(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 theCorresponding Primary Cultures1

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

Laboratoire tic Recherches Dermatologiques. UHR Médecine,UniversitécíeNice-Sophia Aulir/oli.*, tifilo? Nice Cede* 02 fC. B., C. M., A. P.. J. P. O.l: KiochimìcCellulaire.Collège de France. Il Place Marcelin Benheloi. 75231 Paris Cedex 05 ¡M.M. P.¡:and C.I.R.D.. Route de Lucioles. Valbonne Sophia Antipolis 06560 Valhonne ¡M.lì..F. B.¡.France

ABSTRACT

The histogenesis of neuroendocrine carcinomas of the skin is still controversial. To determine the degree of neural differentiation of these neo-

plasias, we studied the expression of intermediate filament proteins intumoral tissues. Expressions of peripherin, the neurofilament proteinNF-L, vimentin, and cytokeratin 8 were analyzed by immunohistochemi-

cal methods on 12 human primary tumors and 3 tumor xenografts onnude mice. Peripherin was detected in 10 primary tumors by immuno-

fluorc.sccncc. The protein and the corresponding messenger RNA wereidentified by two-dimensional gel electrophoresis and Northern analysis inextracts of an immunofluorescence-negative tumor. Peripherin, NF-L, and

cytokeratin 8 were detected in tumoral cells, whereas vimentin was foundexclusively in the stroma. The histológica! and ultrastructural propertiesof the original cells of neuroendocrine carcinomas of the skin, as well ascoexpression of peripherin, cytokeratin 8, and neurofilament polypcptides,were preserved in tumor xenografts and their primary cultures in vitro.These results bring new elements to the knowledge of the biology ofneurocndocrine carcinomas of the skin and indicate that peripherin constitutes a marker for tumor identification.

INTRODUCTION

NECS ' are a distinct entity of tumors also designated as Merkel cell

tumors, because of similarities to the cutaneous neuroendocrine Merkel cells ( 1). These tumors, in addition to features characteristic ofepithelial differentiation, express a program of neuroendocrine differentiation, including synthesis of dense-cored neurosecretory granules,neuropeptide hormones, and neuron-specific enolase (2. 3). Neverthe

less, because of the lack of unequivocal lineage markers, the histogenesis of NECS remains controversial ( I ).

Intermediate filament proteins are considered useful markers oftissue differentiation, in spite of exceptions occurring in both neoplas-

tic and diseased states (4). Particular attention has been focused on theexpression of intermediate filament proteins by NECS (5). It has beenestablished that most NECS simultaneously express cytokeratins, neurofilament proteins (5, 6, 7), and a cytoskeletal component initiallydesignated IT protein and subsequently identified as a novel type ofcytokeratin (CK 20) (5, 8). Peripherin. an intermediate filament protein with an apparent molecular weight of 57,000, was recently described (9, 10). This type III intermediate filament protein is detectedin neuronal populations originating from different lineages (neuraltube, neural crest, placodes) and exerting different functions (moto-neurons, sensory and autonomie neurons) (11-13). The common char-

Received 7/13/92: accepted 12/21/92.The cosls of publication of this article were defrayed in pan by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from INSERM (CJF 9(M)9), 1'Associalion pour

la Recherche contre le Cancer (ARC), the Fondation de France, and the Conseil RégionalPACA.

2 To whom requests for reprints should be addressed, at Service de Dermatologie.Hôpital 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.monoclonal antibody: PBS. phosphate-buffered saline. pH 7.2; poly(A)* mRNA. poly-

adenylated mRNA: cDNA, complementary DNA; NF-L. low-molecular-weight ncurofil-ament suhunit.

acteristic of these neurons is the presence of axons lying, at leastpartially, outside the axis constituted by the encephalon and the spinalmedulla ( 12. 13). This suggests that peripherin may play a role in therecognition of the axonal pathway.

