Expression of cadherins and alpha-catenin in primary epithelial tumors of the liver

GASTROENTEROLOGY 1996;110:1137–1149

Expression of Cadherins and a-Catenin in Primary Epithelial Tumors of the Liver RENATA KOZYRAKI,* JEAN–YVES SCOAZEC,* JEAN–FRANC¸OIS FLEJOU,‡,§ ANTONIA D’ERRICO,㛳 PIERRE BEDOSSA,Ø BENOIˆT TERRIS,‡,§ MICHELANGELO FIORENTINO,㛳 ANNIE–FRANCE BRINGUIER,* WALTER F. GRIGIONI,㛳 and GE´RARD FELDMANN* *Laboratoire de Biologie Cellulaire and INSERM Unite´ 327, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris, Paris, France; ‡Service Central d’Anatomie et de Cytologie Pathologiques, Ho ˆpital Beaujon, Clichy, France; §INSERM Unite´ 410, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris, Paris, France; 㛳Istituto di Anatomia e Istologia Patologica, Policlinico S. Orsola, Bologna, Italy; and ØService Central d’Anatomie et de Cytologie Pathologiques, Ho ˆpital de Bice ˆtre, Le Kremlin-Bice ˆtre, France

Background & Aims: Cadherins and their associated molecules, such as a-catenin, have been shown recently to play a pivotal role in epithelial carcinogenesis. Methods: The expression of E-cadherin, N-cadherin, and a-catenin in 10 normal samples, 28 focal nodular hyperplasias, 9 liver cell adenomas, 65 hepatocellular carcinomas, and 9 cholangiocarcinomas was studied by immunohistochemistry and Western blotting. Results: In the normal liver, hepatocytes expressed Ecadherin and a 129-kilodalton cadherin identified by the anti-N-cadherin antibody GC4. The expression level of a-catenin was low. Bile duct cells expressed only E-cadherin and showed high levels of a-catenin. The expression of cadherins and a-catenin was preserved in focal nodular hyperplasia. In liver cell adenomas, cadherins and a-catenin were heterogeneously expressed. In hepatocellular carcinomas, cadherin and acatenin expression was frequently reduced or absent. Alterations in cadherin expression correlated with large tumor size, low grade of histological differentiation, and occurrence of capsular and vascular invasion. In cholangiocarcinomas, neoplastic cells inconstantly expressed E-cadherin and a-catenin. Conclusions: Alterations of cadherin and a-catenin expression are frequent in liver cell adenomas and primary liver carcinomas. Their incidence in hepatocellular carcinomas is of prognostic significance.

C

adherins form a superfamily of specialized membrane glycoproteins mediating calcium-dependent cell-cell adhesion and expressed in a tissue-specific manner.1 – 4 Among the various cadherins susceptible to be expressed by epithelial cells, the most largely distributed are as follows: E-cadherin, present in all simple, glandular, and stratified epithelial cells tested so far5,6; N-cadherin, detected in various epithelial cell subsets such as lens cells7 and human tubular renal cells8; and P-cadherin, characteristic of the proliferative compartment of stratified and simple epithelia.6 / 5e0b$$0005

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Much interest has focused recently on the possible role of cadherins in epithelial oncogenesis.9 – 11 Impairment of cadherin-mediated adhesion is likely to constitute one of the main factors leading to the reduced cell-cell adhesion characteristic of neoplastic cells. Alterations in cadherin function in neoplastic cells may result from either quantitative or qualitative abnormalities. Quantitative decrease in the expression of cadherins at the surface of neoplastic cells is frequently observed, both in vivo and in vitro.5,6,12 – 15 Qualitative alterations of cadherins usually result from the impairment of their normal interaction with the actin cytoskeleton, which is necessary to their adhesive properties.1 – 3 This interaction is mediated by specialized molecules, including a-catenins, b-catenin, and plakoglobin.16 – 21 In neoplastic cells, catenins, like cadherins, may present various types of quantitative22 – 26 or qualitative alterations27 – 30 that alter the adhesive functions of cadherins and likely contribute to epithelial carcinogenesis. Few data5,31 are currently available about the pattern of expression of cadherins and their associated proteins in the primary epithelial tumors of the liver. However, the study of the various types of benign and malignant liver tumors may be particularly relevant to address the role of cadherins and their associated proteins during the neoplastic process. In contrast to the few examples of benign epithelial tumors studied so far,5,32,33 benign epithelial tumors of the liver present a negligible or low risk of neoplastic transformation34,35; therefore, this study may help to evaluate the relations between cadherin expression and cell proliferation in the absence of ongoing neoplastic process. Hepatocellular carcinomas, which constitute one of the most frequent types of carcinoma on a worldwide basis,36 present a high incidence of allelic losses in the region of the chromosome 16 known to 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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contain the E-cadherin gene.37 – 39 This suggests a particular role for E-cadherin in the process of hepatocarcinogenesis. Moreover, hepatocellular carcinomas present an unusually large spectrum of growth patterns,40 – 42 which offers the opportunity to evaluate the relation between cadherin expression and histological differentiation. Finally, the comparison of hepatocellular carcinomas and cholangiocarcinomas, which derive from hepatocytes and biliary epithelial cells, respectively, may help to evaluate the value of the various subclasses of cadherins as cell lineage markers. Therefore, we analyzed the expression of E-cadherin, N-cadherin, and a-catenin in the normal liver tissue and in a large series of primary liver epithelial tumors by using immunohistochemical and Western blotting techniques. The aims of this study were to search for quantitative and qualitative alterations in cadherin expression in the various types of benign and malignant liver tumors and to correlate the pattern of cadherin expression in malignant liver tumors with clinicopathologic parameters.

