[CANCER RESEARCH 51. 6328-6337. December I. 199I|

E-Cadherin Expression in Squamous Cell Carcinomas of Head and Neck: Inverse

Correlation with Tumor Dedifferentiation and Lymph Node MetastasisJörgH. Schipper, Uwe H. Frixen, JürgenBehrens, Andreas Unger, Klaus Jahnke, and Walter Birchmeier1

Institut fürZellbiologie (Tumorforschung) [J. H. S., U. H. F., 1. B., W. B.], and Department of Otorhinolaryngology [J. H. S., A. U., K. J.J, Medical School,University of Essen, 4300 Essen I, Germany

ABSTRACT

Tissue sections of 32 squamous cell carcinomas (SCCs) of the headand neck were investigated for the expression of the epithelium-specificcell adhesion molecule E-cadherin. We found that E-cadherin expressionis inversely correlated both with the loss of differentiation of the tumorand with lymph node metastasis. The well-differentiated SCCs expressedE-cadherin, often as strongly as the normal stratified epithelium (12cases were tested); the moderately differentiated SCCs expressed intermediate amounts of E-cadherin or were heterogeneous (15 cases wereanalyzed); whereas the poorly differentiated SCCs were all E-cadherin-negative (five cases were investigated). Furthermore, seven of eightinfiltrated lymph nodes of SCCs were E-cadherin-negative. These dataindicate that the loss of the cell adhesion molecule E-cadherin in factplays an important role in the progression of human squamous cellcarcinomas, i.e., that down-regulation of expression is associated withdedifferentiation and metastasis of the tumor cells in vivo.

INTRODUCTION

Head and neck carcinomas of the upper aerodigestive tractare the sixth most frequent human tumors diagnosed worldwide, and their tendency is increasing (1-3). Roughly, threelevels of tumor locations are recognized, the nasopharynx, thepharynx (including floor of mouth, oropharynx, and hypophar-ynx), and the larynx. The carcinomas originate in the mucosawhich lines the inner surfaces of the cavities of the aerodigestivetract, and 80-90% are SCCs,2 followed by adenocarcinomas

and others (3, 4). According to the WHO, squamous cellcarcinomas of head and neck can be subdivided by grade ofepithelial differentiation into well, moderately, and poorly differentiated types (2, 3, 5). It has been suggested that thesegrades of differentiation are associated with patient survival,whereby lower differentiation means shorter survival (2, 6).Little is known about risk factors and the multistep process oftumorigenesis in SCCs of the head and neck; nicotine andalcohol abuse play a role (1,3, 7). Furthermore, human papil-loma-virus and Epstein-Barr virus involvement has been reported for nasopharynx and larynx carcinomas, respectively (8,9). Recently, amplification, overexpression, and mutation of anumber of oncogenes have been described in head and neckcancer (10-17). An important factor for the success of clinicaltherapy of SCCs of the head and neck is the state of theinvolvement of lymph node metastasis (2, 18). In this process,carcinoma cells detach from the primary tumor mass, migratethrough the surrounding connective tissue, invade neighboringlymphoid vessels, and colonize the nearest lymph nodes. Inundisturbed squamous epithelia this does not occur, since theindividual cells are interconnected by a complex network of

Received 7/3/91; accepted 9/23/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Institut fürZellbiologie, University of Essen, Virchowstrasse 173, D-4300 Essen 1, Germany.

2The abbreviations used are: SCC, squamous cell carcinoma; cDNA, complementary DNA; SSC, standard saline citrate (0.15 m NaCl, 0.015 m Na-citrate,pH 7.0).

cell-cell adhesions. Only the disruption of such mutual interactions may allow the carcinoma cells to migrate away fromthe primary tumor (19, 20).

Our laboratory has previously studied epithelial differentiation and invasion in vitro with respect to the expression andfunction of the epithelium-specific cell-cell adhesion moleculeE-cadherin. We have demonstrated that nontransformed Ma-din-Darby canine kidney epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited bythe addition of antibodies against E-cadherin; the separatedcells then assume a fibroblast-like (i.e.. de-differentiated) mor

phology and invade collagen gels and embryonal heart tissue.Furthermore, epithelial cells transformed with Harvey and Mo-loney sarcoma viruses were found to be constitutively fibroblast-like invasive, and they do not express E-cadherin (21). We alsoconfirmed this correlation by examining various human celllines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioidphenotype were noninvasive and expressed E-cadherin, whereascarcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of dedif-ferentiated breast carcinoma cells could be prevented by transfaction with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin monoclonalantibodies (22). These findings indicate that the selective lossof E-cadherin expression can generate dedifferentiation andinvasiveness of human carcinoma cells in vitro.

