Acta Histochem. Cytochem. Vol.29 No.4 305-310 (1996)

Immunohistochemical Expression of E-Cadherin in Salivary Glands and

Their Tumors

Kazuto Yamada1, Wataru Kudeken1, Shinichiro Sumitomo1, Yasunori Muramatu1,

Takeshi Shiba1, Yoshiaki Takai1, Masahiko Mori1 and Paul M.Speight2

'Department of Oral & Maxillofacial Surgery, Asahi University School of Dentistry and 2Department of OralPathology, Eastman Dental Institute, University of London

Received for publication September 20,1996 and in revised form December 20, 1996

E-cadherin is a calcium dependent cell adhe-sion molecule, important in cell to cell interac-

tions in epithelial tissues. The present studyexamined E-cadherin expression in normal

salivary glands, pleomorphic adenoma, War-thin's tumor, mucoepidermoid carcinoma,

acinic cell carcinoma, adenoid cystic car-cinoma and adenocarcinoma of salivary

glands in paraffin sections using amonoclonal antibody (5H9). This study is

firstly reported E-cadherin expression in thesalivary gland and tumors.

In normal submandibular and parotid gland,E-cadherin staining was confined to the cell

membrane of acinar and ductal cells. In ex-cretory ducts, high columnar cells showed

the strongest reaction at the basal aspectand ductal basal cells were weakly positive

or negative, the basement membrane zone

lacked staining. No E-cadherin reaction was

observed in modified myoepithelial cells

and plasmacytoid cells. In pleomorphic

adenoma and mucoepidermoid carcinoma,

E-cadherin was positive at the lateral cell mem-

brane or whole cell membrane of duct-like

structure and epidermoid cells. Acinic cell

carcinoma was negative for E-cadherin. In

adenoid cystic carcinoma, the expression

was only found in cribriform areas. In

adenocarcinoma, the staining was seen in

the cell membrane of duct like structures

and occasional cells showed a strong

cytoplasmic reaction. Positive E-cadherin

staining in salivary gland tumors was higher

in benign tumors than in malignant tumors.

Key words: Salivary gland tumor, Cell adhesion molecule, E-cadherin

I. Introduction

Cell to cell interactions, mediated through variousadhesion molecules, play a key role in cell regulation [16,24,25,30]. Cadherins are a family of calcium-dependenttransmembrane glycoproteins which are important in inter-cellular adhesion [8]. E-cadherin mediates homotypiccell to cell adhesion in epithelium [24,25], and is expressedin all stratified squamous epithelial cells except forkeratinized cells [21]. Reduction of E-cadherin has beendemonstrated in carcinomas of the esophagus, breast,stomach, female genitalia, colon [21], oral mucosa and

gingiva [5] and loss of E-cadherin expression is associatedwith abnormal stratification [29]. Cadherins are also im-

portant in the development and organization of cells in em-bryogenesis and morphogenesis in thyroid [31], pancreas,

gastrointestinal tract [22], skin and appendages [7]. Thereare no reports of E-cadherin expression in salivary glandand their tumors. In this report, we describe E-cadherinstaining patterns of benign and malignant salivary glandtumors. Many salivary gland tumors consist of luminaland non-luminal tumor cells as well as modified myoepi-thelial cells, which show complex histochemical proper-ties. The aim of the present study was to determinewhether E-cadherin-mediated cell to cell adhesion may bealtered in salivary gland tumors.

II. Materials and Methods

MaterialsNormal submandibular gland (n=2), normal parotid

gland (n=2), pleomorphic adenoma (n=20) in the parotidglands and oral minor glands, Warthin's tumor (n=8) inthe parotid gland, mucoepidermoid carcinoma (MEC:n=9) in the submandibular and the palatal gland, aciniccell carcinoma (n=2) in the parotid gland, adenoid cystic

305

Correspondence to: Dr. Kazuto Yamada, Department of Oral and

Maxillofacial Surgery, Asahi University School of Dentistry, 1851,

Hozumi-cho, Motosu-gun, Gifu 501-02, Japan.

306 Yamada et al.

carcinoma (ACC:n=10) in the palatal gland and adenocar-

cinoma (n=1) in the parotid gland were used for

immunohistochemical detection of E-cadherin. The tissue

specimens were fixed in 10% formalin and embedded in

paraffin. Sections, cut at 4ƒÊm, were mounted on

aminopropyltriethoxysilane (APES) coated slides, and

stained with haematoxylin and eosin for histopathologic

orientation, and by an immunohistochemical method.