Materials and Methods Tissue Material Surgical specimens were obtained from January 1991 to June 1994 from the Departments of Pathology of Hoˆpital Beaujon (Clichy, France), Hoˆpital Antoine Be´cle`re (Clamart, France), and Policlinico S. Orsola, (Bologna, Italy). In all cases, tissue samples were immediately snap frozen in isopentane prechilled in liquid nitrogen and stored at 080⬚C until use. Normal liver tissue. Fragments from normal liver tissue were obtained from 15 patients during surgical resection for liver metastases. Samples were taken at a distance from the tumor in macroscopically nonaffected tissue. In each case, the absence of significant histological lesion was verified before inclusion in the study group. Tumor-like lesions and benign tumors of the liver.

Surgical specimens were obtained from 37 patients with tumor-like lesions and benign tumors of the liver. The study group comprised 28 patients with focal nodular hyperplasia (6 men and 22 women; age range, 21–67 years; mean, 35.6 years) and 9 patients with liver cell adenoma (9 women, age range, 23–45 years; median, 32 years). Malignant primary liver tumors. The study group comprised 65 patients with hepatocellular carcinoma (58 men and 7 women; age range, 18–71 years; median, 54.4 years). Identified risk factors were as follows: chronic infection by the hepatitis B virus (20 patients), chronic infection by the hepatitis C virus (18 patients), alcoholic cirrhosis (11 patients), and hemochromatosis (3 patients). Tumor size was õ3 cm in 11 patients (16.9%) and õ5 cm in 24 patients (36.9%). The degree of tumor differentiation, graded according to Edmonson’s classification,40 was as follows: grade I in 4 patients (6.3%), grade II in 28 patients (43%), grade III in 28 patients

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(43%), and grade IV in 5 patients (7.7%). Tumors were infiltrative in 13 patients (20%) and encapsulated in 52 patients (80%). Capsular effraction was present in 43 of the 52 patients with encapsulated tumors (82.7%), satellite nodules were present in 31 patients (47.7%), and vascular invasion was detected in 48 patients (73.8%). The study group also included 9 patients with cholangiocarcinoma (6 men and 3 women; age range, 45–65 years; median, 54.9 years).

Antibodies E-cadherin expression was analyzed by using the mouse monoclonal antibody HECD-16 (Takara, Kyoto, Japan). To study the expression of members of the N-cadherin subclass, we used the mouse monoclonal antibody GC4 (Sigma Chemical Co., St. Louis, MO), raised against chicken heart N-cadherin and reactive with the N-terminal part of the extracellular domain of the molecule.43,44 The rat monoclonal antibody a18, specifically directed against the epithelial isoform of a-catenin, was a generous gift from Akira Nagafuchi (Institute for Informative Physiology, National Institute for Physiological Sciences, Okazaki, Japan).45

Immunohistochemical Technique An indirect immunoperoxidase technique was applied to 4-mm-thick, acetone-fixed cryostat sections of frozen tissue. Briefly, sections were incubated 60 minutes with the primary antibody in appropriate dilution, washed in phosphate-buffered saline (PBS), and then incubated 60 minutes with goat polyclonal species-specific peroxidase-labeled adsorbed antimouse or anti-rat immunoglobulin F(abⴕ)2 antibodies (Immunotech, Marseille-Luminy, France). After washing in PBS, peroxidase activity was shown according to Graham and Karnovsky.46 Sections were then lightly counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Controls were obtained by omitting the first antibody, replaced by PBS or by incubation with isotypic immunoglobulins. All controls were negative. In accordance with previously published criteria,47 tumors were considered homogeneously positive when ú90% of tumor cells were reactive for the antigen tested, heterogeneously positive when 10%–90% of the cells were labeled, and negative when õ10% of the cells were stained. The staining intensity of tumor cells was compared with that of normal hepatocytes only when peritumoral liver tissue was present on the same section. In some experiments, sections of paraformaldehyde-fixed normal liver tissue were prepared for ultrastructural immunohistochemistry according to methods described previously48 to analyze the plasma membrane distribution of cadherins.