In this study we have investigated the expression of the cell-cell adhesion molecule E-cadherin in SCCs of the head andneck and have assessed the results with histopathological differentiation and lymph node metastasis.

MATERIALS AND METHODS

After clinical staging and biopsy, squamous cell carcinoma tissue ofthe head and neck was surgically removed from previously untreatedpatients. Lymph nodes were removed by neck dissection. The materialwas immediately frozen in liquid nitrogen. A separate portion was usedfor routine histopathological grading by the Department of Pathology,University of Essen Medical School. Frozen tissue sections were prepared in a Frigocut 2800-E microtome (Reichert-Jung, Nussloch, Germany). The tumor tissue was initially localized by staining with tolui-dine blue, and then serial sections of 6 M»>were cut and stained forimmunofluorescence (after ethanol fixation at —¿�20°C)by in situ hybrid

ization (after formaldehyde fixation) and with hematoxylin or hema-toxylin/eosin. Infiltrated lymph nodes were sectioned and stained in asimilar manner, except that lO-jim sections were used.

Immunofluorescence staining of primary tumors, infiltrated lymphnodes, and cell lines of SCCs was performed with the anti-E-cadherinmonoclonal antibody 6F9 as described previously (21, 22) or with arabbit polyclonal anti-pancytokeratin antibody (K.40, a generous gift ofDr. F. Ramaekers, Maastricht, The Netherlands). As second antibodies,fluorescein isothiocyanate-conjugated goat anti-mouse and rhodamineisothiocyanate-conjugated goat anti-rabbit IgG (Dianova) were used. In

situ hybridizations were performed as previously described (23, 24).Briefly, antisense and sense riboprobes of a 386-base pair human E-

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E-CADHERIN EXPRESSION IN HEAD AND NECK CARCINOMAS

cadherin cDNA (HC6-1; Ref. 22) in the Bluescript vector were producedby T3 and T7 RNA polymerase (Stratagene), respectively, using "S-

labeled UTP and CTP (Amersham). Hybridization of sections wasperformed at 47°Cin 50% formamide-2x SSC-5 HIMEDTA-10 HIM

dithiothreitol-10 mM 2-mercaptoethanol-10 miviTris-HCl, pH 7.5, -10

mM NaH2PO4, pH 6.8, containing 150 ng/m\ salmon sperm DNA and10 MMS-ATP. Sections were washed with 50% formamide-2x SSC for2 h and then with 50% formamide-O.lx SSC for 12 h. Slides weredipped into Kodak NTP-2 emulsion diluted 1:1 and exposed for 7-14days at 4°C.For Western blot analysis of tissue extracts, 25 frozensections of 20 ^m were extracted for 30 min at 4°Cwith 3% Triton X-

100 in L-CAM assay buffer-100 mM phenylmethylsulfonyl fluoride,

and the extracts were processed as described previously (21, 22).

RESULTS

E-Cadherin Expression in SCC Cell Lines and Tissues of Headand Neck. Well-, moderately, and poorly differentiated SCCcell lines were examined for E-cadherin expression by bothimmunofluorescence and Western blotting. A well-differentiated cell line expressed E-cadherin, whereas the moderatelyand poorly differentiated lines were negative (Figs. 1 and 2).

In the main part of this work, E-cadherin expression wasexamined on tissue sections of 32 SCCs of the head and neck.Generally, well-differentiated SCCs expressed E-cadherin,often as intensely as the normal stratified squamous epithelium(Fig. 3, a and A; Fig. 4 b,f,f, Table 1). A moderately differen-

Fig. 1. E-cadherin expression in SCC celllines of the head and neck by immunofluorescence. a and b, well-differentiated carcinomacell line FaDu from the hypopharynx; c and d,moderately differentiated carcinoma cell lineBA from the larynx; e and / poorly differentiated carcinoma cell line WE from the larynx.a, c, and e, immunofluorescence staining; b, d,and /, corresponding phase contrast image.The well-differentiated carcinoma cell line expresses E-cadherin, whereas the moderatelyand poorly differentiated lines are negative.Bar, 30 urn.

