Peripherin, a Neuronal Intermediate Protein, Is Stably Expressed by Neuro

(CANCER RESEARCH 53. 1175-1181. March I. 199.1] Peripherin, a Neuronal Intermediate Protein, Is Stably Expressed by Neuro- endocrine Carcinomas of the Skin, Their Xenograft on Nude Mice, and the Corresponding Primary Cultures1

C. Baudoin, G. Meneguzzi, M. M. Portier, M. DÃ©marchez,F. Bernerd, A. Pisani, and J. P. Ortonne2

Laboratoire tic Recherches Dermatologiques. UHR MÃ©decine,UniversitÃ©cÃeNice-Sophia Aulir/oli.*, tifilo? Nice Cede* 02 fC. B., C. M., A. P.. J. P. O.l: KiochimÃ¬cCellulaire. CollÃ¨ge de France. Il Place Marcelin Benheloi. 75231 Paris Cedex 05 Â¡M.M. P.Â¡:and C.I.R.D.. Route de Lucioles. Valbonne Sophia Antipolis 06560 Valhonne Â¡M.lÃ¬..F. B.Â¡. France

ABSTRACT acteristic of these neurons is the presence of axons lying, at least partially, outside the axis constituted by the encephalon and the spinal The histogenesis of neuroendocrine carcinomas of the skin is still con medulla ( 12. 13). This suggests that peripherin may play a role in the troversial. To determine the degree of neural differentiation of these neo- recognition of the axonal pathway. plasias, we studied the expression of intermediate filament proteins in tumoral tissues. Expressions of peripherin, the neurofilament protein Since NECS display patterns of neuronal differentiation and since NF-L, vimentin, and cytokeratin 8 were analyzed by immunohistochemi- recent results demonstrate the expression of peripherin and NF-L in cal methods on 12 human primary tumors and 3 tumor xenografts on endocrine tissues during ontogenesis and in cell lines isolated from nude mice. Peripherin was detected in 10 primary tumors by immuno- endocrine tumors ( 14), we looked for the expression of such proteins fluorc.sccncc. The protein and the corresponding messenger RNA were in NECS. Cell extracts prepared from NECS before and after xe- identified by two-dimensional gel electrophoresis and Northern analysis in nografting on nude mice and from primary cultures of these tumor extracts of an immunofluorescence-negative tumor. Peripherin, NF-L, and cells were examined by indirect immunofluorescence, two-dimen cytokeratin 8 were detected in tumoral cells, whereas vimentin was found sional gel electrophoresis. and immunoblotting for the presence of exclusively in the stroma. The histolÃ³gica! and ultrastructural properties of the original cells of neuroendocrine carcinomas of the skin, as well as intermediate filament proteins. We also monitored the expression of coexpression of peripherin, cytokeratin 8, and neurofilament polypcptides, peripherin mRNA on Northern blots. were preserved in tumor xenografts and their primary cultures in vitro. These results bring new elements to the knowledge of the biology of MATERIALS AND METHODS neurocndocrine carcinomas of the skin and indicate that peripherin con stitutes a marker for tumor identification. Tissues. Specimens of 12 cases of NECS (II primary tumors and 1 local recurrence) were obtained by surgery. Small pieces from each sample were INTRODUCTION either immediately frozen in liquid nitrogen and stored at -80Â°C. fixed either in Bouin's fixative or in I07r buffered formalin and embedded in paraffin for NECS ' are a distinct entity of tumors also designated as Merkel cell conventional light microscopy, or fixed in glutaraldehyde for electron micros tumors, because of similarities to the cutaneous neuroendocrine Mer copy. kel cells ( 1). These tumors, in addition to features characteristic of All tumors were analyzed by light microscope examination and indirect epithelial differentiation, express a program of neuroendocrine differ imiminofluorescence (coexpression of cytokeratin. neurofilament proteins, and entiation, including synthesis of dense-cored neurosecretory granules, neuron specific enolase). Five tumors were examined by electron microscopy. neuropeptide hormones, and neuron-specific enolase (2. 