Western Blot Analysis Western blot analysis was performed essentially as described previously.48,49 Tissue pieces weighing 10–50 mg were homogenized with a Dounce homogenizer (Treff, Degersheim, Switzerland) in 1 mmol/L ice-cold sodium carbonate buffer, pH 8.2, containing 2 mmol/L CaCl2 and the following protease

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inhibitors: 0.25 mg/mL pepstatin, 1.25 mg/mL leupeptin, 1.25 mg/mL antipain, 0.25 mg/mL aprotinin, and 0.5 mmol/L phenylmethylsulfonyl fluoride. All reagents were purchased from Sigma Chemical Co. The resulting homogenates were centrifugated for 15 minutes at 10,000g at /4⬚C. Pellets were resuspended in 0.1 mol/L Tris HCl, pH 6.8, containing 2 mmol/L CaCl2 . Reducing buffer containing sodium dodecyl sulfate and dithiotreitol (lane marker reducing buffer; Pierce Chemical Co., Rockford, IL) was finally added at 1:5 dilution. Approximately 100 mg protein per lane was applied to a 6% or 9% polyacrylamide slab gel. After electrophoretical separation, proteins were blotted overnight onto a nitrocellulose sheet (Schleicher and Schu¨ll, Feldbach, Switzerland). After quenching of filters for 1 hour at room temperature with 5% nonfat dried milk diluted in PBS containing 0.1% Tween 20 (Merck, Darmstadt, Germany), antigens were shown with an indirect immunoperoxidase technique. Filters were incubated for 1 hour at room temperature with the primary antibody diluted in PBS, washed for 15 minutes in PBS, and then incubated for 1 hour at room temperature with sheep species-specific peroxidase-labeled anti-mouse immunoglobulin antibody (Amersham, Little Chalfont, England) or goat species-specific peroxidase-labeled anti-rat immunoglobulin F(abⴕ)2 antibody (Immunotech) diluted 1:1000 in PBS. After washing, peroxidase activity was shown by enhanced chemoluminescence (ECL Western blotting detection system; Amersham). Blots were finally exposed to autoradiography films. For comparison purposes, samples from tumor tissue and from the corresponding peritumoral liver tissue were processed in the same way and run in parallel. In some experiments, normal brain tissue obtained during surgical resections of intracranial aneurysms (kindly provided by Dominique He´nin, Service Central d’Anatomie et de Cytologie Pathologiques, Hoˆpital Beaujon) and normal heart tissue obtained at autopsy (kindly provided by Anne Couvelard, Service Central d’Anatomie et de Cytologie Pathologiques, Hoˆpital Bichat) were processed in the same way and run in parallel to homogenates of normal liver tissue.

Statistical Analysis Statistical analysis was performed by using the x2 test and Fisher’s Exact Test. A P value of õ0.05 was considered significant.

Results Normal Liver Tissue Immunohistochemical analysis. In the normal liver tissue, both hepatocytes and bile duct cells were strongly reactive with the monoclonal antibody HECD1 directed against E-cadherin (Figure 1A). Hepatocytes were constantly reactive with the monoclonal antibody GC4 (Figure 1B), directed against members of the Ncadherin family. Bile duct cells were not labeled by GC4 (Figure 1B). Like E-cadherin, a-catenin was detected in both hepatocytes and bile duct cells (Figure 1C). Its

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apparent expression level was greater in bile duct cells than in hepatocytes. By light microscopic examination, the distribution of cadherins and a-catenin was restricted to the lateral domain of the plasma membrane in both hepatocytes and bile duct cells (Figure 1A–C). Ultrastructural examination confirmed that hepatocyte labeling with anti–Ecadherin antibody and with GC4 antibody (Figure 1D) was restricted to the lateral domain of the plasma membrane. No reactivity of the sinusoidal or canalicular membranes was observed in any patient. Western blot analysis. In homogenates from normal liver tissue, Western blot analysis detected Ecadherin as a main band of 124 kilodaltons (Figure 2A). Western blot analysis showed that the monoclonal antibody GC4 usually detected a unique band in the liver with an apparent molecular weight of 129 kilodaltons (Figure 2A). Direct comparison with normal brain and heart tissues processed in the same way showed that, in the brain, GC4 identified a molecule of slightly lower molecular weight, estimated to 125 kilodaltons, whereas in the heart, the GC4-reactive antigen migrated at 135 kilodaltons (Figure 2B). In view of the tissue-specific differences observed in the electrophoretic pattern of the GC4-reactive antigens and pending the full characterization of the corresponding molecules, we will thereafter refer to the GC4-reactive liver cadherin as hepatocyte Nrelated cadherin. Finally, a-catenin was detected as a unique band of 102 kilodaltons. This apparent molecular weight compared well with the previous determinations made in other tissues (Figure 2C). Focal Nodular Hyperplasia Immunohistochemical results. In the 28 patients with focal nodular hyperplasia examined, E-cadherin was uniformly and strongly expressed by hepatocytes in the hyperplastic parenchyma and by the bile duct cells lining the ductular structures present in the central fibrous scar (Figure 3A). In all cases examined, hepatocytes but not bile duct cells expressed the hepatocyte N-related cadherin identified by the monoclonal antibody GC4. In all cases, a-catenin was uniformly detected along the lateral membranes of hepatocytes and bile duct cells (Figure 3B). Staining intensities were much greater in ductular cells than in hepatocytes. The distribution of cadherins and a-catenin was restricted to the lateral domain of the plasma membrane of epithelial cells. Western blot analysis. Fifteen patients were available for Western blot analysis. In 14 patients, the pattern of reactivity was similar to that of normal liver for E-cadherin (Figure 4), hepatocyte N-related cadherin,