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E-CADHERIN EXPRESSION IN HEAD AND NECK CARCINOMAS

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abedFig. 2. Western blot analysis of cell extracts of SCO cell lines from the head

and neck. Lane a, control extract from differentiated human A-431 vulva carcinoma cells; Lane b, extract from the well-differentiated SCC on cell line FaDu;Lane c, from the moderately differentiated line BA; Lane d, from the poorlydifferentiated line WE. The well-differentiated SCC cell line shows expression ofE-cadherin; the moderately and poorly differentiated lines are negative.

tiated SCC showed homogeneous down-regulation of E-cadherin expression, whereas a poorly differentiated SCC wascompletely negative (Fig. 3, c and d). In another set of tumors,again complete down-regulation of E-cadherin expression wasobserved in a poorly differentiated SCC, whereas overall keratinstaining remained (Fig. 4, d and h). The moderately differentiated tumor of Fig. 4, c, g, and k, was heterogeneous; thecentral cell mass of the tumor island was differentiated andexpressed E-cadherin, whereas the border regions (Fig. 4c,arrows) were dedifferentiated and did not express E-cadherin.All regions of the tumor exhibit keratin staining, and the ratioof cytoplasm to nuclei increased from the center to the border.From 15 SCCs of the head and neck, tissue extracts were alsoanalyzed by Western blotting. These experiments confirmedthe immunofluorescence data; well-differentiated tumors expressed the M, 120,000 band of E-cadherin, poorly differentiated tumors were negative, and the moderately differentiatedtumors were intermediate (Fig. 5).

E-Cadherin Is Generally Absent in Infiltrated Lymph Nodesof SCCs of Head and Neck. Of the 32 SCCs, 17 cases showedinfiltrated lymph nodes in the neck region (Table 1). Lymphnodes from eight cases were analyzed for expression of E-cadherin and cytokeratins (Fig. 6). We found a number of lymphnodes which were infiltrated by a small group of carcinomacells, while others were virtually overgrown by the tumor.However, in all but one of these cases the infiltrated carcinomacells were found to be E-cadherin-negative (Fig. 6, a-f).

A summary of the clinical, histopathological, and E-cadherinexpression data of the 32 SCCs with the data on the involvedlymph nodes are shown in Table 1. The well-differentiatedcarcinomas generally expressed E-cadherin (12 cases weretested, two cases showed a heterogeneous pattern), and themoderately differentiated tumors exhibited intermediate E-cadherin staining or were heterogeneous (15 cases were tested),whereas the poorly differentiated carcinomas were all E-cadherin-negative (five cases were tested). In two cases we did notagree with the pathologists' grading (tumors 11 and 27). Inthese, E-cadherin staining confirmed our own classification,e.g., tumor 27 was clearly poorly differentiated and E-cadherin-negative. Lymph node métastaseswere mostly found in caseswhere the primary tumor was E-cadherin-negative or heterogeneous. Remarkably, in seven of eight cases the infiltratedlymph nodes were E-cadherin-negative. These data suggest thatlymph node metastasis in SCCs of the head and neck is largelyincompatible with E-cadherin expression.

E-Cadherin Expression in SCCs of Head and Neck Analyzedby in Situ Hybridization. E-cadherin niRNA was identified ontissue sections of SCCs by in situ hybridization using a humanE-cadherin antisense riboprobe (produced from the HC6-1cDNA; see "Materials and Methods"). In the well-differentiatedSCCs, E-cadherin expression was confined to islands of tumorcells in the connective tissue (Fig. 7, a and b). However, no E-cadherin mRNA was detected in all of the poorly differentiatedtumors analyzed (Fig. 7, c and d). For comparison, high E-cadherin mRNA signals are shown in normal squamous epithelium of head and neck as well as in a neighboring well-differentiated SCC (Fig. 8a; Fig. 80 shows the control using the senseprobe). E-cadherin mRNA and corresponding protein expression could be seen in enlargements of sections (Fig. 8, c-h).