3). Neverthe Nude Mice Xenografts. Five-7-week-old pathogen-free congenital alhy- less, because of the lack of unequivocal lineage markers, the histo mic Swiss nil/nil mice (Iffa-credo. les Oncins. France), were anesthcli/ed with genesis of NECS remains controversial ( I ). sodium pentobarbital (Nemhutal). Biopsies of 3 primary NECS (M,. Mi, M>). with an average size of 5 mm', were implanted s.c. through an incision at the Intermediate filament proteins are considered useful markers of tissue differentiation, in spite of exceptions occurring in both neoplas- base of the tail. Animals were housed in a sterile environment and tumor growth was monitored twice a week. Tumors which had grown to 1-1.5 cm in tic and diseased states (4). Particular attention has been focused on the diameter were subpassaged as for the first implantation. At each passage, expression of intermediate filament proteins by NECS (5). It has been xenografts were studied by light and electron microscopy. established that most NECS simultaneously express cytokeratins, neu NECS Cell Culture. Tumoral tissues were minced with scissors and cul rofilament proteins (5, 6, 7), and a cytoskeletal component initially tured either on collagen-coated supports or on 3T3 feeder layers in Dulbecco's designated IT protein and subsequently identified as a novel type of modified Eagle's medium (GIBCO) containing 10% fetal calf serum (G1BCO) cytokeratin (CK 20) (5, 8). Peripherin. an intermediate filament pro and 57c inactivated horse serum (GIBCO). Cells were fed with fresh medium tein with an apparent molecular weight of 57,000, was recently de every other day and maintained in culture for at least 6 months. scribed (9, 10). This type III intermediate filament protein is detected Electron and Light Microscopy. Tumor specimens and smears of cultured in neuronal populations originating from different lineages (neural NECS cells were immersed for 5 h at 4Â°in half-strengh Karnovsky fixative in tube, neural crest, placodes) and exerting different functions (moto- cacodylate buffer (pH 7.4) containing 5% sucrose and 0.05% CaCU. After neurons, sensory and autonomie neurons) (11-13). The common char- extensive washing in cacodylate buffer, samples were postfixed in \CAosmium tetroxide for l h at 20Â°C.Ultrathin sections were cut with a diamond knife in a Reichert Ultracut microtome, stained with uranyl acetate and lead citrate, and Received 7/13/92: accepted 12/21/92. The cosls of publication of this article were defrayed in pan by the payment of page examined with a Jeol 1200 EX microscope. For light microscopy, semilhin charges. This article must therefore be hereby marked advertisement in accordance with sections of specimens and smears of cultured cells were dehydrated in cthanol. 18 U.S.C. Section 1734 solely to indicate this fact. embedded in Epon, and stained with hematoxylin-eosin. 1This work was supported by grants from INSERM (CJF 9(M)9), 1'Associalion pour Antibodies. The rabbit anti-peripherin polyclonal antibody was routinely la Recherche contre le Cancer (ARC), the Fondation de France, and the Conseil RÃ©gional PACA. used at a 1/KX)dilution for immunohistochemistry and at a 1/5000 dilution for 2 To whom requests for reprints should be addressed, at Service de Dermatologie. Western blotting. The specificity of this antibody was already documented HÃ´pital Pasteur BP 69-30. Avenue de la Voie Romaine. (K><)02Nice Cedex I. France. ' The abbreviations used area: NECS. neuroendocrine carcinomas of the skin; mAb. (13). monoclonal antibody: PBS. phosphate-buffered saline. pH 7.2; poly(A)* mRNA. poly- The anli-NF-L neurofilament protein mAb (Sigma clone NR4) was used at adenylated mRNA: cDNA, complementary DNA; NF-L. low-molecular-weight ncurofil- a dilution of 1/30 for indirect immunofluorescence and at 1/200 for Western ament suhunit. blotting. The anti-vimentin mAb (Sigma clone 13.2) was used for inumino li 75