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Figure 2. Western blotting characterization of cadherins and a-catenin in the normal human liver tissue. (A ) In two different samples of normal human liver tissue, E-cadherin (E) is identified as a main band of 124 kilodaltons and the hepatocyte N-related cadherin (N) detected by the GC4 monoclonal antibody as a main band of 129 kilodaltons. The faint bands detected at 110 kilodaltons for hepatocyte N-related cadherin are likely to correspond to products of proteolytic cleavage. (B ) Western blotting characterization of the antigens recognized by the GC4 monoclonal antibody in normal liver (l), brain (b), and heart (h) tissues. The apparent molecular weight of the GC4reactive antigen is slightly different according to the tissue: 129 kilodaltons in the liver, 124 kilodaltons in the brain, and 135 kilodaltons in the heart. The faint bands of lower molecular weights detected in each tissue are likely to correspond to products of proteolytic cleavage. (C ) In two different samples of normal human liver tissue, acatenin (cat) is identified as a band of 102 kilodaltons.

and a-catenin. In only 1 patient (Figure 4), an additional band of low molecular weight, estimated to 95 kilodaltons, was detected for E-cadherin. Liver Cell Adenoma Immunohistochemical results. In the 9 patients

examined, E-cadherin (Figure 3C and D) and the hepatocyte N-related cadherin were detectable all along the lateral membranes of tumoral hepatocytes. The apparent expression level of cadherins was usually variable from cell to cell within the same tumor (Figure 3D). In all the patients examined, a-catenin was faintly detected along the lateral membranes of tumoral hepatocytes. Western blot analysis. Three patients were available for Western blot analysis. In the 3 patients, the pattern of reactivity of E-cadherin (Figure 4), hepatocyte N-related cadherin, and a-catenin was identical to that observed in the peritumoral liver tissue. Hepatocellular Carcinoma Immunohistochemical results. Four immunohistochemical patterns of expression of E-cadherin and hepatocyte N-related cadherin were observed in hepato-

cellular carcinomas (Table 1). In 26 patients (40%), neoplastic cells expressed both E-cadherin (Figure 5A) and the hepatocyte N-related cadherin (Figure 5B); the expression was heterogeneous in 14 patients for E-cadherin and in 11 patients for hepatocyte N-related cadherin. In 16 patients (24.6%), only E-cadherin was detectable (Figure 5C and D); seven tumors were uniformly positive, and nine were heterogeneously positive. In 10 patients (15.4%), only the hepatocyte N-related cadherin was detectable; expression was constantly heterogeneous. Finally, in 13 patients (20%), no cadherin expression could be shown on neoplastic cells by immunohistochemistry. The apparent level of expression of E-cadherin and hepatocyte N-related cadherin was frequently fainter on malignant cells than on normal hepatocytes present in the adjacent peritumoral tissue. At the cellular level, the distribution of cadherins over the surface of neoplastic cells varied with the degree of histological differentiation. In well-differentiated, trabecular cases, the distribution of cadherins was preferentially restricted to the lateral membranes (Figure 5A and B). In poorly differentiated cases, the cellular distribution of cadherins was frequently abnormal. Cadherins could be detected not only along the lateral membranes of neoplastic cells but also along the part of the plasma membrane directly facing the tumor stroma (Figure 5D). The expression and distribution of a-catenin usually closely correlated with that of E-cadherin. a-Catenin was strongly detectable in neoplastic cells showing high levels of E-cadherin expression (Figure 5E) but was faint or absent in cells with reduced or undetectable levels of Ecadherin. In many cases, the apparent level of detection of a-catenin in the neoplastic tissue was greater than in the peritumoral liver. Western blot analysis. Thirty-five patients were available for Western blot analysis (Table 2). The following patterns of reactivity were observed for E-cadherin and hepatocyte N-related cadherin (Figure 6): (1) pattern identical to that observed in the peritumoral liver tissue, this pattern was observed in 20 patients for E-cadherin and in 18 patients for hepatocyte N-related cadherin; (2) presence of additional bands undetectable in the normal

