Copyright ©The Histochemical Society, Inc.

An Immunohistochemical Study of the Expression of Adhesion Molecules in Gallbladder Lesions

Yoon-La Choi, Yan Hua Xuan, Young Kee Shin, Seoung Wan Chae, Myeong Cherl Kook, Ro Hyun Sung, Sei Jin Youn, Jae Woon Choi and Seok Hyung Kim

Department of Pathology (YHX,RHS,SHK), Chungbuk National University, College of Medicine, Chungbuk, Korea; Department of Pathology (Y-LC), Samsung Medical Center, Sungkyunkwan University, College of Medicine, Sungkyunkwan, Korea; Department of Pharmacy (YKS), Seoul National University, College of Pharmacy, Seoul, Korea; Department of Pathology (SWC), Sungkyunkwan University, College of Medicine, Sungkyunkwan, Korea; DiNonA, Inc. (YKS), Seoul, Korea; Department of Pathology (MCK), Seoul National University, College of Medicine, Seoul, Korea; Departments of Internal Medicine (SJY) and Surgery (JWC), Chungbuk National University, College of Medicine, and Chungbuk National University, Medical Research Institute (SHK), Chungbuk, Korea; and Department of Pathology (YHX), YanBian University, College of Medicine, YanBian, China

Correspondence to: Seok Hyung Kim, Dept. of Pathology, Chungbuk National University Hospital, 62 Kaesin-dong, Cheongju, Chungbuk, South Korea 361-763. E-mail: platoshkim{at}freechal.com


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 Materials and Methods
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 Literature Cited
 
We investigated the expression of 10 adhesion molecules ({alpha}-catenin, ß-catenin, {gamma}-catenin, CD44, CD44v6, ICAM-1, CD56, CEA, E-cadherin, and CD99) in 46 gallbladder carcinomas, 14 adenomas, 15 low-grade dysplasias, nine intestinal metaplasias, and 20 samples of normal gallbladder epithelium by immunohistochemistry. The expression of adhesion molecules was altered in gallbladder carcinomas and adenomas. In gallbladder carcinomas, increased expression of ICAM-1, CEA, and CD44v6 was observed, together with decreased expression of {alpha}/ß/{gamma}-catenin and CD99. In adenomas, aberrant expression of CD44v6 and CD56, as well as reduced expression of {alpha}/ß/{gamma}- and E-cadherins, was noted. Expression of {alpha}/ß/{gamma}-catenin was reduced in low-grade dysplasia, whereas there was no change in the expression of these adhesion molecules in metaplasia. Expression of ICAM-1, CD99, E-cadherin, and CD56 was correlated with clinical stage. In addition a correlation was noted between expression of ICAM-1 and E-cadherin and lymph node metastasis (p<0.05). These results suggest that altered expression of these adhesion molecules is involved in the progression and metastasis of gallbladder carcinomas.

(J Histochem Cytochem 52:591–601, 2004)

Key Words: gallbladder • catenin • CD44 • CD44v6 • ICAM-1 • CD56 • CEA • E-cadherin • CD99 • immunohistochemistry


    Introduction
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 Introduction
 Materials and Methods
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 Discussion
 Literature Cited
 
CARCINOMA of the gallbladder is a relatively uncommon and poorly understood but highly lethal malignancy that tends to present at an advanced stage. In Korea it is the most common malignancy in the biliary tract and the fifth most common malignancy in the gastrointestinal tract (Piehler and Crichlow 1978Go). Its incidence shows marked geographic variation. For example, it is the single largest cause of cancer death of women in Chile but accounts for less than 0.5% of cancers in women in the United States (Albores–Saavedra and Henson 2000Go).

Carcinogenesis and the formation of metastases are both multistep processes involving complex interactions between tumor cells and their environment (Liotta and Stracke 1988Go). Adhesion is a key component of the metastatic cascade, involving many interactions among tumor cells and between tumor cell and host cells in the processes of angiogenesis, proteolysis, motility, and invasion (Tang and Honn 1994–95Go).