Since NECS display patterns of neuronal differentiation and sincerecent results demonstrate the expression of peripherin and NF-L in

endocrine tissues during ontogenesis and in cell lines isolated fromendocrine tumors ( 14), we looked for the expression of such proteinsin NECS. Cell extracts prepared from NECS before and after xe-

nografting on nude mice and from primary cultures of these tumorcells were examined by indirect immunofluorescence, two-dimen

sional gel electrophoresis. and immunoblotting for the presence ofintermediate filament proteins. We also monitored the expression ofperipherin mRNA on Northern blots.

MATERIALS AND METHODS

Tissues. Specimens of 12 cases of NECS (II primary tumors and 1 localrecurrence) were obtained by surgery. Small pieces from each sample wereeither immediately frozen in liquid nitrogen and stored at -80°C. fixed eitherin Bouin's fixative or in I07r buffered formalin and embedded in paraffin for

conventional light microscopy, or fixed in glutaraldehyde for electron microscopy.

All tumors were analyzed by light microscope examination and indirectimiminofluorescence (coexpression of cytokeratin. neurofilament proteins, andneuron specific enolase). Five tumors were examined by electron microscopy.

Nude Mice Xenografts. Five-7-week-old pathogen-free congenital alhy-mic Swiss nil/nil mice (Iffa-credo. les Oncins. France), were anesthcli/ed with

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

base of the tail. Animals were housed in a sterile environment and tumorgrowth was monitored twice a week. Tumors which had grown to 1-1.5 cm in

diameter were subpassaged as for the first implantation. At each passage,xenografts were studied by light and electron microscopy.

NECS Cell Culture. Tumoral tissues were minced with scissors and cultured either on collagen-coated supports or on 3T3 feeder layers in Dulbecco'smodified Eagle's medium (GIBCO) containing 10% fetal calf serum (G1BCO)

and 57c inactivated horse serum (GIBCO). Cells were fed with fresh mediumevery other day and maintained in culture for at least 6 months.

Electron and Light Microscopy. Tumor specimens and smears of culturedNECS cells were immersed for 5 h at 4°in half-strengh Karnovsky fixative in

cacodylate buffer (pH 7.4) containing 5% sucrose and 0.05% CaCU. Afterextensive washing in cacodylate buffer, samples were postfixed in \CAosmiumtetroxide for l h at 20°C.Ultrathin sections were cut with a diamond knife in

a Reichert Ultracut microtome, stained with uranyl acetate and lead citrate, andexamined with a Jeol 1200 EX microscope. For light microscopy, semilhinsections of specimens and smears of cultured cells were dehydrated in cthanol.embedded in Epon, and stained with hematoxylin-eosin.

Antibodies. The rabbit anti-peripherin polyclonal antibody was routinely

used at a 1/KX)dilution for immunohistochemistry and at a 1/5000 dilution forWestern blotting. The specificity of this antibody was already documented(13).

The anli-NF-L neurofilament protein mAb (Sigma clone NR4) was used at

a dilution of 1/30 for indirect immunofluorescence and at 1/200 for Westernblotting. The anti-vimentin mAb (Sigma clone 13.2) was used for inumino

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M-.t kOI.MKX'RIM-, CARCINOMAS OF THK SKIN KXPRI-.SS PtiRIPHt-.RIN

histochemistry at a 1/100 working dilution. The Troma 1 mAb, recognizingbasic keratin 8. was a kind gift from Dr. Kemler and was used at a I/30 dilution(15).

Fluorescein isothiocyanate-, tetramethyl isothiocyanate-, or rhodamineisothiocyanate-labeled anti-rabbit (1/30 final dilution), anti-rat (1/30 final dilution) or anti-mouse ( 1/50 final dilution) (DAKO) were the second antibodiesfor indirect immunofluorescence. Peroxidase-conjugated anti-mouse or anti-

rabbit IgGs (1/100 final dilution: Diagnostic Pasteur) were the second antibodies for Western blotting.