䉳 Figure 1. Expression of cadherins and a-catenin in the normal human liver tissue. (A ) E-cadherin shown by the monoclonal antibody HECD-1 is detected along the lateral membranes of hepatocytes (arrows) and of bile duct cells (arrowheads). (B ) The GC4-reactive cadherin is detected along the lateral membranes of hepatocytes (arrows) but is absent from adjacent bile duct cells (B). (C ) a-Catenin is detected both in hepatocytes (arrows) and in bile duct cells (arrowheads), but its apparent level of expression is greater in biliary epithelial cells than in hepatocytes. (D ) By ultrastructural immunohistochemistry, the restricted distribution of hepatocyte N-related cadherin, as detected by the GC4 antibody, to the lateral membrane (arrow) of hepatocytes (H ) is confirmed. No labeling is visible along the sinusoidal (arrowheads) and the canalicular (open arrow) domains of the plasma membrane (E, sinusoidal endothelium; C, bile canaliculus). (A–C ) Indirect immunoperoxidase with light nuclear counterstaining. (D ) Electron-microscopic examination and indirect immunoperoxidase without further staining (bar Å 1 mm) (original magnifications: A, 2301; B, 3101; C, 1801; and D, 11,0001).

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Figure 4. Western blotting characterization of E-cadherin in benign epithelial tumors of the liver. Seven representative samples from different patients with focal nodular hyperplasia (FNH) (lanes 1–7) and the three samples of liver cell adenoma (LCA) tested (lanes 1–3) are shown and compared with a sample from normal liver tissue (N). In most patients, E-cadherin is detected as a main band with an apparent molecular weight of 124 kilodaltons. In 1 patient with focal nodular hyperplasia (lane 4), an additional band of 95 kilodaltons is present. Note the faint and heterogeneous reactivity of samples from liver cell adenoma compared with samples from normal liver tissue and from focal nodular hyperplasia.

liver tissue, this pattern was observed in 8 patients for E-cadherin and in 9 patients for hepatocyte N-related cadherin; and (3) absence of detection, this was observed in 7 patients for E-cadherin and in 8 patients for hepatocyte N-related cadherin. The distribution of these various patterns according to the grade of differentiation is given in Table 2. Ecadherin was detected by Western blotting in 2 patients negative by immunohistochemistry. Hepatocyte N-related cadherin was detected by Western blotting in 3 patients negative by immunohistochemistry. In all those patients, abnormal bands of low molecular weights were present. a-Catenin was detected in 27 of the 35 patients tested. It was usually detected as a unique band of 102 kilodaltons, similar to that observed in the normal liver. In well-differentiated hepatocellular carcinomas, the apparent intensity of the detection of a-catenin was usually greater in neoplastic tissue than in the peritumoral liver tissue (Figure 7). Correlations between cadherin expression and clinicopathologic parameters. We tested the relation-

ships between the pattern of cadherin expression and the following clinicopathologic parameters: tumor size, grade of histological differentiation, pattern of growth, capsular effraction, presence of satellite nodules, and vascular invasion (Table 1).

In our study group, 100% of the tumors measuring õ3 cm in diameter coexpressed E-cadherin and hepatocyte N-related cadherin compared with only 27% of tumors of ú3 cm in diameter (Table 1). The difference was statistically significant (x2 test; P õ 0.005). No statistically significant difference was observed in the expression of E-cadherin and hepatocyte N-related cadherin between tumors of õ5 cm or ú5 cm. The immunohistochemical pattern of expression of Ecadherin and hepatocyte N-related cadherin was closely correlated with the grade of histological differentiation (x2 test; P õ 0.001). The percentage of patients expressing the two cadherins was as follows, respectively (Table 1): 100% in grade I tumors, 61% in grade II tumors, 18% in grade III tumors, and 0% in grade IV tumors. The percentage of patients expressing only one of the two cadherins showed by normal hepatocytes was as follows: 0% in grade I tumors, 32% in grade II tumors, 50% in grade III tumors, and 60% in grade IV tumors. Finally, the percentage of patients lacking the two cadherins expressed by normal hepatocytes was as follows: 0% in grade I tumors, 8% in grade II tumors, 32% in grade III tumors, and 40% in grade IV tumors. We also tested individually the correlations between the expression of each cadherin and the histological grade of differentiation. The expressions of both E-cadherin and hepatocyte N-related cadherin were individually correlated with Edmonson’s grade (x2 test; P Å 0.015 and P õ 0.005, respectively). No correlation between the Western blotting pattern of cadherins and the histological grade of differentiation was observed. In particular, the existence of abnormal, supplementary bands was not statistically correlated with Edmonson’s grade. No statistically significant difference was observed in the patterns of expression of E-cadherin and hepatocyte N-related cadherin between infiltrative or encapsulated tumors (Table 1). Among encapsulated tumors, a close correlation was observed between the immunohistochemical pattern of expression of E-cadherin and hepatocyte N-related cadherin and the existence of capsular invasion (x2 test; P õ 0.001). The percentage of patients expressing the two cadherins shown by normal hepatocytes was 100% in the 9 patients without evidence of capsular invasion and only 32.5% (14 of 43 cases) in the 43