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. M-.t kOI.MKX'RIM-, CARCINOMAS OF THK SKIN KXPRI-.SS PtiRIPHt-.RIN histochemistry at a 1/100 working dilution. The Troma 1 mAb, recognizing Seven ug of poly(A) ' mRNA from NEC xenografts and 15 ug of poly(A) ' basic keratin 8. was a kind gift from Dr. Kemler and was used at a I/30 dilution mRNA from primary tumors were fractionated by electrophoresis on 1.2% (15). denaturing agarose gel containing 1.2 M formaldehyde and transferred onto Fluorescein isothiocyanate-, tetramethyl isothiocyanate-, or rhodamine nylon membrane (Hybond N; Amersham). Membranes were subsequently isothiocyanate-labeled anti-rabbit (1/30 final dilution), anti-rat (1/30 final di hybridized in 50% formamide at 42Â°Cwith either peripherin or ÃŸ-actin12P- lution) or anti-mouse ( 1/50 final dilution) (DAKO) were the second antibodies labeled random-primed cDNAs and exposed to hyperfilm MP (Amersham) for indirect immunofluorescence. Peroxidase-conjugated anti-mouse or anti- with intensifying screens. rRNA and 0.24-9.5-kilobase RNA ladder (GIBCO- rabbit IgGs (1/100 final dilution: Diagnostic Pasteur) were the second anti BRL, France) were used as molecular weight standards. bodies for Western blotting. Indirect Immunofluorescence. Drops of NECS cell suspensions were par tially dried on glass slides and fixed in acetone for 10 min at 4Â°C.Five-um- RESULTS thick sections from fro/.en tissues were air dried and fixed in acetone as described previously ( 16). Samples washed 15 min at 4Â°Cin PBS were incu Upon light and electron microscopy examination, the 12 primary bated I for h at 37Â°Cwith the primary antibody diluted in PBS (see "Anti tumors presented the hallmarks of neuroendocrine skin carcinomas bodies"). After extensive washing in PBS, sections and smears were incubated (1). All tumors were positively stained with the anti-cytokeratin 8 with the appropriate second antibodies. Sections stained with a fluorescein antibody and with an antibody raised against the neurofilament protein isothiocyanate conjugate were counterstained with propidium iodide and NF-L. The tumors reacted with the anti-vimentin antibody, but fluo mounted in ÃŒOVcglycerol-paraphenylene diamine. Slides were examined with rescence was specific to the cells of the stroma. The anti-peripherin a Zeiss Universal Microscope with epiillumination using an HBO 50 mercury polyclonal antibody stained 10 of the 12 NECS (Fig. 1; Table 1). The arc lamp. Ill RS epifluorescence condenser, and fluorescein isothiocyanate or antibodies reacted against cytoplasmic proteins: the anti-cytokeratin tetramethyl Â¡sothiocyanate filters. Photographs were taken on Kodak Ektach antibody clearly bound to thin cytoplasmic proteins, whereas the rome 400 film using a Zeiss MC 63 camera system. anti-peripherin and the anti-NF-L antibody did not define any orga Polypeptide Analysis by Bidimensional Gel Electrophoresis and West nized network. All the three antibodies stained paranuclear balls in ern Blotting. NECS tumoral tissues were homogenized and proteins of the crude extracts were separated by two-dimensional gel electrophoresis accord large tumor cells. ing to the method of O'Farrell (17) and modified as described (18). Gels were stained with Coomassie blue. Peptides were then blotted for 45 min onto nitrocellulose membranes (Hybond C; Amersham International UK) at 700 mA Table I Detection of intermediate filaments in human ni'itnientlwrine carcintinuu in a Transphor apparatus (Hoefer Scientific Instruments) as described (19). of the skin Peptides were first visualized on the nitrocellulose membrane by 5 min staining Primary tumors with Ponceau red (0.2% Ponceau red in 3% trichloroacetic acid), located with AntibodyAnti-cytokeratin a marker, and then destained with distilled water. (monoclonal)Anti-NF-L8 Northern Blot Analysis. Total mRNAs were prepared from tumoral tissues according to the method of Chomczynski and Sacchi (20) and poly(A) + (monoclonal)Anti-peripherin (polyclonal)Anti-vimentin mRNAs were purified following standard techniques. (monoclonal)Positive/total12/129/1010/120/12Negative/total0/121/102/1212/12

