Parotid metastasis of Merkel cell carcinoma in a young patient with ectodermal dysplasia. Diagnosis by fine needle aspiration cytology and immunocytochemistry.

Fine needle aspiration (FNA) biopsy was performed on an intraparotid lymph node metastasis of a Merkel cell carcinoma of the eyelid in a 15-year-old girl with antecedent ectodermal dysplasia syndrome. The cytologic appearance of the aspirate and the results of immunocytochemical typing of intermediate filaments on the FNA smears provided a definitive diagnosis. The Romanowsky stain provided an excellent delineation of paranuclear intracytoplasmic "buttons," which appeared to contain both cytokeratin and neurofilaments by immunocytochemical studies. These findings confirm previous data emphasizing the role of light microscopic observations, supplemented by proper immunocytochemical investigations, in the differential diagnosis of metastatic Merkel cell carcinoma in fine needle aspirates.     

Immunohistochemical analyses point to epidermal origin of human Merkel cells

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.     

Identification of Merkel cells in human skin by specific cytokeratin antibodies:

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.     

Merkel cell distribution in human hair follicles of the fetal and adult scalp

The distribution of Merkel cells in fetal and adult terminal hair follicles of human scalp was studied immunohistochemically using cytokeratin (CK) 20 as a specific Merkel cell marker. In hair follicles of adult scalp, abundant Merkel cells were found enriched in two belt-like clusters, one in the deep infundibulum and one in the isthmus region. No Merkel cells were found in the deep follicular portions including the bulb, or in the dermis. In early fetal hair follicles (bulbous peg stage), Merkel cells were only detected in the basal layer of the developing infundibulum but not in deeper follicular areas. In later stages, Merkel cells were also present in the isthmus and bulge. No Merkel cells were seen in the dermis around developing hair follicles. Nerve growth factor receptor was not only present in nerves but was found to be widely distributed within fetal skin. In adult skin, this receptor was localized to the basal cell layers of the outer root sheath of the bulb and the suprabulbar area, but was not detectable in the areas containing Merkel cells. The present study localizing Merkel cells within the permanent hair follicle structures close to their possible stem cells suggests that they have paracrine functions.     

Merkel cell (small cell) carcinoma of the skin: Immunohistochemical and ultrastructural demonstration of distinctive perinuclear cytokeratin aggregates and a possible association with B cell neoplasms

Summary Three cases of Merkel cell (small cell) carcinoma of the skin are presented with immunohistochemistry for epithelial and neuroendocrine antigens. All three cases showed distinctive punctate perinuclear cytoplasmic positivity for cytokeratin which corresponded to aggregates of intermediate filaments, seen ultrastructurally in two cases. Epithelial membrane antigen was also identified in two cases. Only one case showed cytoplasmic positivity for neuron specific enolase, and immunostaining for a battery of polypeptide hormones was negative. The demonstration of cytokeratin perinuclear inclusions provides a distinctive immunohistochemical feature to aid in their diagnosis. Two of the three patients had chronic lymphocytic leukaemia years before the diagnosis of Merkel cell carcinoma. The possible association of lymphoproliferative disorders, particularly B cell tumours, and Merkel cell carcinoma is discussed.     

The sensorineural apparatus of the human eyelid

The present study describes a complex array of sensory nerve terminals in the human eyelid. In many respects this pattern of innervation resembles that previously described in the rhesus monkey, but in other respects it is unique to man. The most prominent nerve terminals are a complex array of lanceolate and circular Ruffini and free nerve endings that envelop the eyelashes. In addition, Merkel cells have not been conclusively identified to date in other nonhuman primate nonsinus hairs. The external root sheath collar contains Merkel cells, and dermal Merkel cells have also been identified close to the collar. The anterior cutaneous surface of the eyelid contains small vellus hairs with typical lanceolate, Ruffini, and free nerve endings resembling those of primate facial skin. Scattered Meissner and scant simple corpuscles as well as scattered free nerve endings (FNEs) can be identified on the occlusal surface of the eyelid. Intraepithelial as well as dermal FNEs were most easily identified in this region in areas lacking other corpuscular receptors. Corpuscular receptors are especially common at the occlusal/conjunctival angle. The inner or conjunctival surface of the eyelid appears to be a glandular epithelium, whereas in the rhesus monkey it is stratified squamous epithelium. This epithelium needs additional study. In summary, the present study confirms the unique sensory neural status of the human eyelid and verifies the presence of Ruffini nerve terminals by light and electron microscopy and of free nerve ending terminals at least by light microscopy, as well as a unique pattern of innervation of the human eyelash.     

In vitro formation of the Merkel cell‐neurite complex in embryonic mouse whiskers using organotypic co‐cultures

A Merkel cell‐neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch‐sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell‐neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell‐neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co‐culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell‐neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co‐cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell‐neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament‐H‐positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell‐neurite complex can be observed under a microscope using our organotypic co‐culture method.     

Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications

The cytokeratins are the intermediate filament proteins characteristic of epithelial cells. In human cells, some 20 different cytokeratin isotypes have been identified. Epithelial cells express between two and ten cytokeratin isotypes and the consequent profile which reflects both epithelial type and differentiation status may be useful in tumour diagnosis. The transitional epithelium or urothelium of the urinary tract shows alterations in the expression and configuration of cytokeratin isotypes related to stratification and differentiation. In transitional cell carcinoma, changes in cytokeratin profile may provide information of potential diagnostic and prognostic significance. The intensification of immunolabelling with some CK8 and CK18 antibodies may underly an active role in tumour invasion and foci of CK17-positive cells may represent proliferating populations. Loss of CK13 is a marker of grade and stage and de novo expression of CK14 is indicative of squamous differentiation and an unfavourable prognosis. However, perhaps the most important recent finding is the demonstration that a normal CK20 expression pattern is predictive of tumour non-recurrence and can be used to make an objective differential diagnosis between transitional cell papilloma and carcinoma. This review will consider cytokeratin expression in urothelium and discuss the application of cytokeratin typing to the diagnosis and prognosis of patients with TCC.     

Synemin down-regulation in human hepatocellular carcinoma does not destabilize cytoskeletons in vivo

Synemin is a large intermediate filament protein that has been identified in all types of muscle cells. It plays a role in human muscle diseases; however, the role of synemin in tumor cell transformation has rarely been investigated. Because hepatocellular carcinoma cells are morphologically different from normal human hepatocytes, we hypothesized that altered synemin expression and cytoskeletal disorganization might underlie this pleomorphic transformation. To test this hypothesis, we studied synemin expression in hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblotting. In addition, we analyzed the expression level and organization of all cytoskeletal elements after synemin knock-down in human Chang liver cells. Previously we found that plectin knock-down in human Chang liver cells causes a reduction in cytokeratin 18 expression with effects on intermediate filament disorganization and altered cellular morphology. In this study we also compared the effects of synemin knock-down and plectin knock-down on the cytoskeleton expression and organization. The results revealed that synemin expression was down-regulated in human hepatocellular carcinoma compared with normal liver, which is similar to the plectin expression. Surprisingly, the expression of cytoskeletal elements (cytokeratin 18, actin and tubulin) was not influenced by synemin knock-down in human Chang liver cells. The organization of cytoskeletal networks was also unaltered after synemin knock-down. In conclusion, both plectin and synemin are down-regulated in human hepatocellular carcinoma in vivo and transformed human liver cell in vitro. However, the mechanism of cell transformation caused by synemin knock-down is different from that of plectin knock-down. Plectin, but not synemin, knock-down provoked liver cell transformation via suppressing cytokeratin 18 expression and disrupting intermediate filament networks. Synemin knock-down did not influence the cytoskeleton expression and organization of human Chang liver cells.     

Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells

Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork that helps maintain the uniform size and shape of cells. As cells of hepatocellular carcinoma are morphologically different from healthy human hepatocytes, we hypothesized that plectin deficiency and cytoskeletal disorganization underlies this pleomorphic transformation. To test this hypothesis we induced apoptosis as the most accessible pathway for creating plectin deficiency status in vivo. We analyzed expression levels and organization of plectin and other cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after staurosporine-induced apoptosis in human Chang liver cells. The results revealed the expression of plectin and cytokeratin 18 were downregulated in hepatocellular carcinoma tissues in vivo. The expression of actin and tubulin, however, were not altered. In vitro analysis indicated that plectin and cytokeratin 18 were cleaved following staurosporine-treatment of human Chang liver cells. Time course experiments revealed that plectin was cleaved 2 h earlier than cytokeratin 18. The organization of plectin and cytokeratin 18 networks collapsed after staurosporine-treatment. Conclusively, degradation of plectin induced by staurosporine-treatment in liver cells resulted in cytoskeleton disruption and induced morphological changes in these cells by affecting the expression and organization of cytokeratin 18.     

Fine needle aspiration cytologic diagnosis of metastatic Merkel cell carcinoma in the parotid gland

A case of Merkel cell (trabecular) carcinoma metastatic to the parotid gland and diagnosed clinically by fine needle aspiration cytology is described. The primary tumor was on the left upper eyelid and had been resected four months earlier. This unusual presentation and the incidence of secondary tumors involving the parotid gland are discussed.     

Syncytial giant cell component. Review of 55 renal cell carcinomas

Different types of multinucleated giant cells (MGC) have been documented in tumors with osteoclast-like appearance, with trophoblastic differentiation or as tumoral malignant giant cells. A new variety of MGC has been described in renal cell carcinoma. In order to study the frequency, nature and significance of this cellular type, we have reviewed our files. To assess the presence, nature and significance of these MGC in renal cell carcinomas and associated histologic subtype. To review all malignant renal tumors diagnosed in the last 5 years in our hospital and to carry out a morphologic and immunohistochemical study in renal cell carcinomas with syncytial type MGC. 55 renal cell carcinomas were reviewed. Clear cell (conventional) renal cell carcinoma was the most common type encountered (40 cases); two of these cases showed syncytial type MGC and low grade malignancy. Microscopically the MGC contained from 5 to 40 nuclei. Immunohistochemically, mononucleated and multinucleated cells were positive for cytokeratin CAM 5.2, cytokeratin AE1/AE3 and weakly positive for vimentin. Histiocytic, muscular, neural markers, beta-HCG and alpha-fetoprotein were negative. The presence of syncytial type MGC in renal cell carcinomas is an exceptional event. Among 55 renal cell carcinomas we found two cases, both of which were of clear cell subtype and low grade malignancy. The MGC proved positive for epithelial markers and probably are the result of mononucleated tumoral cell fusion. We are unaware of the impact of this MGC in the outcome of patients; such cells appear in low grade carcinomas and do not seem to be of dismal prognosis.     