These various adhesion processes are mediated by adhesion molecules belonging to four major families: integrins, cadherins, immunoglobulins, and selectins. Altered expression of adhesion molecules such as {alpha}/ß/{gamma}-catenin, CD44, ICAM-1, CD56, and E-cadherin has been implicated in various stages of tumor progression and metastasis (Tang and Honn 1994–95Go). A striking feature of metastatic cells is the considerable flexibility of their adhesive interactions with other cells and with components of the extracellular matrix (ECM). Of these adhesion molecules, expression of {alpha}/ß/{gamma}-catenins, CD44, ICAM-1, E-cadherin, and CEA has been studied intensively in carcinomas of the gastrointestinal tract, and their diagnostic usefulness has been evaluated (Van Aken et al. 2001Go). A correlation has been reported between the expression of adhesion molecules such as E-cadherin, {alpha}/ß/{gamma}-catenin, and ICAM-1 and the clinical stage or grade of cancers of the breast, stomach, and esophagus (Van Aken et al. 2001Go). However, there have been few such studies of gallbladder carcinomas.

We therefore investigated the expression of a number of cell adhesion molecules ({alpha}/ß/{gamma}-catenin, CD44, CD44v6, ICAM-1, CD56, E-cadherin, CEA, and CD99) in gallbladder carcinomas and in their precursor lesions.


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 Materials and Methods
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Patients, Tissue Samples, and Reagents
A total of 84 cases of gallbladder carcinoma and its related lesions, which consisted of 46 carcinomas, 14 adenomas, 15 low-grade dysplasias, and nine intestinal metaplasias, were retrospectively identified from the surgical pathology files of Chungbuk National University Hospital. Histological slides were reviewed to analyze the pathological parameters, including tumor size, histological grading, depth of invasion, and presence of nodal metastasis. The 46 gallbladder carcinomas (age 49–85 years; average age 66.1 years; 24 women and 22 men) comprised 10 early cases (pTis 6, pT1 4), and 36 advanced cases (pT2 20, pT3 11, pT4 5). Of these, 24 cases (52.2%) were classified as well-differentiated, 17 cases (40%) as moderately differentiated, and five cases (10.9%) as poorly differentiated adenocarcinomas. TNM staging according to the staging system of the American Joint Committee on Cancer (AJCC) was used (Greene et al. 2002Go).

All archival materials were routinely fixed in 10% neutral-buffered formalin and embedded in paraffin. Four-µm sections were prepared on silane-coated slides (Sigma; St Louis, MO).

The immunostaining kit and all antibodies were from DiNonA (Seoul, Korea) and are listed in Table 1.


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Table 1

Primary antibodies used in this study

 
Immunohistochemical Staining Procedure
Tissue sections on microslides were deparaffinized with xylene, hydrated in serially diluted alcohol, and the sections immersed in 3% H2O2 to quench endogenous peroxidase activity. The sections were then microwaved in 10 mM sodium citrate (pH 6.0) or in 1 mM EDTA (pH 8.0) for 15 min for antigen retrieval. After antigen retrieval, the avidin and biotin were applied consecutively to slides to eliminate endogenous biotin related background staining (Kim et al. 2002Go). The sections were then incubated with primary antibodies for 60 min and, after three successive rinsings with washing buffer, were further incubated with biotinylated goat anti-mouse Abs (DiNonA), for 20 min. After rinsing, the tissue sections were incubated with the HRP-conjugated streptavidin (DiNonA) for 20 min at room temperature. The slides were washed and the chromogen was developed for 5 min with liquid 3,3'-diaminobenzidine (DiNonA). The slides were then counterstained with Meyer's hematoxylin, dehydrated, and mounted with Canada balsam for examination. As a rinsing solution, distilled water with 0.1% Tween-20 was used (Kim et al. 2003Go).

Evaluation of Results of IHC Staining
The scoring method of Sinicrope et al. (1995)Go was applied to evaluate the IHC staining intensity and the proportion of stained epithelial cells. Membrane staining and nuclear staining were considered independently. The staining intensity was subclassified as 1, weak, 2, moderate, and 3, strong. The numbers of positive cells were expressed as the percentage of the total number of epithelial cells and assigned to one of five categories: 0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4, >75%. The percentage of positivity of tumor cells and the staining intensity were multiplied to produce an immunoreactive score (IS) for each tumor specimen. Each lesion was examined and scored separately by two pathologists, and cases with discrepant scores were discussed until unity was achieved.