䉳 Figure 3. Expression of cadherins and a-catenin in benign epithelial tumors of the liver. A and B show the pattern of reactivity of E-cadherin and a-catenin in a patient with focal nodular hyperplasia. (A ) E-cadherin is detected in the hepatocytes of the hyperplastic parenchyma (arrows) as well as in the bile duct cells lining the ductular structures scattered in the central fibrous scar (arrowheads). (B ) As in the normal liver, the apparent expression level of a-catenin is lower in hepatocytes (arrows) than in bile duct cells (arrowhead). C and D show the pattern of reactivity of E-cadherin in 2 patients with liver cell adenoma. E-cadherin is constantly expressed, but its apparent level of expression is either (C ) homogeneous or (D ) heterogeneous within a given tumor. Indirect immunoperoxidase with light nuclear counterstaining (original magnifications: A, 751; B, 2101; C, 951; and D, 2501).

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Table 1. Correlations Between the Immunohistochemical Pattern of Expression of Cadherins and the Clinicopathologic Parameters in 65 Patients With Hepatocellular Carcinoma

Tumor size

Histological grade of differentiation

õ3 cm ú3 cm I Coexpression of E-cadherin and hepatocyte N-related cadherin (n Å 26) Expression of E-cadherin alone (n Å 16) Expression of hepatocyte N-related cadherin alone (n Å 10) Absence of immunoreactive cadherins (n Å 13)

II

11

15

4 17

0

16

0

0

10

0

13

III

Pattern of growth

Capsular effraction

Satellite nodules

Vascular invasion

IV Infiltrative Encapsulated Absent Present Absent Present Absent Present

5

0

3

23

9

14

15

11

11

15

5

10

1

3

13

0

13

7

9

1

15

0

4

4

2

3

7

0

7

6

4

1

9

0

2

9

2

4

9

0

9

6

7

4

9

patients with evidence of capsular invasion. There was no statistically significant difference in the pattern of expression of cadherins between solitary tumors and tumors with satellite nodules. The number of tumors coexpressing E-cadherin and hepatocyte N-related cadherin was greater among tumors without evidence of vascular invasion (11 of 17 patients [71%]) than among tumors with vascular emboli (15 of 48 patients [38.5%]) (Table 1). The difference was statistically significant (x2 test; P Å 0.032). Cholangiocarcinoma Immunohistochemical results. E-cadherin was expressed in 7 of the 9 patients examined (Figure 5F). The labeling was restricted to the lateral membranes of neoplastic cells (Figure 5F). Two patients presented no detectable expression of E-cadherin. No expression of the hepatocyte N-related cadherin was detected in any patient. a-catenin was faintly detected in the majority of neoplastic cells in all the patients examined. Western blot analysis. Three patients were available for Western blot analysis. In accordance with the corresponding immunohistochemical results, only Ecadherin and a-catenin were detected. E-cadherin was observed in the form of a unique band of 127 kilodaltons

and a-catenin as a unique band of 102 kilodaltons. No qualitative alteration was observed.

Discussion Our study shows that, in the normal state, hepatocytes and bile duct cells express different combinations of cadherins. In contrast to bile duct cells, which express only E-cadherin, hepatocytes express at least two different members of the cadherin superfamily: E-cadherin and a 129-kilodalton cadherin identified by the monoclonal antibody GC4 directed against N-cadherin. However, the pattern of electrophoretic migration of the GC4reactive molecule in the liver is different from that observed in other tissues containing typical N-cadherin, such as the brain or the heart. Therefore, future work is necessary to determine whether the GC4-reactive molecule identified in the human liver is a tissue-specific variant of N-cadherin or the product of a different but closely related gene. Pending its full characterization, we therefore have referred to this molecule as hepatocyte N-related cadherin. It is interesting to note that the coexpression of at least two distinct cadherins by hepatocytes have been shown previously in various animal species. In the mouse liver as in the human liver, E-cadherin is coexpressed with an N-cadherin analogue.50 In the