Indirect Immunofluorescence. Drops of NECS cell suspensions were partially dried on glass slides and fixed in acetone for 10 min at 4°C.Five-um-

thick sections from fro/.en tissues were air dried and fixed in acetone asdescribed previously ( 16). Samples washed 15 min at 4°Cin PBS were incubated I for h at 37°Cwith the primary antibody diluted in PBS (see "Antibodies"). After extensive washing in PBS, sections and smears were incubated

with the appropriate second antibodies. Sections stained with a fluoresceinisothiocyanate conjugate were counterstained with propidium iodide andmounted in ÃŒOVcglycerol-paraphenylene diamine. Slides were examined with

a Zeiss Universal Microscope with epiillumination using an HBO 50 mercuryarc lamp. Ill RS epifluorescence condenser, and fluorescein isothiocyanate ortetramethyl ¡sothiocyanate filters. Photographs were taken on Kodak Ektachrome 400 film using a Zeiss MC 63 camera system.

Polypeptide Analysis by Bidimensional Gel Electrophoresis and Western Blotting. NECS tumoral tissues were homogenized and proteins of thecrude extracts were separated by two-dimensional gel electrophoresis according 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 ontonitrocellulose membranes (Hybond C; Amersham International UK) at 700 mAin a Transphor apparatus (Hoefer Scientific Instruments) as described (19).Peptides were first visualized on the nitrocellulose membrane by 5 min stainingwith Ponceau red (0.2% Ponceau red in 3% trichloroacetic acid), located witha marker, and then destained with distilled water.

Northern Blot Analysis. Total mRNAs were prepared from tumoral tissuesaccording to the method of Chomczynski and Sacchi (20) and poly(A) +

mRNAs were purified following standard techniques.

Seven ug of poly(A) ' mRNA from NEC xenografts and 15 ug of poly(A) '

mRNA from primary tumors were fractionated by electrophoresis on 1.2%denaturing agarose gel containing 1.2 M formaldehyde and transferred ontonylon membrane (Hybond N; Amersham). Membranes were subsequentlyhybridized in 50% formamide at 42°Cwith either peripherin or ß-actin12P-

labeled random-primed cDNAs and exposed to hyperfilm MP (Amersham)with intensifying screens. rRNA and 0.24-9.5-kilobase RNA ladder (GIBCO-

BRL, France) were used as molecular weight standards.

RESULTS

Upon light and electron microscopy examination, the 12 primarytumors presented the hallmarks of neuroendocrine skin carcinomas(1). All tumors were positively stained with the anti-cytokeratin 8

antibody and with an antibody raised against the neurofilament proteinNF-L. The tumors reacted with the anti-vimentin antibody, but fluorescence was specific to the cells of the stroma. The anti-peripherin

polyclonal antibody stained 10 of the 12 NECS (Fig. 1; Table 1). Theantibodies reacted against cytoplasmic proteins: the anti-cytokeratin

antibody clearly bound to thin cytoplasmic proteins, whereas theanti-peripherin and the anti-NF-L antibody did not define any orga

nized network. All the three antibodies stained paranuclear balls inlarge tumor cells.

Table I Detection of intermediate filaments in human ni'itnientlwrine carcintinuu

of the skin

Primary tumors

AntibodyAnti-cytokeratin

8(monoclonal)Anti-NF-L(monoclonal)Anti-peripherin(polyclonal)Anti-vimentin

(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 withantibodies 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.

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NEUROF.NDOCRINF. CARCINOMAS OF THF. SKIN EXPRESS PKRIPHFRIN

b7I

*^-.-•'s»1

:^: U-«,! '«•: :*••£

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 granulesand paranuclear globular fibrous bodies composed of intermediate filaments. Tumor cells grown in vitra, present the paranuclear globular fibrous bodies and dense-cored neurosccretorygranules characteristic of NECS it: X 5,000; <AX 25,(KH)>.