For the proper comparison of expression, both the frequency of expression and the mean value of the IS were analyzed. For frequency of expression, different criteria for positivity were employed for each molecule owing to the great variation in the expression of each molecule in normal gallbladder epithelium, which ranges from 0% (CD44v6, ICAM-1, CD56) to 90% (ß/{gamma}-catenin, E-cadherin). In {alpha}/ß/{gamma}-catenin, CD44, and E-cadherin, we defined the cases in which tumor cells were positive in more than 50% of total tumor cells at moderate intensity as positivity. In contrast, in CD44v6, ICAM-1, CD56, CD99, and CEA, the cases with more than 5% positive cells were defined as positivity.

Statistical Analysis
Statistical analyses were performed using the Fisher's exact test, Pearson's {chi}2 test, Mann–Whitney test, Kruskal–Wallis test, and Tukey HSD, and the Duncan test as post hoc test. For comparison of means of immunoreactive scores, the Mann–Whitney test, Kruskal–Wallis test, Tukey HSD, and Duncan test were used. The association of the expression rate with clinicopathological factors was assessed by cross-tabulation, and the significance of differences was determined by the Fisher's exact test and Pearson's {chi}2 test. A p value less than 0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS software (SPSS; Chicago, IL).


    Results
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 Materials and Methods
 Results
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 Literature Cited
 
Expression of E-cadherin and {alpha}/ß/{gamma}-Catenins
A large number of the gallbladder lesions and normal epithelia had high levels of {alpha}-catenin, ß-catenin, {gamma}-catenin, and E-cadherin (Table 2). We noted diffuse and strong membrane staining for {alpha}/ß/{gamma}-catenins and E-cadherin in almost all the normal gallbladder mucosa and a significant reduction in the proportion of carcinomas, adenomas, and low-grade dysplasias expressing {alpha}/ß/{gamma}-catenin (p<0.05) (Table 2). E-cadherin was also less frequently expressed in adenomas (p<0.01) (Table 2). Nuclear staining for ß-catenin, in addition to membrane staining, was also observed in 3/46 carcinomas and 6/14 adenomas, and its nuclear expression was significantly higher in adenoma than in carcinoma (p<0.001). Furthermore, all nuclear ß-catenin expression in carcinoma was restricted to grade 1 (well-differentiated) carcinomas, whose morphology was very similar to that of adenomas except for invasion.


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Table 2

Frequency of membrane expression for 10 cell surface adhesion molecules in carcinoma, adenoma, low-grade dysplasia, metaplasia, and normal gallbladder epitheliuma

 
In addition, we found that changes in the frequency and intensity of expression of these molecules were correlated with clinicopathological parameters (Tables 3 and 4). E-cadherin expression was more prevalent in well-differentiated adenocarcinomas (p<0.05) (Table 3), and ß-catenin expression was more common in the younger group (p<0.05) (Table 3). Table 4 shows the association between clinicopathological parameters and IS of these molecules according to staining intensity and proportion of cells stained. The mean IS for E-cadherin was significantly higher in node-negative cases, at earlier stages (stage 0, I), and in well-differentiated adenocarcinomas (p<0.05) (Table 4). In addition, the average ß-catenin IS was higher in well-differentiated adenocarcinomas (p<0.05) (Table 4).


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Table 3

Clinicopathological features and frequency of membrane expression for 10 surface adhesion molecules in gallbladder carcinomaa

 

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Table 4

Clinicopathological features and mean of membrane immunoreactivity scores of 10 surface adhesion molecules in gallbladder carcinomaa

 
ICAM-1 Expression
In the normal gallbladder mucosa, ICAM-1 is expressed in vascular endothelial cells, lymphocytes, and fibroblasts in the stroma and is not expressed on epithelial cells. However, we did detect its expression in a proportion of the neoplastic glands of carcinomas (18/46; 31%) and adenomas (2/14; 14.3%) (Table 2; Figure 2G). Both the frequency and intensity of ICAM-1 expression, as assessed by the mean IS, were higher in carcinomas than in the other conditions (p<0.001) (Table 2; Figure 1) . In addition, the extent of expression in carcinomas was closely correlated with clinicopathological parameters. We detected expression more frequently in poorly differentiated carcinomas, node-positive cases, and in advanced stages (stage II, III, IV) (p<0.05) (Table 3). ICAM-1 expression was also more prevalent in female patients (p<0.05) (Table 3), and the intensity of immunostaining was higher in poorly differentiated carcinomas, node-positive cases, and female patients (p<0.05) (Table 4).