䉴 Figure 5. Expression of cadherins and a-catenin in malignant epithelial tumors of the liver. A–E show the pattern of reactivity of cadherins and a-catenin in hepatocellular carcinomas of various grades of differentiation. (A and B ) In a well-differentiated hepatocellular carcinoma (grade II of the Edmonson’s classification), both (A ) E-cadherin and (B ) hepatocyte N-related cadherin are strongly and homogeneously detected. (C and D ) In a poorly differentiated hepatocellular carcinoma (grade III of the Edmonson’s classification), only (D ) E-cadherin is strongly detected on neoplastic cells, whereas only a few neoplastic cells (arrows) express detectable levels of (C ) hepatocyte N-related cadherin. (D ) Note that, in this patient, E-cadherin is detected not only along the lateral membranes of tumor cells (arrows) but also along the part of the plasma membrane facing the tumor stroma (arrowheads). (E ) The expression of a-catenin in a patient with well-differentiated hepatocellular carcinoma (grade II of the Edmonson’s classification) is shown. (F ) The expression of E-cadherin by neoplastic cells in a patient with cholangiocarcinoma characterized by the presence of tubular formations scattered within an abundant stroma is shown. Indirect immunoperoxidase with light nuclear counterstaining (original magnifications: A, 801; B, 851; C, 1701; D, 2101; E, 1301; and F, 1801).

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Table 2. Distribution of Western Blotting Patterns for ECadherin and Hepatocyte N-Related Cadherin According to the Grade of Histological Differentiation in Hepatocellular Carcinomas Edmonson’s grade

Normal pattern E-cadherin Hepatocyte N-related cadherin Existence of additional bands E-cadherin Hepatocyte N-related cadherin Absence of detection E-cadherin Hepatocyte N-related cadherin

I

II

III

IV

2 2

11 8

5 4

2 4

1 1

2 5

3 3

2 0

0 0

2 2

5 6

0 0

Figure 7. Western blotting characterization a-catenin (cat) in hepatocellular carcinoma. The detection of a-catenin in two samples of welldifferentiated hepatocellular carcinoma (grade II of the Edmonson’s classification) (T) is compared with that observed in the samples from the corresponding peritumoral tissue (N). No difference is observed in the apparent molecular weight, but the apparent level of detection is much greater in the tumor (T) than in the peritumoral tissue (N).

NOTE. n Å 35 patients.

rat liver, E-cadherin is coexpressed with LI-cadherin, a nonclassical cadherin specific for hepatocytes and intestinal epithelial cells.51 In the chicken liver, the avian homologue of E-cadherin L-CAM is coexpressed with Bcadherin,52 the product of the K-CAM gene.53 Differences in tissue distributions and electrophoretic patterns suggest that rat LI-cadherin, chicken B-cadherin, and human and mouse hepatocyte N-related cadherins are different molecules. This implies important species-specific differences in the pattern of expression of cadherins by hepatocytes. The differences in cadherin expression observed between hepatocytes and bile duct cells in the normal human liver tissue are associated with differences in the apparent level of expression of the epithelial form of acatenin. We detected a higher expression level of acatenin in bile duct cells than in hepatocytes. Because a-catenin is preferentially concentrated in adherens junctions,54 this difference may be related to the low develop-

Figure 6. Western blotting characterization of E-cadherin (E) and hepatocyte N-related cadherin (N) in seven representative samples of hepatocellular carcinoma (lanes 1–7) compared with a sample from peritumoral liver tissue (NL). The following various patterns of reactivity are shown: pattern comparable with that observed in the normal liver tissue (lanes 4–7 for E-cadherin and lanes 1–5 and 7 for hepatocyte N-related cadherin); presence of additional bands of higher (lanes 2 and 3 for E-cadherin and lane 7 for hepatocyte N-related cadherin) and lower (lane 4 for hepatocyte N-related cadherin) apparent molecular weight; and absence of detection (lane 1 for E-cadherin and lane 6 for hepatocyte N-related cadherin).

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ment of adherens junctions in hepatocytes compared with bile duct cells, as suggested by previous ultrastructural55 and cell fractionation56 studies. The two types of benign liver epithelial tumors included in our study, focal nodular hyperplasia and liver cell adenoma, retain most of the characteristics shown by normal hepatocytes and bile duct cells. Cells of the hepatocyte lineage constantly coexpress E-cadherin and hepatocyte N-related cadherin and present a low expression level of a-catenin. In contrast, cells of the bile duct lineage, such as those present in the central scar of focal nodular hyperplasia, express only E-cadherin and show high levels of a-catenin. However, in liver cell adenoma, alterations in the pattern of cadherin expression could be observed. In this type of tumor, the apparent expression levels of both E-cadherin and hepatocyte N-related cadherin usually presented a high degree of heterogeneity from one cell to another. The occurrence of quantitative and qualitative alterations in the pattern of cadherin expression has been shown previously in other examples of benign epithelial tumors, such as colic adenomas46 or breast hyperplasias,5 and has been considered as a further sign of the precancerous nature of these lesions. This interpretation is unlikely for benign epithelial tumors of the liver because malignant transformation is an exceptional event in the course of liver cell adenomas.34,35,57 An alternative hypothesis is that the alterations in the pattern of cadherin expression observed in benign liver cell adenomas are related to the increased proliferative activity characteristic of these lesions.35 The low expression level of cadherins observed in liver cell adenomas may indicate a reduced level of cell-cell adhesion, which is known to be an essential prerequisite to the initiation of cell proliferation. Therefore, our findings raise the possibility that the alterations of cadherin expression observed in epithelial tumors may be, at least in part, nonWBS-Gastro