In order to follow a possible phenotypic evolution of tumoral tissues during proliferation, tumor expiants were xenografted on nudemiee. Implants were passaged in vivo every 4 months, on average, forat least 3 years. Tumoral growth was slightly enhanced in animalsgrafted with expiants from xenografts obtained after a high number of

passages. Animals were sacrificed when the xenograft reached a sizeof 2 cm and the excised tumor tissues underwent macroscopic examination. Xenografts were always localized in the dermis, with noevidence of invasive proliferation. Neoplastic cells were arranged insolid sheets and cellular cords formation were faintly visible at (he

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NEUROENDOCRINE CARCINOMAS OF THE SKIN EXPRESS PERIPHERIN

Table 2 Detection of peripherin expression in xenografted and primary NECS

M, M2" M," NEC-1

Indirect immunofluorescenceTwo-dimensional SDS/PAGE'

Western blottingNorthern blotting

ND

NDND

" NECS grafted on athymic nude mice." Primary NECS.' SDS/PAGE. sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

tumor periphery. At the microscopic level, the tumoral cells displayeda homogeneous shape and high rates of mitoses. As assessed byelectron microscopy, the majority of the xenograft tumor cells retainedthe ultrastructural features typical of NECS, including membrane-bound dense-cored neuroendocrine-like granules, paranuclear whorl-

like intermediate filament aggregates and occasional rudimentaryjunctions (Fig. 2). The general immunohistological picture drawnfrom studies performed on three tumor xenografts was similar to thatpreviously obtained from the studies performed on the primary tumors. With the exception of the tumor xenograft M, which wasnegative for the expression of peripherin, the other two tumors wereindistinguishable from the corresponding original tumors. Results aresummarized in Tables I and 2.

To verify whether the phenotypic and biochemical properties ofNECS cells were stably maintained in an artificial environment, primary cultures were established in vitro from tumor expiants. Whencultured on plastic, all the tumor cells maintained a round shape andgrew as loosely packed floating aggregates, amorphous and irregularin outline (Fig. 3). Indirect immunofluorescence performed on smearsrevealed that each cell expressed a pattern of cytoskeletal proteinssimilar to that observed in the corresponding tumors. Cells could be

stained by the anti-peripherin, the anti-NF-L, and the anti-cytokeratinantibodies but not by the anti-vimentin antibody. Interestingly, andwith the exception of the M, tumor-derived cultures, peripherin ex

pression was stronger in cultured cells than in the original tumor cells.Double immunofluorescence labeling for peripherin and NF-L re

vealed a partial overlap of the cytoplasmic staining. However, the twotypes of intermediate filaments were constantly detected in the para-

nuclear balls, clearly conspicuous in large fractions of the cellularpopulation (Fig. 3, c and d).

We then performed the biochemical characterization of NECS intermediate filament polypeptides. A primary NECS tumor (NECS 1)and two NECS xenografts (MrM2) were analyzed by two-dimen

sional gel electrophoresis and subsequent immunostaining. Peripherin, NF-L, and cytokeratin 8 were clearly found coexpressed inNECS 1 and in the xenograft M2 (Fig. 4, A, A- A" and C, C'- c"). The

amount of these two proteins was not stoichiometric, since in thexenograft M, NF-L was visualized as a small spot, whereas peripherin

remained undetectable. Interestingly, the amount of vimentin found inthe stroma was proportional to the amounts of peripherin and NF-L

detected in tumoral tissues (Fig. 4). Although the imunofluorescencestudies show that vimentin is expressed only in stroma, this proteinwas detected in all gels, this because of the unavoidable presence ofstroma cells in the tumor samples. An important observation is thatboth peripherin and vimentin migrated at a pH¡lower than that expected (9). The cause for this altered migration is under investigation.

Lack of immunological staining cannot be regarded as an absolutecriterion for excluding the possible synthesis of low amounts of peripherin. We therefore examined on xenografts whether absence ofimmunological staining of peripherin could be correlated with theabsence of specific RNA transcripts. Poly(A) ' mRNAs were prepared

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 inDulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 5% inactivated horse serum, Staining with méthylèneblue («);immunostaining with anticytokeratin8 antibody (h) double immunostaining with antiperipherin antibody (c)', and with anti NF-L antibody (d). X 200.

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NF-L»- .