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Figure 2

Immunostaining of gallbladder carcinoma using antibodies that recognize each adhesion molecule. Tissues and antibodies used are indicated as follows: adenocarcinoma with anti-{alpha} catenin (A), adenocarcinoma with anti-ß-catenin (B), adenocarcinoma with anti-{gamma}-catenin (C), adenocarcinoma with anti-CD44 (D), adenocarcinoma with anti-CD44v6 (E), adenoma with anti-CD404v6 (F), adenocarcinoma with anti-ICAM-1 (G), adenocarcinoma with anti-CD56 (H), adenoma with anti-CD56 (I), adenocarcinoma with anti-CD99 (J), adenocarcinoma with anti-CEA (K), and adenocarcinoma with anti-E-cadherin (L). Prominent membrane staining is demonstrated with each antibody.

 


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Figure 1

Membrane expression of 10 adhesion molecules in gallbladder carcinoma and its related lesions. The mean of immunoreactivity scores for each adhesion molecule was demonstrated. CA, carcinoma; AD, adenoma; LD, low-grade dysplasia; MP, metaplasia; NL, normal.

 
Expression of CD44s and CD44v6
We noted some membrane staining of CD44s in gallbladder epithelia and infiltrating lymphocytes in the various pathological lesions. However, there was no significant difference in CD44 expression between the various lesions and normal epithelia (Table 2; Figure 1). In contrast, there was a dramatic difference in CD44v6 expression between the tumor groups (carcinoma and adenoma) and the non-tumor groups (normal, metaplasias, and low-grade dysplasias) (Table 2). CD44v6 was expressed at a significant level only in the neoplastic glands of carcinomas (12/46; 26.1%) and adenomas (6/14; 42.9%), and the difference between the expression in these tumor groups and in non-tumor groups was statistically significant in terms of both frequency and mean IS (p<0.05) (Table 2; Figures 2E and 2F) . However, CD44v6 expression in carcinomas did not show any correlation with any clinicopathological parameters.

CD56 and CD99 Expression
In normal gallbladder, CD56 is expressed in ganglion cells and infiltrating NK-cells as well as in a subset of activated T-lymphocytes but is not expressed in epithelial cells. However, a basolateral pattern of CD56 expression was observed in the epithelial cells of 10.9% and 50% of the carcinomas and adenomas, respectively (Table 2; Figures 2H and 2I). Both the frequency and the mean IS were significantly higher in adenomas (50%; 3.07 ± 3.56) than in the other conditions, including carcinomas (p<0.001) (Table 2; Figure 1). Expression correlated with clinical stage of the gallbladder carcinoma, being substantially more prevalent in the earlier stages (0, I) (p<0.05) (Table 3). However, there was no correlation between CD56 expression and the other clinicopathological parameters.

Relatively weak CD99 immunoreactivity was observed in the epithelial cells of almost every normal gallbladder, with strong membrane staining in infiltrating lymphocytes and capillary endothelial cells. CD99 expression was observed less frequently in carcinomas (p<0.01) (Table 2), and the extent of its expression was found to correlate with histological differentiation and clinical stage of the carcinomas (Tables 3 and 4). Both the frequency and the mean IS were lower in poorly differentiated (20% and 1 ± 2.24, respectively) than in well-differentiated carcinomas (83.3% and 3.92 ± 3.56, respectively) (p<0.01) (Tables 3 and 4). Similarly, expression of CD99 was downregulated in advanced-stage (II, III, IV) compared with early-stage carcinoma (0, I) in terms of both frequency and IS (Tables 3 and 4).