DISCUSSION

In the present study we have examined the expression of thecell-cell adhesion molecule E-cadherin on tissue sections ofhuman SCCs of the upper respiratory and digestive tract. Invitro experiments had shown previously that the absence or lossof function of E-cadherin leads to the disappearance of epithelial characteristics of the cells and generates higher invasivenessfor extracellular matrices and embryonal heart tissue (21, 22).We show here that all poorly differentiated SCCs of the headand neck did not express E-cadherin, whereas the moderatelydifferentiated tumors expressed E-cadherin in an intermediateor heterogeneous fashion. Well-differentiated SCCs expressedE-cadherin often as strongly as the normal stratified squamousepithelium. Remarkably, lymph node métastaseswere generallyE-cadherin-negative, irrespective of whether they originatedfrom poorly or moderately differentiated SCCs. These datasuggest that E-cadherin plays a major role as a differentiationfactor and invasion suppressor in epithelial tissues in vivo.

Several technical prerequisites made the present study feasible. Throughout we have obtained fresh human tumor materialin close consultation with the surgeon (K. J.), which was clinically and histopathologically well characterized and was fromuntreated patients who had not undergone radio- or chemotherapy or previous surgical treatment. We then did carefulpresectioning of the material to first obtain a broad outline ofthe tumor structure, before we finally went into the detailedserial section analysis. We are also familiar with the propertiesof the anti-E-cadherin antibodies and the E-cadherin cDNAprobe, which we have prepared and tested ourselves (22). Fur-

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Fig. 3. E-cadherin expression in SCC tissues of the head and neck, a, normal stratified squamous epithelium from the hypopharynx; b, well-differentiated carcinomafrom the larynx: c, moderately differentiated carcinoma from the hypopharynx; a. poorly differentiated carcinoma of the larynx. The well-differentiated carcinomatissue shows a high expression of E-cadherin comparable with that of the normal epithelium, whereas the moderately differentiated carcinoma showed a weak signal.No E-cadherin expression is detectable in the poorly differentiated carcinoma. Tissues are from patients also listed in Table 1: a, case 1; />,case 2; c, case 22; d, casell.Bar, 40 jim.

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E-CADHERIN EXPRESSION IN HEAD AND NECK CARCINOMAS

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Fig. 4. Double immunofluorescence of E-cadherin and keratins in SCC tissues of the head and neck. Serial sections of SCCs were stained for E-cadherin (a-d) forcytokeratins (e-h), and with hematoxylin (il), a, e, and /. normal stratified epithelium of the pharynx; b,f, ;uid /. well-differentiated SCC of the larynx; c, g, and k,moderately differentiated SCC of the hypopharynx; ¡I.h, and /, poorly differentiated SCC of the hypopharynx. Note that the carcinoma tissues showed a gradual lossof E-cadherin with the loss of differentiation, but that the keratin staining was largely unchanged. The tumor in c, g, and k is heterogeneous; the central portionexpressed E-cadherin and showed large cytoplasm, whereas the border area (arrows) was E-cadherin-negative and showed marginal cytoplasm. Tissues are frompatients also listed in Table 1: a, case 12; b, case 5; c, case 2; d, case 30. Bar, 50 urn.

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Table 1 Properties of the squamous cell carcinomas of head and neck

E-cadherinexpression in

Case Age/sex/site0 TNM*

InfiUratedDifferentiation Primary lymph

grade' tumorrf node'

1234567891011121314IS161718192021222324252627282930313266/f/H69/m/L45/m/H43/f/H64/m/L57/m/L67/m/L52/m/H57/m/H67/f/O64/m/O77/m/F39/f/O51/f/O50/m/L60/f/O57/m/L61/m/O55/f/O51/f/L62/m/H62/m/H66/f/F72/m/O47/m/F57/m/O54/f/L62/f/O65/m/H47/m/H49/f/L50/m/HT2NOMOT3NOMOT3NOMOT2N1

MOT3N1MOT3

NOMOT3NOMOT4NOMOT4NOMOT4NOMOTIN3MOT2N1

MOT4NOMOT2N1

MOTisT2

NOMOT2NOMOT3NOMOT4NOMOT2N1

MOT2N1MOT2N2bMOT3N2cMOT2N2bMOT4N2bMOT2

N3MOT4N2cMOT2

NOMOT3N1MOT2N2bMOT4N1MOT3

N3 MOWell

++Well++Well++Well++Well++Well+Well+Well+Well+Well+Well+/-Wei/+/-Moderately++Moderately++/-Moderately+Moderately+Moderately+Moderately+/—Moderately+Moderately+Moderately+/—Moderately+Moderately