Fig. I. Immunofluorescence microscopy of cutaneous neuroendocrine carcinoma cells using antibodies against intermediate filament proteins. Specimens were treated with antibodies directed against cylokeratin 8 (a), vimentin (b), peripherin (r), or NF-L protein (d). Note the specific labeling of tumor (a, c, d) and stroma cells (b). X 200. 1176

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Fig. 2. Electron microscopy of cutaneous ncurocndocrine carcinoma cells. NECS xenograft. if, x 5,(HK);/?. X K).(XK))The tumoral cells exhibit dense-cured neurosecretory granules and paranuclear globular fibrous bodies composed of intermediate filaments. Tumor cells grown in vitra, present the paranuclear globular fibrous bodies and dense-cored neurosccretory granules characteristic of NECS it: X 5,000; .

In order to follow a possible phenotypic evolution of tumoral tis passages. Animals were sacrificed when the xenograft reached a size sues during proliferation, tumor expiants were xenografted on nude of 2 cm and the excised tumor tissues underwent macroscopic exam miee. Implants were passaged in vivo every 4 months, on average, for ination. Xenografts were always localized in the dermis, with no at least 3 years. Tumoral growth was slightly enhanced in animals evidence of invasive proliferation. Neoplastic cells were arranged in grafted with expiants from xenografts obtained after a high number of solid sheets and cellular cords formation were faintly visible at (he 1177

Table 2 Detection of peripherin expression in xenografted and primary NECS stained by the anti-peripherin, the anti-NF-L, and the anti-cytokeratin M, M2" M," NEC-1 antibodies but not by the anti-vimentin antibody. Interestingly, and Indirect immunofluorescence with the exception of the M, tumor-derived cultures, peripherin ex Two-dimensional SDS/PAGE' ND pression was stronger in cultured cells than in the original tumor cells. Double immunofluorescence labeling for peripherin and NF-L re Western blotting ND Northern blotting ND vealed a partial overlap of the cytoplasmic staining. However, the two " NECS grafted on athymic nude mice. types of intermediate filaments were constantly detected in the para- " Primary NECS. ' SDS/PAGE. sodium dodecyl sulfate/polyacrylamide gel electrophoresis. nuclear balls, clearly conspicuous in large fractions of the cellular population (Fig. 3, c and d). We then performed the biochemical characterization of NECS in tumor periphery. At the microscopic level, the tumoral cells displayed termediate filament polypeptides. A primary NECS tumor (NECS 1) a homogeneous shape and high rates of mitoses. As assessed by and two NECS xenografts (MrM2) were analyzed by two-dimen electron microscopy, the majority of the xenograft tumor cells retained sional gel electrophoresis and subsequent immunostaining. Periph the ultrastructural features typical of NECS, including membrane- erin, NF-L, and cytokeratin 8 were clearly found coexpressed in NECS 1 and in the xenograft M2 (Fig. 4, A, A- A" and C, C'- c"). The bound dense-cored neuroendocrine-like granules, paranuclear whorl- like intermediate filament aggregates and occasional rudimentary amount of these two proteins was not stoichiometric, since in the junctions (Fig. 2). The general immunohistological picture drawn xenograft M, NF-L was visualized as a small spot, whereas peripherin from studies performed on three tumor xenografts was similar to that remained undetectable. Interestingly, the amount of vimentin found in previously obtained from the studies performed on the primary tu the stroma was proportional to the amounts of peripherin and NF-L mors. With the exception of the tumor xenograft M, which was detected in tumoral tissues (Fig. 4). Although the imunofluorescence negative for the expression of peripherin, the other two tumors were studies show that vimentin is expressed only in stroma, this protein indistinguishable from the corresponding original tumors. Results are was detected in all gels, this because of the unavoidable presence of summarized in Tables I and 2. stroma cells in the tumor samples. An important observation is that To verify whether the phenotypic and biochemical properties of both peripherin and vimentin migrated at a pHÂ¡lower than that ex NECS cells were stably maintained in an artificial environment, pri pected (9). The cause for this altered migration is under investigation. mary cultures were established in vitro from tumor expiants. When Lack of immunological staining cannot be regarded as an absolute cultured on plastic, all the tumor cells maintained a round shape and criterion for excluding the possible synthesis of low amounts of pe grew as loosely packed floating aggregates, amorphous and irregular ripherin. We therefore examined on xenografts whether absence of in outline (Fig. 3). Indirect immunofluorescence performed on smears immunological staining of peripherin could be correlated with the revealed that each cell expressed a pattern of cytoskeletal proteins absence of specific RNA transcripts. Poly(A) ' mRNAs were prepared similar to that observed in the corresponding tumors. Cells could be from a primary tumor (NECS-1 ), from the xenograft M2, and from the