CEA Expression
We demonstrated apical membrane staining of CEA in most carcinomas (31/46; 68.9%), but expression was relatively rare in the other pathological entities and in normal gallbladder epithelia (Table 2; Figure 2K). Overexpression in carcinoma was statistically significant in terms of both frequency and intensity of immunostaining (p<0.001) (Table 2; Figure 1). Moreover, the increased expression of CEA in carcinoma was closely related to the extent of histological differentiation (Tables 3 and 4).


    Discussion
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 Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
In this study we examined the expression of 10 adhesion molecules in the various pathological entities of gallbladder (carcinoma, adenoma, low-grade dysplasia, and metaplasia) and the relation between the expression of these molecules and clinicopathological parameters. We detected considerable changes of expression in the various pathological lesions, as summarized in Table 5. In carcinoma, we noted reduced expression of {alpha}-catenin, ß-catenin, {gamma}-catenin, and CD99, as well as increased expression of CD44v6, ICAM-1 (CD54), and CEA (Table 5). In adenoma, expression of {alpha}-catenin, ß-catenin, {gamma}-catenin, and E-cadherin was reduced, whereas that of CD44v6 and CD56 was increased. Of these changes, the characteristic overexpression of CD56 in adenoma is unique. The expression of {alpha}/ß/{gamma}-catenins was significantly decreased in low-grade dysplasia, but we found no alteration in the expression of any of these molecules in metaplasia (Table 5).


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Table 5

Summary of IHC study of 10 adhesion molecules in gallbladder carcinoma and related precursor lesions

 
There were therefore many alterations in the expression of adhesion molecules in carcinoma and adenoma but only a few in low-grade dysplasia and metaplasia. The two neoplastic lesions, carcinoma and adenoma, shared some changes, such as those in {alpha}-catenin, ß-catenin, {gamma}-catenin, and CD44v6. However, the abnormal expression of CD56 and E-cadherin was unique to adenoma. Therefore, despite the similarity in expression pattern between carcinoma and adenoma, there are considerable differences. A possible explanation of these findings is that carcinomas are heterogeneous entities, some deriving from adenomas and others from dysplasias. To date, there has been no agreement about the origin of gallbladder carcinoma. A number of studies have shown that it is often preceded by dysplasia and carcinoma in situ (Albores–Saavedra et al. 1993Go), whereas adenoma has also been suggested to play an important role in its origin, primarily because remnants of adenomas have been detected in invasive carcinomas (Kozuka et al. 1982Go; Kijima et al. 1989Go). Our results suggest that at least some carcinomas originate from adenomas.

E-cadherin (120 kD) is a classical cadherin forming the key component of adherens junctions that provide lateral adhesion between epithelial cells. It is bound to the actin cytoskeleton by a series of underlying proteins, the catenins ({alpha}, ß, and {gamma}) (Shiozaki et al. 1996Go). The resulting E-cadherin/catenin complexes serve not only to maintain adherens junctions but also to transduce signals to the nucleus and the cytoskeleton, either directly or through connections with many other complexes. Changes in the membrane expression of E-cadherin/catenin ({alpha}, ß, and {gamma}) complexes have been implicated in several malignant neoplasms, e.g., breast carcinoma, gastric carcinoma, colorectal carcinoma, and cervical adenocarcinoma (Van Aken et al. 2001Go). However, there have been few studies of the expression of {alpha}/ß/{gamma}-catenins or E-cadherin in gallbladder carcinoma (Sasatomi et al. 1996Go; Yanagisawa et al. 2001bGo; Chang et al. 2002Go; Kohya et al. 2003Go). The pattern of membrane expression that we observed in adenoma and carcinoma is consistent with the finding of decreased expression of ß-catenin in moderately and poorly differentiated adenocarcinoma (Yanagisawa et al. 2001bGo). With respect to nuclear staining for ß-catenin, it was largely restricted to adenoma (6/14) and was rare in other conditions (carcinoma 3/46; low-grade dysplasia 0/15; metaplasia and normal 0/29). This pattern is also compatible with those observed in previous studies (Yanagisawa et al. 2001bGo; Chang et al. 2002Go). This result implies that ß-catenin-related molecular events may be implicated in the pathogenesis of adenoma in gallbladder, as is true for adenoma in colon. However, when we consider the rare nuclear ß-catenin expression in gallbladder carcinoma, which is clearly in contrast to colon cancer, it is possible that the oncogenesis of gallbladder carcinoma is different from that of colon carcinoma.