KIT receptor activation by autocrine and paracrine stem cell factor stimulates growth of merkel cell carcinoma in vitro

The co‐expression of KIT receptor and its ligand stem cell factor (SCF) has been reported in biopsy specimens of Merkel cell carcinoma (MCC). However, the functional role of SCF/KIT in the pathogenesis of this aggressive tumor has not been elucidated. The present study reports expression and effects of SCF and KIT in the Merkel cell carcinoma cell line MCC‐1 in vitro. SCF and KIT were endogenously co‐expressed in MCC‐1 cells. Exogenous soluble SCF modulated KIT receptor mRNA and protein expression, stimulated growth of MCC‐1 cells, upregulated endogenous activation of KIT, AKT, and of extracellular signal‐regulated kinase (ERK) 1/2 signaling pathway. On the contrary, an inhibitory antibody that neutralized the KIT ligand binding site, reduced growth of MCC‐1 cells, as did high doses of the KIT kinase inhibitors imatinib and nilotinib. Also, inhibitors of KIT downstream effectors, U0126 that blocks MEK1/2 as well as wortmannin and LY294002 that inhibit phosphatidylinositol 3‐kinase‐dependent AKT phosphorylation, inhibited the proliferation of MCC‐1 cells. These data support the hypothesis that KIT is activatable by paracrine or autocrine tumor cell‐derived SCF and stimulates growth of Merkel cell carcinoma in vitro. Blockade of KIT and the downstream signaling cascade at various levels results in inhibition of Merkel cell carcinoma growth in vitro, suggesting targets for therapy of this cancer. J. Cell. Physiol. 226: 1099–1109, 2011. © 2010 Wiley‐Liss, Inc.     

The distribution and origin of keratin 20-containing taste buds in rat and human

Sections of tissues containing lingual and extra-lingual taste buds were evaluated with monoclonal antibodies against cytokeratins. In the caudal third of the rat's tongue, keratin 20 immunoreactivity was restricted to taste buds, whereas keratins 7, 8, 18, and 19 were expressed in vallate and foliate taste buds and in cells of salivary ducts that merge with these taste epithelia. Hence, antibodies against keratin 20 most clearly distinguished differentiated taste cells from all other cells. In rat epiglottis, taste buds and isolated bipolar cells were keratin-20-positive. In rat nasopalatine papilla and palate, antibodies against keratin 20 identified Merkel cells, none of which was near to the keratin-20-negative taste buds. Nor were Merkel cells present at epiglottal taste buds or the keratin-20-negative fungiform taste buds or elsewhere in rat tongue. Hence, Merkel cells make no contribution to rat fungiform, epiglottal, nasopalatine, or palatal taste buds. Human and rat keratin-20-positive tissues are reported to be endodermal derivatives with the exception of Merkel cells and luminal urothelial cells. In rats the distribution of keratin-20-positive taste buds was in full agreement with the classical view that the posterior third of the tongue is derived from endoderm (keratin-20-positive taste buds), whereas the anterior two-thirds of the tongue is derived from stomadeal ectoderm (keratin-20-negative taste buds). The equally intense keratin 20 immunoreactivity of human fungiform and vallate taste buds violates this traditional rostro-caudal segregation and suggests that endodermally derived tissues may be present in the tip of the human tongue.     

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

Summary The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm-Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.     

Merkel cells in the stratified squamous nonkeratinized epithelium of the human oesophagus

The aim of our research was to identify Merkel cells in stratified squamous nonkeratinized epithelium of the human oesophagus. We chose the middle and lower part of the oesophagus. For identification of Merkel cells we used antibodies against simple-epithelial cytokeratines, especially anti CK 20, because it is providing the highest degree of specificity. Our investigation showed increased number of CK 20- positive Merkel cells in lower (distal) part of the oesophagus, approximately 60%. In the middle part of the oesophagus there were 30% of CK20-positive Merkel cells.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Using two human tumour cell lines, T24 bladder carcinoma and Molt-4 leukemia, flow-cytometric DNA analysis of pure and mixed cell populations was performed using cellular cytokeratin content to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratin. Using cytokeratin content to gate DNA analysis, the same specificity and sensitivity of cellular DNA content and distribution measurement could be achieved by single-pass FCM analysis of a mixture of the two cell types as was seen when analysing pure populations of the two cell lines. This technique has broad applicability to FCM analysis of mixed populations composed of cells from different tissues of origin.