++Moderately+/——¿�Moderately

+/—Moderately+/——¿�Moderately*

—¿�—¿�Poorly—¿�Poorly—¿�Poorly—¿�Poorly-Poorly

—¿� —¿�

to indirect suppression of E-cadherin gene expression. Concerning the first possibility, a functionally inactive E-cadherinwas recently created by the introduction of a point mutationinto the cDNA coding for the extracellular domain (28). Furthermore, a new tumor suppressor gene has recently beenidentified by allelic loss on chromosome 16q, bands 22.1-23.2,which is affected in most of the dedifferentiated liver carcinomas (29) as well as in breast and prostate carcinomas (30, 31).E-cadherin is a good candidate for this possible invasion suppressor gene, since it is located on chromosome 16q, band 22.1(32). Second, epithelial-specific regulatory elements have recently been identified in the E-cadherin gene promoter, whichare active in certain differentiated but not dedifferentiatedbreast carcinoma cell lines (33). It is not yet known whether16q mutations in fact exist in head and neck tumors or whetherthe postulated epithelial specific promoter elements play a role.Recently, various other components have been described whichmight be involved in invasion and metastasis (deleted in coloncarcinoma, carcinoembryonic antigen, CD44, and nm 23; cf.Refs. 34-37). Deleted in colon carcinoma, carcinoembryonicantigen, and CD44 also seem to influence the adhesive properties of the tumor cells.

E-cadherin expression was also studied recently in other typesof carcinomas. A highly undifferentiated hepatocellular carci-

°Site of tumor: F, floor of mouth; O, oropharynx; H, hypopharynx; L, larynx.* TNM staging (25) as performed by the Department of Otorhinolaryngology,

Medical School, University of Essen. T, tumor size; N, lymph node infiltration;NO, no infiltration; NI, one infiltrated lymph node <3 cm; N2b, several infiltratedlymph nodes <6 cm on one side; N2c, same, but on both sides; N3, all infiltratedlymph nodes >6 cm; MO, no distant metastasis; Tis, carcinoma in situ.

' Tumor grading as determined by the Department of Pathology, Medical

School. University of Essen.d E-cadherin expression as determined by immunofluorescenee. ++, homoge

neously high expression as in Fig. ib, comparable with the staining of normalstratified squamous epithelium; +, homogeneously weak expression as in Fig. .V;—¿�,no detectable expression; ++/—, +/—,heterogeneous staining.

' Only infiltrated lymph nodes were analyzed.1 According to our hematoxylin/eosin staining, we would classify this tumor

as moderately differentiated.* The area of this tumor we have analyzed was clearly poorly differentiated.

thermore, the results were verified by different methods onidentical material from a selected number of patients, i.e.,immunofluorescenee, in situ hybridization, and Westernblotting.

In all cases of poorly differentiated SCCs of the head andneck we found complete down-regulation of E-cadherin proteinand mRNA expression as measured by the different techniques.Examination of the infiltrated lymph nodes of SCCs showedthat they were generally E-cadherin-negative. The results suggest that preferentially E-cadherin-negative carcinoma cells migrate from the primary tumors and colonize the neighboringlymph nodes. Alternatively, local lymphatic vessels might havebetter access to E-cadherin-negative tumor tissue, and by thisE-cadherin-deficient cells might be preferentially mobilized.The one exception (the lymph nodes of tumor 23 in Table 1)could be due to other factors (e.g., motility factors) which canmobilize carcinoma cells without affecting E-cadherin expression (26, 27). The underlying molecular mechanism for E-cadherin down-regulation in the SCCs is not known. It couldbe due either to mutations in the E-cadherin structural gene or

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aFig. 5. Western blot analysis of tissue extracts of selected SCCs of the head

and neck. Lane a, cell extract of the differentiated human A-431 vulva carcinomacell line (control); lanes b-d, tissue extract of a well differentiated (/>).a moderatelydifferentiated (c), and a poorly differentiated (d) SCC. The well-differentiatedcarcinoma shows high, the moderately differentiated weak, and the poorly differentiated no expression of E-cadherin. Arrows, M, 120,000 band of E-cadherin.The extracts are from SCCs also listed in Table 1: *, case 2, c, case 18; d, case31.