The relationship between E-cadherin expression and lymph node metastasis has been studied in other organs. However, conflicting results have been obtained. Reduced expression of E-cadherin was reported to correlate with lymph node metastasis in gastric cancer, breast cancer, and esophageal cancer, whereas in colorectal carcinoma {alpha}-catenin levels rather than those of E-cadherin were reported to correlate with lymph node metastasis (Oka et al. 1992Go,1993Go; Gofuku et al. 1999Go). However, we found that the correlation between lower expression of E-cadherin and lymph node metastasis in gallbladder carcinoma did not apply to the {alpha}/ß/{gamma}-catenins. Oka and co-workers (1992)Go(1993Go) have reported a correlation between lower E-cadherin expression and higher-grade breast and gastric carcinomas, consistent with our results. Interestingly, downregulation of E-cadherin expression has been shown to be involved in loss of cell-to-cell contact, which is a key initial step in tumor progression and the generation of metastatic variants (Tang and Honn 1994–95Go).

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a 90-kD cell surface-bound glycoprotein belonging to the immunoglobulin superfamily. It mediates adhesion-dependent cell–cell and cell–ECM interactions. Moreover, it plays an important role in cell adhesion and locomotion in inflammation and also in malignant disease (Dustin and Springer 1991Go). ICAM-1 is normally expressed by cytokine-activated endothelial cells but has also been detected in various malignant neoplasms such as pancreatic (Schwaeble et al. 1993Go), colon (Dippold et al. 1993Go), and gastric cancers (Nasu et al. 1997Go), and its levels of expression correlate positively with the metastatic potential of neoplastic cells (Nasu et al. 1997Go; Anastassiou et al. 2000Go). However, no investigation of ICAM-1 expression in gallbladder cancer has been published. Despite the difference in organs, the findings just referred to are consistent with our observation that expression of ICAM-1 is positively correlated with lymph node metastasis. Moreover, in our study ICAM-1 expression turned out to be significantly higher in advanced cases (stage III, IV) than in earlier cases (stage 0, I) (p<0.05).

The neural cell adhesion molecule (NCAM, CD56), a membrane-bound glycoprotein, functions in development and in contact-mediated interactions among neural cells. CD56 (NCAM) is present in neuroendocrine and neurectodermal cells and tumors. Nevertheless, it is not absolutely specific to neuroendocrine differentiation because expression has been found in a minority of breast cancers, non-endocrine lung cancers (Zoltowska et al. 2001Go), colon carcinomas (Roesler et al. 1997Go), and endometrial carcinomas (Arck et al. 2000Go). In this study we observed CD56 expression only in gallbladder carcinoma and adenoma, and the frequency of CD56 expression was particularly high in the latter. Such a high frequency of CD56 expression has not been documented in adenomas of other gastrointestinal organs, such as stomach and colon. The control of CD56 expression in gallbladder adenoma and its implications for progression of the tumor remain to be clarified. However, CD56 is reported to be an important adhesion molecule in the colon and to act as a tumor suppressor (Roesler et al. 1997Go), whereas a reduction in its expression has been shown to induce pancreatic tumor cell metastasis (Perl et al. 1999Go). CD56 may therefore play a central role in the cascade of tumor progression as a tumor suppressor.

CD44 molecules are adhesion molecules involved in cell-to-cell and cell-to-matrix interactions. They are a group of transmembrane glycoproteins encoded by a single CD44 gene located on human chromosome 11. CD44 protein exists in many isoforms generated by alternative splicing. A standard 90-kD form (CD44s) is present on cells of normal epithelial and hematopoietic origin. In contrast, the alternatively spliced CD44 variants, ranging in size from 120 to 150 kD, are expressed predominantly on cells and tumors of epithelial origin and are designated as CD44v (variant). CD44 variant expression is associated with tumor metastasis and disease progression in patients with several types of tumor, such as gastric (Saito et al. 1998Go), gallbladder (Yamaguchi et al. 2000Go; Yanagisawa et al. 2001aGo), and breast carcinoma (Berner et al. 2003Go).