Immunohistochemical method

Deparaffinized sections were treated with 1.0%

hydrogen peroxide in methanol (10min) to block endo-

genous peroxidase. They were then treated with 0.1%

proteinase K (Sigma) for 5min at 37•Ž. The primary

antibody, mouse monoclonal antibody (dilution: 1/5;

EuroPath LTD. U.K.; 5H9) raised against human

E-cadherin, was applied for 2 hr at room temperature.

After washing with PBS, biotinylated secondary antibody

(1/200; Dako. Denmark) was applied for 30 min at room

temperature. After a further wash and incubation with

peroxidase conjugated streptavidin biotin peroxidase com-

plex (Dako ABC kit), the reaction product was visualized

with 3, 3'diaminobenzidine tetrahydrochroride with 0.05%

hydrogen peroxide. Sections were counterstained with

methyl-green.

The sections for negative controls were incubated

without the primary antibody. Positive controls used the

presence of oral epithelium overlaying minor salivary

gland tumors, which showed normal E-cadherin expres-

sion in the cell membrane. Specificity for mouse

monoclonal antibody (5H9) for E-cadherin was already

tested in Westan blotting [6,15,19] and the staining of E-

cadherin was compared with frozen sections and the same

distribution was obtained in paraffin and frozen sections in

squamous epithelial tissue.

III. Results

Normal salivary glandImmunostaining of E-cadherin was confined to the

cell membrane of acinar cells and all the ducts (Fig.1-A,B). Myoepithelial cells were negatively stained. Basal in-foldings in striated duct cells and the lateral cell membraneof intercalated duct cells were strongly positive forE-cadherin staining (Fig.1-B,C). In excretory ducts, thestaining of the cell membrane of luminal cells was strongbut ductal basal cells were very weak or negative. E-cadherin expression towards the basal aspect of columnarductal cells was particularly strong (Fig.1D).

Pleomorphic adenomaSeventeen of twenty cases (85%) were positive for

E-cadherin. E-cadherin staining was localized to the cellmembrane of luminal tumor cells in duct like structuresand squamous metaplastic cells (Fig.2A). No, or veryweak reaction product was found in modified and neo-

plastic myoepithelial cells (Fig.2B).

Warthin's tumorSeven of eight cases (87.5%) were positive for E-

cadherin. E-cadherin was strongly expressed on the cellmembranes of luminal tumor cells and basal tumor cellswere weakly stained and/or sometimes negative. Base-ment membrane was negative (Fig.2C).

Mucoepidermoid carcinoma (MEC)Five of nine cases (55.5%) were positive for

E-cadherin. E-cadherin positive reaction was found inepidermoid cells, intermediate cells and mucous cells. E-cadherin was localized at all cell membranes despite oftheir histopathological variation as mucous or epidermoidcell (Fig.2D).

Acinic cell carcinomaAll cases showed negative staining for E-cadherin.

Adenoid cystic carcinoma(ACC)Three of ten cases (30%) of ACC were positive for E-

cadherin. The E-cadherin reaction was seen on the cellmembrane of cells in cribriform areas.

AdenocarcinomaA case of adenocarcinoma was positive in the cell

membrane of a part of duct like structure and was alsolocalized in the cytoplasm of occasional tumor cells.

IV. Discussion

E-cadherin is a 124 kd transmembrane protein inepithelial cells which mediates homotypic cell to cell adhe-sion [24,25]. E-cadherin binds to actin, which modulatesthe shape of cells through the intermediation of catenins[10]. This structure suggested that E-cadherin regulatesthe segmentation, organization and differentiation ofcells. The present study shows, for first time, theE-cadherin staining pattern in the normal salivary glandas well as salivary gland tumors. It has already been shownthat E-cadherin localizes to the cell membrane of variousepithelia [9,13,21]. In squamous epithelium, E-cadherinis expressed in all layers of stratified squamous epitheliumexcept for the keratinized layer [21]. In mammary gland,E-cadherin was expressed in the intercellular areas ofluminal cells of both the interlobular, a intralobular ter-minal ducts and ductules. Myoepithelial cells showed amuch weaker reaction at cell-cell borders in paraffin sec-tions. In some specimens, the myoepithelial cells of themammary gland were negative despite the paraffin sections[13]. Glandular cells of the cervix, endometrium and fallo-pian tube showed strong E-cadherin expression at cell-to-cell borders [9]. In renal epithelium, E-cadherin wasexpressed in distal tubules and collecting ducts usingfrozen sections [11,17,26].

