10.1245/s10434-006-9114-9
Annals of Surgical Oncology 14:94-99 (2007)
© 2007 Society of Surgical Oncology
Expression of Liver–Intestine Cadherin and Its Correlation with Lymph Node Metastasis in Gastric Cancer: Can It Predict N Stage Preoperatively?
Sung-Soo Park, MD1,
Sang-Hee Kang, MD1,
Joong-Min Park, MD1,
Jong-Han Kim, MD1,
Sang-Cheul Oh, MD2,
Ju-Han Lee, MD3,
Yang-Seok Chae, MD3,
Seung-Joo Kim, MD1,
Chong-Suk Kim, MD1 and
Young-Jae Mok, MD1
1 Department of Surgery, Korea University College of Medicine, Seoul, Korea
2 Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea
3 Department of Pathology, Korea University College of Medicine, Seoul, Korea
Correspondence: Address correspondence and reprint requests to: Young-Jae Mok, MD; E-mail: kugspss{at}kumc.or.kr
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ABSTRACT
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Background: Reliable method to predict lymph node metastasis is not yet available. In the present study, therefore, we examined LI-cadherin expression in human gastric cancer and attempted to find its relationship with clinicopathologic data, especially with lymph node metastasis. We also analyzed the expression in preoperative biopsy specimen to uncover its possibility of prognostication for lymph node metastasis.
Methods: The paired preoperative endoscopic biopsy and postoperative resected specimens from 208 patients who had surgically been treated for gastric cancer were retrospectively analyzed immunohistochemically for expression of LI-cadherin.
Results: There were 47 (22.6%) and 161 (77.4%) tumors which had positive and negative LI-cadherin expression, respectively. LI-cadherin expression was significantly correlated with tumor histology and lymph node metastasis: Furthermore, reduced expression of LI-cadherin was closely associated with tumor progression and lymph node metastasis in human gastric carcinoma. LI-cadherin expressions in both resected tumor and preoperative endoscopic tissues were found to be independent factors associated with lymph node metastasis.
Conclusions: There is a close association between reduced expression of LI-cadherin and lymph node metastasis in human gastric cancer. Immunohistochemical study of LI-cadherin is relatively simple compared to sentinel node navigation surgery, and it could be a practical prediction method for lymph node metastasis in patients with this malignancy.
Key Words: Gastric cancer • Liver–intestine cadherin • Lymph node metastasis • Immunohistochemisty
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INTRODUCTION
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Because of advances in diagnostic and operative technologies, including minimally invasive treatment such as endoscopic mucosal resection and laparoscopic surgery for gastric cancer, surgical treatment has changed to stage-oriented tailored therapy. In order to decide appropriate extent of surgery, it is essential to evaluate preoperative clinical stage, and depth of tumor invasion can be accessed preoperatively by using endoscopic ultrasonography and endoscopy.1,2 However, any reliable method for prediction of lymph node metastasis, another important determinant for prognosis, has not yet been found. Therefore, in order not to over- or under-treat patients, it is extremely essential to develop a method or find a biologic marker that can preoperatively predict lymph node metastasis and minimize recurrent potential of individual tumors after surgery.
Adhesion molecules are considered to be important factors during the process of invasion or metastasis. Cadherins represent a family of cell adhesion molecules which mediate Ca2+-dependent adhesion of adjacent cells in a homophilic manner,3,4 and distinct members of the cadherin superfamily have been shown to function as tumor invasion suppressors.5
Liver–intestine cadherin (LI-cadherin) represents a novel type of cadherin within the cadherin superfamily, and it is distinguished from other cadherins by structural and functional features. In mouse and human, LI-cadherin is selectively expressed on the basolateral surface of enterocytes and goblet cells in the small and large intestine, and it is capable of mediating Ca2+-dependent homophilic cell–cell adhesion independent of interactions with cytoskeleton.6 However, the adhesive property of LI-cadherin in cancer remains to be elucidated.