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Fig. 6. E-cadherin expression by immunofluorescence in infiltrated lymph nodes of SCCs of the head and neck, a, c, and e. E-cadherin expression; b. d. and /corresponding staining of cytokcralins. The lymph node in a and b is infiltrated by small groups of E-cadherin-negative carcinoma cells (the cytokeratin-positive cellsare marked by arrows in b). and the lymph node in c and </ is virtually overgrown by E-cadherin-negative carcinoma cells. <•and /, the only case with E-cadherin-positive lymph nodes. Lymph nodes are from cases 12 (a and b), 27 (c and d), and 23 (e and f) (see Table 1). Bar, 40 jim.

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Fig. 7. E-cadherin expression as analyzed by in situ hybridization of SCC tissues of the head and neck, a and ft,well-differentiated SCC; c and </,poorly differentiatedSCC. Tissues were from patients listed in Table I: a, case S; c, case 31. Sections were hybridized with E-cadherin antisense riboprobes as described in "Materials andMethods." After autoradiography, sections were counterstained with hematoxylin to reveal the location of cell nuclei and epithelial structures. The sections were thenanalyzed by darkfield (a and c) and brightfield (b and d) illumination. E-cadherin niKN'A is strongly expressed in the tumor islands of the well-differentiated and not

expressed in the poorly differentiated carcinoma. , borderline between carcinoma (cui and connective tissue (cl). Bar, 40 /mi.

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ct

Fig. 8. /n s/V«hybridization of E-cadherin in normal epithelium and carcinoma tissue of the head and neck. Serial sections of both normal epithelium (ep) and aneighboring well-differentiated SCC (ca) separated by connective tissue (ct) were hybridized with E-cadherin antisense (a) and sense (A) riboprobes. c-h, enlargementsof characteristic regions of the normal epithelium (* in a) and the SCC l v in a), c and /'. staining with the antisense riboprobe; </and g, immunofluorescence staining

wilh antibody 6F9; e and h, staining with hematoxylin. Tissue is from patient 3 in Table 1. Bars: b, 530 ^m; r, 45 um;/ 90 ^m.

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noma classified as grade IV completely lacked E-cadherinexpression (38). In gastric adenocarcinomas, cases of E-cadherin down-regulation were also found, although at a muchlower frequency than in SCCs, and the lymph node métastaseswere E-cadherin-positive (39). Clearly, more types of carcinomatissues must be examined to obtain the overall picture of therole of E-cadherin in the process of differentiation and metastasis of carcinomas in vivo.

ACKNOWLEDGMENTS

We thank Dr. J. K. Field (Liverpool) for critically reading themanuscript, Dr. T. P. U. Wustrow (Munich) for the generous gift ofthe head and neck carcinoma cell lines, Eva Sonnenberg (Köhn)fordemonstrating the technique of in situ hybridization, Beate Voss forexpert technical support, and B. Lelekakis for excellent secretarialwork. J. H. Schipper was supported by a fellowship from the DeutscheForschungsgemeinschaft. This study was supported by the DeutscheKrebshilfe e. V.

REFERENCES

1. Parkin, D. M., Laara, E., and Muir, C. S. Estimates of the worldwidefrequency of 16 major cancers in 1980. Int. J. Cancer, 41: 184-197, 1988.

2. Million, R. R., Cassisi, N. J., and Clark, J. R. Cancer of the head and neck.In: V. T. DeVita, S. Hellman, and S. A. Rosenberg (eds.). Cancer: Principlesand Practice of Oncology, Ed. 3, pp. 488-590. Philadelphia: J. B. LippincottCo., 1989.

3. Meyer-Breiting, E., and Burkhardt, A. Tumors of the Larynx. Berlin: Springer-Verlag, 1988.

4. Noltenius, H. Tumor-Handbuch: Pathologie und Klinik der menschlichenTumoren. Munich: Verlag Urban und Schwarzenberg, 1987.

5. Wahi, P. N., Cohen, B., Luthra, U. K., and Torloni, H. Histológica! Typingof Orala and Oropharyngeal Tumours. Geneva: WHO, 1971.

6. Glanz, H. K. Carcinoma of the larynx: growth, p classification and gradingof squamous cell carcinoma of the vocal cords. Adv. Otorhinolaryngol., 32:1-123, 1984.

7. Graham, S., Metlin, C, Marshall, J., Priore, R., Rzepka, T., and Shedd, D.Dietary factors in the epidemiology of cancer of the larynx. Am. J. Epidemici., 123: 675-680, 1981.