Pe- --Vi

(3

A

r

A' A"

NF-L.

-.Vi

B B' B' B'"

at:

ßVi

- ViDC' C"

Fig. 4. Identification by two-dimensional gel electrophoresis and Western blotting of cytoskeletal components of NECS cells. Portions of Coomassie blue-siained two-dimensionalgels 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) andafter 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.ß.corresponding 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 peripherincDNA clone isolated from a murine neuroblastoma Agt 11 cDNAlibrary by immunoscreening (10). As shown in Fig. 5, hybridization ofNorthern blots with the |12P]cDNA revealed the presence of a specific

2-kilobase transcript both in the xenograft M, and. as expected, in theperipherin-positive tumors M2 and NECS I. Surprisingly the amount

of peripherin transcripts in M, was estimated to be at least 3 timeshigher than that detected in M2. The different rates of transcription inthe xenografts were confirmed by rehybridization of the blot with aß-actinprobe. In contrast to the xenograft M2, the M, tumor exhibitedhigher levels of transcription for peripherin than for ß-actin.The

intensity of the hybridization bands was comparable in the NECS 1preparation.

DISCUSSION

In this report we demonstrate that NECS cells express peripherin.Synthesis of the protein was first monitored in human tumors byindirect immunofluorescence using a specific antibody that does notcross-react with other cytoskeletal components (9). A strong labelingof tumor cells indicated the presence of peripherin (Fig.Io) coex-pressed with cytokeratins and the small neurofilament NF-L protein

(Fig. 3.C and d). This observation reveals the complexity of thecytoskeletal structures of NECS, which display patterns common bothto simple and stratified epithelia and to neural cells. Cytokeratin andNF-L neurofilament protein appear as thin cytoplasmic fibrils and

perinuclear plaques. Accordingly to previous observations (6, 16, 21 ),perinuclear plaques were brightly stained by anti-cytokeratin and anti-

neurofilament antibodies, whereas fibrils reacted predominantly withanti-cytokeratin antisera. We could not determine by double labeling

techniques whether cytokeratin and neurofilament proteins are foundwithin the same intermediate filaments or whether cytokeratin filaments and neurofilaments are distinct but closely intermingled. It isalso not clear whether peripherin is a constituent of NECS heteropoly-

mer filaments, or whether it provides a distinct filament structure. Theanti-peripherin antibody strongly stained the perinuclear plaques, thussuggesting the presence of peripherin in the aggregates. The coex-

pression of the three different types of intermediate filament proteinswas confirmed by double immunofluorescence experiments.

The nature of the paranuclear aggregates, alternatively termed fibrous bodies or perinuclear aggregates, is unknown. Ultrastructurally,they correspond to whorl-like intermediate filaments and could arise

from a depolymerization of the intermediate filament network possibly caused by overphosphorylation of cytoskeletal components. In-

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1

BFig. 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 hybridized with a peripherin cDNA probe (A ). Lune t was loaded with 7 ug of poly (A) + mRNAextracted from M¡;hiñes2 and .îwere loaded with 15 ug of poly (A)~ mRNAs from the

xenograft Mi and the primary tumor NEC-], respectively. Upon dehybridization. thenitrocellulose sheet was rehybridized with a ß-actinprobe (ß).The two probes recognizea 2-kilobase mRNA.

deed, it is now admitted that phosphorylation of vimentin and that ofthe amino-terminal domain of NF-L induce a depolymerization ofintermediate filaments in vitro (22-24). Moreover, altered phosphor

ylation patterns of vimentin and peripherin would be compatible withthe more acidic pH¡sof these proteins extracted from NECS cells seenby isoelectric focusing (Fig. 4).