Fig. 3. Light and immunofluorescence microscopy of cutaneous neuroendocrine carcinoma cells in primary culture. Appearance of subconfluent NECS tumor cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 5% inactivated horse serum, Staining with mÃ©thylÃ¨neblue(Â«);immunostaining with anticytokeratin 8 antibody (h) double immunostaining with antiperipherin antibody (c)', and with anti NF-L antibody (d). X 200. 1178

NF-LÂ»- .

Pe- - -Vi (3 A' A" A r NF-L.

-.Vi

B' B B' B'"

at:

Vi ÃŸ - ViD C' C"

Fig. 4. Identification by two-dimensional gel electrophoresis and Western blotting of cytoskeletal components of NECS cells. Portions of Coomassie blue-siained two-dimensional gels loaded with an extract of a primary tumor (NEC-1) (A ), or with extracts from the NECS grafted on athymic nude mice MI (fi), and MT (C). Gels are shown before (A-C) and after Western blotting on il nitrocellulose membrane and immunodetection with the monoclonal antibody against NF-L (A', B', C' ), the polyclonal against peripherin. (A", fi", C" ) and the monoclonal against vimentin (A"', B'".C'"). NF-L, low-molecular-mass neurofilament protein; Pe. peripherin; Vi, vimentin; ViD, vimentin-derived peptides; a.ÃŸ.corre sponding subunits of tubulin. The slightly decorated spots on the left side of A'" and C'" are due to degradation products of lamins.