8. Bryan, R. L., Bevan, I. S., Crocker, J., and Young, L. S. Detection of HPV6and 11 in tumors of the upper respiratory tract using the polymerase chainreaction. Clin. Otolaryngol., IS: 177-180, 1990.

9. Fahraeus, R., Rymo, L., Thim, J. S., and Klein, G. Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barrvirus. Nature (Lond.), 345:447-449, 1990.

10. Field, J. K., and Spandidos, D. A. Expression of oncogenes in human tumorswith special reference to the head and neck region. J. Oral, l'alimi., 16: 97-

107, 1987.11. Saranath, D., Panchal, R. G., Naiv, R., Mehta, A. R., Sanhavi, V., Sumegi,

J., Klein, G., and Deo, M. G. Oncogene amplification and squamous cellcarcinoma of the oral cavity. Jpn. J. Cancer Res., SO:430-437, 1989.

12. Somers, K. D., Cartwright, S. L., and Schechter, G. L. Amplification of thei'/if-2 gene in human head and neck squamous cell carcinomas. Oncogene, 5:

915-920, 1990.13. Volling, P., Jungehulsing, M., Tesch, H., and Steunert, E. Onkogene bei

Plattenepithelkarzinomen in Kopf-Hals-Bereich. Laryng. Rhino!. Otol., 66:160-164, 1988.

14. Ishitoya, J., Toriyama, M., Oguchi, N., Kitamura, V., Ohshima, M., Asano,K., and Yamamoto, T. Gene amplification and overexpression of EGF-receptor in squamous cell carcinoma of the head and neck. Br. J. Cancer, 59:559-562, 1989.

15. Kasid, U., Pfeiffer, A., Weichselbaum, R. R., Dritschilo, A., and Mark, G.G. The ra/oncogene is associated with a radiation resistant human laryngealcancer. Science (Washington DC), 237: 1039-1041, 1987.

16. Merritt, W. D., Weissler, M. C., Turk, B. F., and Gilmer, T. M. Oncogeneamplification in squamous cell carcinoma of the head and neck. Arch.Otolaryngol. Head Neck Surg., 116: 1394-1398, 1990.

17. Greenhalgh, D. A., Welty, D. J., Player, A., and Yuspa, A. S. Two oncogenes,

\-fos and v-r<w,cooperate to convert normal keratinocytes to squamous cellcarcinoma. Proc. Nati. Acad. Sci. USA, 87: 643-647, 1990.

18. Grandi, C., Aloisio, N., Moglia, D., Prodecca, S., Sala, S., Salvatori, P. E.,and Molinar, R. Prognostic significance of lymphatic spread in head andneck carcinomas: Therapeutic implications. Head Neck Surg., 8: 67-73,1985.

19. Birchmeier, W., Behrens, J., Weidner, M., Frixen, U., Sachs, M., andVandekerckhove, J. Molecular and cellular aspects of tumor cell invasion.In: Origin of Human Cancer: A Comprehensive Review. Cold Spring Harbor,NY: Cold Spring Harbor Laboratory Press, in press, 1991.

20. Takeichi, M. Cadherin cell adhesion receptors as a morphologic regulator.Science (Washington DC), 251:1451-1455, 1991.

21. Behrens, J., Marcel, M. M., Van Roy, F. M., and Birchmeier, W. Dissectingtumor cell invasion: epithelial cells acquire invasive properties after the lossof uvomorulin-mediated cell-cell adhesion. J. Cell Biol., IOS: 2435-2447,1989.

22. Frixen, U., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., Löchner,D., and Birchmeier, W. E-cadherin-mediated cell-cell adhesion preventsinvasiveness of human carcinoma cells. J. Cell Biol., 113: 173-185, 1991.

23. Dony, C., and Gruss, P. Specific expression of the Hoxl.3 horneo box genein murine embryonic structures originating from or induced by the mesoderm.EMBO J.. 6: 2965-2975. 1987.

24. Sonnenberg, E., Gödecke, A., Walter, B., Bladt, F., and Birchmeier, C.Transient expression of the roj-1 protooncogene during mouse embryogen-esis. EMBO J., 12, in press, 1991.