In normal salivary gland, E-cadherin was localized tothe cell membrane of acinar and ductal cells, similar tothat of the mammary gland. It is interesting to note that

E-Cadherin Expression of Salivary Gland Tumors 307

A

C

B

DFig.1. Normal submandiular gland. A: E-cadherin is seen on cell membrane of acinar cell and ductal segments. (•~100). B&C: Higher

magnification of A. E-cadherin staining is found in cell membrane of all acinar cells, intercalated duct cells, striated duct cells. Immunoreac-

tivity of E-cadherin is strong in intercalated duct cell membrane and basal infoldings of striated duct cells (•~200). D: E-cadherin staining

of excretory duct. High columnar cells (luminal cells), stain intensely in basal aspect and positively in cell membrane. Basal cells do not show

for E-cadherin (•~200).

308 Yamada et al.

ACB

DFig.2. Salivary gland tumors. A: Pleomorphic adenoma. E-cadherin reaction is seen in cell membrane of luminal tumor cells, and is unstained

in modified myoepithelial cells (•~100). B: Pleomorphic adenoma. The neoplastic myoepithelial cells in these myoepithelioma-like cells

show negative to slight immunoreactivity for E-cadherin (•~100). C: Warthin's tumor, E-cadherin is seen in cell membrane of luminal cells

(•~200). D: Mucoepidermoid tumor, E-cadherin staining is seen in cell membrane of all tumor cells, both of epidermoid cells and mucous

cells (•~100).

the site of basal infolding of striated duct cells was strong-ly positive. In excretory ducts, there are charactristicfeatures that the highest reaction of E-cadherin existed inbasal aspect of high columner cells while negative or veryweak staining in the ductal basal cells, suggesting thosetwo cells obtained different cellular functions. It is possi-ble that the stage of cellular differentiation may be a factorin the expression of E-cadherin, and ductal basal cells were

posible progenitor cells of salivary gland tumors [12,14].In pleomorphic adenoma, E-cadherin expression was

lacking in modified or neoplastic myoepithelial cellslocated on the outer zone of tubulo-ductal structures or inchondroid structure. Immunohistochemical studies of themodified and neoplastic myoepithelial cells have shownthem to express epithelial(cytokeratin) and mesenchymal

(vimentin) markers as well as S-100 proteins, showing that

they have complex cellular properties and may not be true

epithelial cells [15, 20]. Pignatelli suggested that develop-

ment of fully differentiated glandular structures seems to

require other adhesion molecules, including integrin(ƒ¿2 ƒÀ1)

or growth factors [18].

In Warthin's tumor as in pleomorphic adenoma, E-

cadherin staining was confined to the cell membranes of

luminal cells, while malignant salivary tumors, MEC,

ACC, acinic cell carcinoma and adenocarcinoma, had a

heterogeneous population of tumor cells with or without

E-cadherin expression. All these tumors contain cells

which may be counterparts of neoplastic myoepithelial

cells of pleomorphic adenoma [3,4]. A lack of cell to cell

contacts in neoplastic myoepithelial cells of pleomorphic

adenoma, which often secrete excessive amounts of

extracellular matrix proteins may be related to the down

E-Cadherin Expression of Salivary Gland Tumors 309

regulation of E-cadherin in those cells.Loss of E-cadherin leads to the dissociation of cells

from coherent tissue [1,27], and several experimentalstudies have shown a relationship between down regula-tion of E-cadherin expression in tumor cells and acquisi-tion of an invasive phenotype [2,6,23,28]. In the presentstudy, we were able to demonstrate loss of E-cadherin ex-

pression in a number of malignant salivary gland tumorswith an aggressive behaviour. The results of the presentshow that E-cadherin is expressed in normal salivary

glands and may be loss in salivary malignant tumors.

V. Acknowledgment

This work is supported in part by Grants-in aid fromthe Ministry of Education, Science and Culture, Govern-ment of Japan and from Miyata Research Fund, AsahiUniversity.

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