In this study, to evaluate LI-cadherin expression and its relationship with clinicopathologic data in gastric cancer, especially lymph node metastasis, we examined LI-cadherin expression in both preoperative endoscopic biopsy and postoperative resected tumor specimens by using polyclonal antibody.
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PATIENTS AND METHODS
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Patients and Specimens
The paired preoperative endoscopic biopsies and postoperative resected specimens were obtained from 208 patients who underwent surgical treatment for histologically proven gastric adenocarcinoma at the Department of Sugery, Korea University College of Medicine, from January 2001 to December 2002. None of the patients had received chemotherapy or radiation therapy prior to undergoing resection of the primary tumors. Metastatic lymph nodes were also harvested during gastrectomy. As many perigastric lymph nodes as possible were collected by means of a careful manual palpation, and all the specimens of 208 patients were found to have more than 15 lymph nodes for accurate N staging.7
Immediately after removal, the tissues were fixed in 10% buffered formalin and embedded in paraffin. A section from each specimen block was stained with hematoxylin and eosin for histological evaluation, and representative blocks were also used for immunohistochemical studies. The specimens were classified by the tumor-node-metastasis (TNM) classification, recommended by the International Union Against Cancer,8 and other clinicopathological parameters (tumor location, histological type, lymphatic, venous and neural invasion) were assessed according to the criteria proposed by the Japanese Research Society for Gastric Cancer.9
Immunohistochemistry
Paraffin-embedded 4 µ m-thick tissue sections were stained for the LI-cadherin by using primary rabbit polyclonal anti-LI-cadherin antibody (Santa Cruz Biotechnology, CA, USA). Thus, all sections were first deparaffinized by using a series of xylene baths and then rehydrated using a graded series of alcohol. To retrieve the antigenicity, the tissue sections were autoclaved once in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 min to block endogenous peroxidase activity and then incubated in 2.5% normal goat serum to reduce nonspecific finding. Sections were incubated with primary anti-LI-cadherin antiserum (1:100) for 30 min at room temperature. The sections were then processed by standard avidin–biotin immunohistochemical techniques according to the manufacturer’s recommendations (Dako, Denmark). Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used for counterstaining. Adjacent tissue sections with normal-appearing epithelium served as a positive internal control.
Evaluation of Immunohistochemical Staining
The LI-cadherin expression of the tumor cells was evaluated according to the proportion of positively stained cells. When more than 10% of the carcinoma cells were positively stained, the case was classified as ‘positive LI-cadherin expression’. On the other hand, the case was classified as ‘negative LI-cadherin expression’ when less than 10% of the tumor cells were positively stained. All the hematoxylin and eosin-stained and immunohistochemical slides were assessed by two independent observers (Chae YS and Lee JH) who had no prior knowledge on the clinicopathologic information of the patients.
Statistical Analysis
Statistical analyses were performed using the SPSS 11.0 for Windows (SPSS, USA). Continuous variables were expressed as means ± SD. LI-cadherin expression was assessed for associations with various clinicopathologic parameters using the following statistical tests: the Mann–Whitney test for age, maximum tumor size, and number of retrieved lymph nodes; and the chi-square test or Fisher’s exact test for the remaining parameters. Significant factors identified on univariate analysis were subjected to multivariate analysis using forward stepwise logistic regression. P value of less than .05 was considered statistically significant.
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RESULTS
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Expression of LI-Cadherin in Normal Stomach and Gastric Carcinomas
Expression of LI-cadherin was immunohistochemically examined in 208 patients with gastric carcinoma. In normal gastric epithelial cells, LI-cadherin immunoreactivity was present only at the portion of intestinal metaplasia (Fig. 1a
). In carcinoma tissues, however, LI-cadherin expression was detected in almost all the cancer cells of well-differentiated cancer nests with tight cell–cell adhesion (Fig. 1b), whereas the expression was detected only in some cells of infiltrative and poorly differentiated cancer nests (Fig. 1c).