25. Hermanek, P., and Sobin, L. H. UICC TNM Classification of MalignantTumors, Ed. 4. Berlin: Springer Verlag, 1987.

26. Weidner, K. M., Behrens, J., Vandekerckhove, J., and Birchmeier, W. Scatterfactor: molecular characteristics and effect on the invasiveness of epithelialcells. J. Cell Biol., ///: 2097-2108, 1990.

27. Jouanneau, J., Gavrilowic, J., Cavuelle, D., Jaye, M., Moens, G., Cavuelle,J-P., and Thiery, J. P. Secreted or non-secreted forms of acid fibroblastgrowth factor produced by transfected epithelial cells influence cell morphology, motility invasive potential. Proc. Nati. Acad. Sci. USA, SS: 2893-2897,1991.

28. Ozawa, M., Engel, J., and Kemler, P. Single amino acid substitutions in oneCa2* binding site of uvomorulin abolish the adhesive function. Cell, 63:1033-1038, 1990.

29. Tsuda, H., Zhang, W., Shimosato, Y., Yokata, J., Terada, M., Sugimura, T.,Miyamura, T., and Hirohashi, S. Alíeleloss on chromosome 16 associatedwith progression of human hepatocellular carcinoma. Proc. Nati. Acad. Sci.USA, «7:6791-6794, 1990.

30. Sato, T., Tanigami, A., Yamakawa, K., Akiyama, F., Kasumi, F., Sakamoto,G., and Nakamura, Y. Allelotype of breast cancer: cumulative alíelelossespromote tumor progression in primary breast cancer. Cancer Res., 50:7184-7189, 1990.

31. Carter, B. S., Ewing, C. M., Ward, W. S., Treiger, B. F., Aalders, T. W.,Schalken, J. A., Epstein, J. I., and Isaacs, W. B. Allelic loss of chromosomes16q and lOq in human prostate cancer. Proc. Nati. Acad. Sci. USA, 87:8751-8755, 1990.

32. Natt, E., Magenis, R. E., Zimmer, J., Mansouri, A., and Scherer, G. Regionalassignment of the human loci for uvomorulin (UVO) and chymotrypsinogenB (CTRB) with the help of two overlapping deletions on the long arm ofchromosome 16. Cytogenet. Cell Genet., 50: 145-148, 1989.

33. Behrens, J., Lowerick, O., Klein-Hitpass, L., and Birchmeier, W. The E-cadherin promoter: functional analysis of a GC-rich region and an epithelialspecific palindromi^ regulatory element. Proc. Nati. Acad. Sci. USA, inpress, 1991.

34. Fearon, E. R., Cho, K. R., Nigro, J. M., Kern, S. E., Simons, J. W., Ruppert,J. M., Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W., andVogelstein, B. Identification of a chromosome 18q gene that is altered incolorectal cancers. Science (Washington DC), 247:49-56, 1990.

35. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., and Stan-ners, C. P. Carcinoembryonic antigen, a human tumor marker, functions asan intercellular adhesion molecule. Cell, 57: 327-334, 1989.

36. Gunthert, U., Hofmann, M., Rudy, W., Reber, S., Zöller,M., Haussmann,L, Matzku, S., Wenzel, A., Ponta, H., and Herrlich, P. A new variant ofglycoprotein. CD44 confers metastatic potential to rat carcinoma cells. Cell,65:13-24, 1991.

37. Leone, A., Flatow, U., King, C. R., Sandee, M. A., Margulies, L. M. K.,Liotta, L. A., and Steeg, P. S. Reduced tumor incidence, metastatic potential,and cytokine responsiveness of nm 23 transfected melanoma cells. Cell, 65:25-35, 1991.

38. Shimoyama, Y., and Hirohashi, S. Cadherin intercellular adhesion moleculein hepatocellular carcinomas: loss of E-cadherin expression in an undiffer-entiated carcinoma. Cancer Lett., 57: 131-135, 1991.

39. Shimoyama, Y., and Hirohashi, S. Expression of E- and P-cadherin in gastriccarcinomas. Cancer Res., 51: 2185-2192, 1991.

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1991;51:6328-6337. Cancer Res   Jörg H. Schipper, Uwe H. Frixen, Jürgen Behrens, et al.   Lymph Node Metastasisand Neck: Inverse Correlation with Tumor Dedifferentiation and E-Cadherin Expression in Squamous Cell Carcinomas of Head

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