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specific changes associated with increased cell proliferation. In the two main types of malignant tumors of the liver, hepatocellular carcinomas and cholangiocarcinomas, neoplastic epithelial cells retain some of the characteristics of their normal counterparts. In particular, the expression of the hepatocyte N-related cadherin, absent from normal bile duct cells, was detected only in hepatocellular carcinomas and not in cholangiocarcinomas. This suggests that, like integrins,58 cadherins behave as cell lineage markers and may prove to be useful for the diagnosis of the various types of liver carcinomas and for the differential diagnosis of liver metastases. In contrast to previous studies5,31 based on a limited number of patients, we observed a high incidence of quantitative and qualitative alterations of cadherin expression in hepatocellular carcinomas. This is in line with the demonstration of a frequent loss of heterozygosity in the region of chromosome 16 (16q22.1 to 23.2) known to contain the E-cadherin gene.37 – 39 In our series, alterations in cadherin expression in hepatocellular carcinomas consisted of the following: (1) decreased or absent expression of one or the two cadherins expressed by normal hepatocytes; (2) abnormal distribution at the surface of neoplastic cells, including the expression along the basal domain of the plasma membrane; and (3) occurrence of abnormal isoforms detected by Western blotting analysis. The significance of such abnormal isoforms, previously described in other types of carcinomas, is likely to be variable. Lowmolecular-weight bands may correspond to products of proteolytic cleavage, whereas high-molecular-weight bands probably correspond to intracellular precursors of mature molecules. As reported previously, in most other types of carcinomas,5,6,32,59,60 and in particular in those of the breast,47,61–63 the prostate,64 and the digestive tract,24,31,65,66 we observed that the immunohistochemical pattern of cadherin expression in hepatocellular carcinoma is correlated with several clinicopathologic parameters of prognostic significance. In particular, we observed that the expression by neoplastic cells of the two cadherins present on normal hepatocytes is correlated with a tumor size of õ3 cm, a high degree of histological differentiation, and the absence of capsular effraction and vascular invasion. In several large previous series of hepatocellular carcinomas,67 – 72 all these parameters have been associated with a better prognosis. The absence of correlation observed in our study between the pattern of cadherin expression and the existence of a capsule is in line with the previous demonstration that capsule formation depends on the reaction of the adjacent liver tissue and is not related to specific properties of neoplastic cells.73 / 5e0b$$0005

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In our study, the expression level of epithelial a-catenin in liver carcinomas was usually closely correlated with that of E-cadherin. Moreover, the apparent level of expression of a-catenin in neoplastic hepatocytes was frequently greater than that observed in normal hepatocytes, as suggested by both immunohistochemical and Western blotting results. Our results contrast with observations performed in some other types of carcinomas, including those of the breast, the prostate, and the esophagus,22 – 26 in which the loss of expression of a-catenin is a frequent event that is likely to represent a main cause of decreased cell-cell adhesion in the neoplastic tissue. Therefore, our study suggests that the respective role of cadherins and catenins in carcinogenesis may vary according to the tissue of origin. In conclusion, alterations of cadherins and a-catenin expression may occur in both benign and malignant tumors of the liver. Among benign tumors, altered cadherin expression has been observed only in liver cell adenomas, most likely as a nonspecific change that is possibly related to increased cell proliferation. In hepatocellular carcinomas and cholangiocarcinomas, quantitative and qualitative abnormalities of cadherins and a-catenin expression are frequent. In hepatocellular carcinomas, the occurrence of altered cadherin expression correlates with clinicopathologic parameters of prognostic significance, including tumor size, degree of histological differentiation, and existence of capsular and/or vascular invasion.

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Received September 22, 1995. Accepted November 29, 1995. Address requests for reprints to: Jean-Yves Scoazec, M.D., Laboratoire de Biologie Cellulaire, INSERM Unite´ 327, Faculte´ de Me´decine Xavier Bichat, BP 416, 75870 Paris, Cedex 18, France. Fax: (33) 144-859-279. Supported in part by a grant from the Fonds de Recherche de la Socie´te´ Nationale Franc¸aise de Gastroente´rologie and by grant 93.02330.PF from the Consiglio Nazionale delle Ricerche (Italy). The collaboration between the Laboratoire de Biologie Cellulaire (Universite´ Paris, Paris, France) and the Istituto di Anatomia e Istologia Patologica (Universita` di Bologna, Italy) is supported by a joint grant from the Galileo program, held by the Ministe`re des Affaires Etrange`res (France) and the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (Italy).

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