xenograft M,, negative for this protein. The probe was a peripherin (Fig. 3.C and d). This observation reveals the complexity of the cDNA clone isolated from a murine neuroblastoma Agt 11 cDNA cytoskeletal structures of NECS, which display patterns common both library by immunoscreening (10). As shown in Fig. 5, hybridization of to simple and stratified epithelia and to neural cells. Cytokeratin and Northern blots with the |12P]cDNA revealed the presence of a specific NF-L neurofilament protein appear as thin cytoplasmic fibrils and 2-kilobase transcript both in the xenograft M, and. as expected, in the perinuclear plaques. Accordingly to previous observations (6, 16, 21 ), peripherin-positive tumors M2 and NECS I. Surprisingly the amount perinuclear plaques were brightly stained by anti-cytokeratin and anti- of peripherin transcripts in M, was estimated to be at least 3 times neurofilament antibodies, whereas fibrils reacted predominantly with higher than that detected in M2. The different rates of transcription in anti-cytokeratin antisera. We could not determine by double labeling the xenografts were confirmed by rehybridization of the blot with a techniques whether cytokeratin and neurofilament proteins are found ÃŸ-actinprobe. In contrast to the xenograft M2, the M, tumor exhibited within the same intermediate filaments or whether cytokeratin fila higher levels of transcription for peripherin than for ÃŸ-actin.The ments and neurofilaments are distinct but closely intermingled. It is intensity of the hybridization bands was comparable in the NECS 1 also not clear whether peripherin is a constituent of NECS heteropoly- preparation. mer filaments, or whether it provides a distinct filament structure. The anti-peripherin antibody strongly stained the perinuclear plaques, thus suggesting the presence of peripherin in the aggregates. The coex- DISCUSSION pression of the three different types of intermediate filament proteins In this report we demonstrate that NECS cells express peripherin. was confirmed by double immunofluorescence experiments. Synthesis of the protein was first monitored in human tumors by The nature of the paranuclear aggregates, alternatively termed fi indirect immunofluorescence using a specific antibody that does not brous bodies or perinuclear aggregates, is unknown. Ultrastructurally, cross-react with other cytoskeletal components (9). A strong labeling they correspond to whorl-like intermediate filaments and could arise of tumor cells indicated the presence of peripherin (Fig.Io) coex- from a depolymerization of the intermediate filament network possi pressed with cytokeratins and the small neurofilament NF-L protein bly caused by overphosphorylation of cytoskeletal components. In- 1179