The results obtained on NECS cells were confirmed by the analysisof xenografted tumors. Even after a high number of serial heterotrans-

plantations, xenografts were morphologically indistinguishable fromthe original tumors, as evidenced by the presence of neurosecretorygranules, neuron-specific enolase, and neuropeptides (data not

shown). The histológica! and ultrastructural properties of the originalNECS cells were also preserved in NECS cells derived from primarycultures of xenografts upon expansion in vitro. Coexpression of peripherin. cytokeratins, and neurofilament polypeptides was maintained at elevated rates, while synthesis of vimentin remained unde-

tectable. In culture, NECS cells adhered poorly to plastic substratesand showed average mean doubling times exceeding 5 days. Attemptsto improve adhesion by coating dishes and seeding cells on fibroblastfeeder layers were unsuccessful, as well as adjunction of mitogenicfactors to media (epidermal growth factor, neuronal growth factor,basic fibroblast growth factor, from horse serum) to stimulate growth.Similar observations had been previously made on the neuroendocrineskin carcinoma cell line MKL-1 (25) and on primary cultures of a

lymph node metastasis (26).Injection of NECS cells induced tumors in mice after latency pe

riods of 3-5 months, without occurrence of detectable metastasis. The

histológica! and ultrastructural characteristics of NECS found again inall the analyzed tumors gave evidence of the stability of the phenotype(not shown).

The biological role of peripherin is still unclear. The protein mayplay a role in the recognition of the axonal pathway (27, 28) and itsexpression provides an additional stigma of the neural differentiationof NECS. Because of striking morphological similarities, it has beenproposed that NECS could arise from Merkel cells ( 1). Definite his-

togenetic relationships between these two cellular types and studies onthe expression of peripherin by normal human Merkel cells are lacking; nevertheless, the possible Merkelian origin of NECS is supportedby immunofluorescence and immunoelectron microscope analyses ofpig snout, suggesting that intraepidermal Merkel cells express peripherin.4 Endocrine cells express neural markers that are detected at key

4 J. P. Ortonnc. unpublished observations.

stages of ontogenesis (27) and expression of neural-specific genes

during the development has been reported for chromaffin cells andß-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 inMerkel cells of adult pigs, it is conceivable that neuroendocrine tumors and their corresponding primary cultures could maintain synthesis of neural polypeptides already expressed by normal cells fromwhich they originate. On the other hand, cancerization may induceactivation of suppressed functions. In adult chromaffin cells andß-cellsof Langerhans islets and in the corresponding tumors, neural-

specific genes are inactive. However, reactivation of peripherin andNF-L filament protein synthesis in the tumor-derived pheochromocy-

toma PC 12 cell line and in the ¡nsulinomaRIN5F cells was demonstrated (14). Moreover, in primary cultures of normal endocrine cells,enhanced expression of neural intermediate filaments appears to becorrelated with the absence of intercellular interactions preventingextending processes (31, 32).

Peripherin constitutes an additional marker for the identification ofthe histogenesis of NECS. Of 12 tumors, 10 were positive for im-munostaining with a peripherin-specific antibody. Moreover, when

further analyzed by Northern blot, an apparently negative tumor displayed a low level of expression of peripherin mRNA. In this context,the relevance of peripherin as a marker for differential diagnosis, e.g.,to discriminate between primary NECS and secondary location ofneuroendocrine tumors of extracutaneous origin is still to be evaluated. It is well established that rat pheochromocytoma and mouseneuroblastoma cells express peripherin. however, no data are yetavailable from other human neuroendocrine tumors of epithelial type,such as pulmonary neuroendocrine tumors, the medullary carcinomaof the thyroid, islet cell tumors of pancreas, and the carcinoid of smallintestine. Further investigations on the distribution of peripherin in awide variety of neuroendocrine cells and related tumors are thereforerequired.

ACKNOWLEDGMENTS

We thank O. Partouche for her skillful technical assistance. C. Minghelli forthe photographic work. E. Van Ohherghen for critical reading of the manuscript, and D. Sripati for helpful discussion.

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1993;53:1175-1181. Cancer Res   C. Baudoin, G. Meneguzzi, M. M. Portier, et al.   Nude Mice, and the Corresponding Primary Culturesby Neuroendocrine Carcinomas of the Skin, Their Xenograft on Peripherin, a Neuronal Intermediate Protein, Is Stably Expressed

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