In this study we observed CD44v6 expression only in neoplastic lesions, i.e., carcinoma and adenoma, and not in normal epithelial tissue, metaplasia, or low-grade dysplasia. This is consistent with other reports that have documented restriction of CD44v6 expression to neoplastic cells (Ishida 2000Go; Yanagisawa et al. 2001aGo). However, we failed to find any association with clinical or histological parameters of the tumors such as metastasis to lymph nodes, histological differentiation, or tumor stage. This appears to be contrary to other reports that demonstrate a correlation between CD44v6 expression in gallbladder carcinoma and extent of histological differentiation (Yanagisawa et al. 2001aGo). However, in the case of colorectal adenocarcinoma there is some disagreement regarding the clinical significance of CD44v6 expression (Ishida 2000Go). Several authors report an association of CD44v6 expression with advanced stage and poor prognosis in colorectal carcinoma (Mulder et al. 1997Go), whereas other investigators failed to find a correlation between expression and progression of colorectal carcinoma (Ishida 2000Go).

CEA has been reported to be present in both benign and malignant gallbladder epithelia (Dowaki et al. 2000Go; Kanthan et al. 2000Go). In the present study the expression of CEA was significantly increased in gallbladder carcinoma compared with the other lesions, consistent with these reports. CEA expression was correlated with histological differentiation (p<0.05) but not with clinical stage or lymph node metastasis, contrary to the findings of Dowaki et al. (2000)Go. However, there have been very few studies of the clinical significance of CEA expression in gallbladder carcinoma, and further study of this question is called for.

CD99 (Mic2) is a 32-kD transmembrane glycoprotein involved in cell-cell adhesion during hematopoietic cell differentiation (Hahn et al. 1997Go), apoptosis of immature thymocytes (Bernard et al. 1997Go), and transport of transmembrane proteins (Choi et al. 1998Go). It is expressed in most human tissues, especially in cortical thymocytes, pancreatic islet cells, and Leydig and Sertoli cells. It is also found in tumor cells, including Ewing's sarcoma/primitive neuroectodermal tumors (Folpe et al. 2000Go), lymphoblastic lymphoma/leukemia (Dworzak et al. 1999Go), some rhabdomyosarcomas (Folpe et al. 2002Go), some ovarian tumors (Choi et al. 2000Go), and in stomach cancer (Jung et al. 2002Go). Our results suggest that loss of CD99 expression is a common event in the evolution of gallbladder carcinomas. Downregulation of CD99 was significantly associated with poorly differentiated and advanced-stage tumor cells, in agreement with previous reports in gastric cancer (Jung et al. 2002Go). Loss of CD99 expression may alter the cytoskeletal system and the expression of other adhesion molecules and may thus lead to morphological and behavioral changes of the tumor cells. Kim et al. (2000)Go have reported that the loss of CD99 plays a critical role in the formation of Hodgkin's and Reed–Sternberg lesions. These results indicate that CD99 is related to gallbladder tumor progression.

In conclusion, we have first examined the expression of adhesion molecules such as {gamma}-catenin, ICAM-1, CD56, and CD99 expression in gallbladder tumors. There were extensive changes in the expression of various adhesion molecules in carcinoma and adenoma, some of which were related to clinicopathological parameters. Our results suggest that downregulation of {alpha}/ß/{gamma}-catenin is a relatively early event in tumor progression, whereas aberrant expression of ICAM-1 and downregulation of CD99 and E-cadherin occur later.

The expression of CD44v6 and CEA is relatively specific to neoplastic lesions, but its implications for tumor progression remain to be determined. Knowledge of the expression profile of adhesion molecules in gallbladder lesions obtained by IHC should be of considerable help in the diagnosis of neoplastic gallbladder lesions and the prediction of outcomes.


    Acknowledgments
 
We are very grateful to Kwang Ju Lee and Ji-Hye Yun for their technical assistance, including slide cutting and immunohistochemistry.


    Footnotes
 
Received for publication October 13, 2003; accepted January 22, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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