stages of ontogenesis (27) and expression of neural-specific genes 1 during the development has been reported for chromaffin cells and ÃŸ-cellsof Langerhans islets (14. 30). Expression of neural-specific genes by Merkel cells during development has not been investigated, but since peripherin is detected by indirect immunofluorescence in Merkel cells of adult pigs, it is conceivable that neuroendocrine tu mors and their corresponding primary cultures could maintain syn thesis of neural polypeptides already expressed by normal cells from which they originate. On the other hand, cancerization may induce activation of suppressed functions. In adult chromaffin cells and ÃŸ-cellsof Langerhans islets and in the corresponding tumors, neural- B specific genes are inactive. However, reactivation of peripherin and NF-L filament protein synthesis in the tumor-derived pheochromocytoma PC 12 cell line and in the Â¡nsulinomaRIN5F cells was demon Fig. 5. Northern hlot analysis of niRNA purified from NECS tumors. The poly{AP RNAs from a primar\' tumor (NEC-1 ) and from the xenografts Mi and M? were hybrid strated (14). Moreover, in primary cultures of normal endocrine cells, ized with a peripherin cDNA probe (A ). Lune t was loaded with 7 ug of poly (A) + mRNA enhanced expression of neural intermediate filaments appears to be extracted from MÂ¡;hiÃ±es2 and .Ã®wereloaded with 15 ug of poly (A)~ mRNAs from the xenograft Mi and the primary tumor NEC-], respectively. Upon dehybridization. the correlated with the absence of intercellular interactions preventing nitrocellulose sheet was rehybridized with a ÃŸ-actinprobe (ÃŸ).The two probes recognize extending processes (31, 32). a 2-kilobase mRNA. Peripherin constitutes an additional marker for the identification of the histogenesis of NECS. Of 12 tumors, 10 were positive for im- deed, it is now admitted that phosphorylation of vimentin and that of munostaining with a peripherin-specific antibody. Moreover, when the amino-terminal domain of NF-L induce a depolymerization of further analyzed by Northern blot, an apparently negative tumor dis intermediate filaments in vitro (22-24). Moreover, altered phosphor played a low level of expression of peripherin mRNA. In this context, ylation patterns of vimentin and peripherin would be compatible with the relevance of peripherin as a marker for differential diagnosis, e.g., the more acidic pHÂ¡softhese proteins extracted from NECS cells seen to discriminate between primary NECS and secondary location of by isoelectric focusing (Fig. 4). neuroendocrine tumors of extracutaneous origin is still to be evalu The results obtained on NECS cells were confirmed by the analysis ated. It is well established that rat pheochromocytoma and mouse of xenografted tumors. Even after a high number of serial heterotrans- neuroblastoma cells express peripherin. however, no data are yet plantations, xenografts were morphologically indistinguishable from available from other human neuroendocrine tumors of epithelial type, the original tumors, as evidenced by the presence of neurosecretory such as pulmonary neuroendocrine tumors, the medullary carcinoma granules, neuron-specific enolase, and neuropeptides (data not of the thyroid, islet cell tumors of pancreas, and the carcinoid of small shown). The histolÃ³gica! and ultrastructural properties of the original intestine. Further investigations on the distribution of peripherin in a NECS cells were also preserved in NECS cells derived from primary wide variety of neuroendocrine cells and related tumors are therefore cultures of xenografts upon expansion in vitro. Coexpression of pe required. ripherin. cytokeratins, and neurofilament polypeptides was main tained at elevated rates, while synthesis of vimentin remained unde- ACKNOWLEDGMENTS tectable. In culture, NECS cells adhered poorly to plastic substrates and showed average mean doubling times exceeding 5 days. Attempts We thank O. Partouche for her skillful technical assistance. C. Minghelli for the photographic work. E. Van Ohherghen for critical reading of the manu to improve adhesion by coating dishes and seeding cells on fibroblast script, and D. Sripati for helpful discussion. feeder layers were unsuccessful, as well as adjunction of mitogenic factors to media (epidermal growth factor, neuronal growth factor, REFERENCES basic fibroblast growth factor, from horse serum) to stimulate growth. Similar observations had been previously made on the neuroendocrine 1. Gould. V., Moll. R., Moll. I., Lee. !.. and Franke, W. W. Biology of disease. Neu- roendocrine (Merkell cells of the skin: hyperplasias. dysplasias. and neoplasms. Lab. skin carcinoma cell line MKL-1 (25) and on primary cultures of a Invest.. 52: 334-353. 1985. lymph node metastasis (26). 2. Sibley. R. K., Dehner. L. P.. and Rosai. J. Primary neuroendocrine (Merkel cell?) carcinoma of the skin: I. Aclinicopathologic and ultrastructural study of 43 cases. Am. Injection of NECS cells induced tumors in mice after latency pe J. Surg. Palhol.. 9: 95-108, 1985. riods of 3-5 months, without occurrence of detectable metastasis. The 3. Sibley. R. K.. and Dahl. D. Primary neuroendocrine (Merkel cell?) carcinoma of the histolÃ³gica! and ultrastructural characteristics of NECS found again in skin: II. An immunocytochemical study of 21 cases. Am. J. Surg. Pathol., 9: 109-116, all the analyzed tumors gave evidence of the stability of the phenotype 1985. 4. Osborn. M.. and Weber. K. Tumor diagnosis by intermediate filament typing: a novel (not shown). tool for surgical pathology. Lab. Invest., 48: 372-374. 1983. The biological role of peripherin is still unclear. The protein may 5. Moll. R.. and Franke. W. W. Cytoskeletal differences between human neuroendocrine tumors: a cytoskeletal protein of molecular weight 46.000 distinguishes cutaneous play a role in the recognition of the axonal pathway (27, 28) and its from pulmonary neuroendocrine nenoplasms. Differentiation. M): 165-175. 1985 . expression provides an additional stigma of the neural differentiation 6. Moll. R.. Osbom. M.. Hartschuh, W.. Moll, !.. Mahrle, G., and Weber. K. Variability of NECS. Because of striking morphological similarities, it has been of expression and arrangement of cytokeratin and ncurofilamcnts in cutaneous neuroendocrine carcinomas (Merkel cell tumors): Immunocytochemical and biochemical proposed that NECS could arise from Merkel cells ( 1). Definite his- analysis of twelve cases. 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