. The male n.IX-X neuron is a more complex and hypertrophied version of the female n.IX-X cell. The number of primary dendrites is the same for both sexes, but males have more total dendritic segments. The overall dendritic length of male n.IX-X neurons is two to three times that of the female. Males have longer dendritic segments between all branch points. Male and female frogs differ in levels of circulating androgens; neurons of n.IX-X are targets for androgenic steroids. To determine if androgen can affect dendritic morphology in adult females, we examined Golgi-impregnated cells in n.IX-X from ovariectomized females treated with testosterone for 1 month. The total number of dendritic segments was reduced by androgen treatment due to reduction in the number of higher order dendritic segments; the number of primary dendritic segments was unchanged. Androgen treatment may induce resorption of higher order dendritic branches. The overall dendritic length of androgen-treated female n.IX-X neurons was unchanged, and dendritic segments were longer. Thus, although androgen can alter dendrites of n.IX-X cells in adult females, this short-term treatment does not produce a masculine dendritic architecture.