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FIG. 1. Immunohistochemical expression of liver–intestine (LI) cadherin in normal gastric epithelium (a) and gastric carcinomas (b, c). LI-cadherin was present only in the portion of intestinal metaplasia of normal gastric epithelium (a). LI-cadherin was strongly expressed in well-differentiated tumor nests (b), but absent in poorly differentiated tumor nests (c) (original magnification 200x).
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Relationship Between LI-Cadherin Expression and Clinicopathological Features
According to our criteria described above, 47 (22.6%) of 208 cases were classified as having positive LI-cadherin expression, whereas the remaining 161 cases (77.4%) were classified as having negative LI-cadherin expression. As shown in Table 1, negative LI-cadherin expression was significantly correlated with gender (P = .025), tumor size (P = .003), histologic type (P < .001), T stage (P < .001), lymph node metastasis (P < .001), lymphatic (P = .023) and neural invasion (P = .001), and pTNM stage (P < .001) in univariate analysis. However, multivariate analysis revealed that histologic type (P = .002) and lymph node metastasis (P = .001) were statistically significant.
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TABLE 1. Relationship between clinicopathological features and liver–intestine (LI) cadherin expression in patients with gastric carcinoma
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Factors Associated with Lymph Node Metastasis
In univariate analysis, lymph node metastasis in gastric cancer was found to be related with maximum tumor size (P < .001), histologic type (P = .001), T stage (P < .001), number of retrieved nodes (P = .008), and lymphatic, venous and neural invasion (P < .001, = .001, < .001, respectively) (Table 2). Multivariate analysis using the logistic regression model was then performed to identify which of the factors significantly influenced lymph node metastasis. As seen in Table 3, maximum tumor size (P = .016), T stage (P = .004), and lymphatic (P < .001) and venous invasion (P = .006) were found to be independently correlated with lymph node metastasis. Moreover, LI-cadherin expressions in not only resected tumor (P = .048), but also in preoperative endoscopic tissues (P = .042), were shown to be independent factors associated with lymph node metastasis (Table 3).
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TABLE 2. Clinicopathological characteristics and the expression of LI-cadherin in relation to lymph node metastasis
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DISCUSSION
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In mouse, the novel cadherin is expressed solely in liver and intestine; therefore it has been designated as liver–intestine (LI) cadherin. Furthermore, the structure of LI-cadherin is different from those of the classical cadherins in that the extracellular domain of LI-cadherin consists of seven instead of five cadherin type repeats, and that the His-Ala-Val motif in the N-terminal domain is absent. The second major structural difference concerns the cytoplasmic portion of LI-cadherin: whereas the cytoplasmic tail is highly conserved among the classical cadherins and it consists of 150–160 amino acid residues, that of LI-cadherin has only 20 amino acid residues and it displays no significant homology to classical cadherins.10 Because of its relatively short cytoplasmic domain, LI-cadherin does not interact with the catenin network or with the actin cytoskeleton.11 However, LI-cadherin does mediate the Ca2+-dependent cell–cell adhesion in transfected Drosophila S2 cells.10
Human carcinomas generally show reduced cell–cell adhesiveness, thus resulting in destruction of the histological structure and allowing carcinoma cells to invade or metastasize. Indeed, cadherin-mediated cell adhesion has amply been demonstrated to play a critical role in the behavior of carcinoma cell.12
As for the cut-off value in evaluating the LI-cadherin expression, the significance of the adhesion molecules for invasion and metastasis of cancer tissue appears to depend on how little they are expressed. Therefore, we considered that pure negative for LI-cadherin was more important than the proportion of positively stained cells. Consequently, we determined the negative expression as being those tissues having less than 10% of stained cells.
The expression of LI-cadherin has been reported in gastric adenocarcinoma,13 ductal adenocarcinoma of pancreas,14 and colorectal carcinoma.15,16 All the investigators mentioned above observed that the LI-cadherin was expressed in the well-differentiated adenocarcinoma cells, but this expression was reduced in the dedifferentiated adenocarcinoma cells, in good agreement with our present results. It is of interest to note that the multivariate analysis revealed that the LI-cadherin expression was significant for only the histologic type and lymph node metastasis, but not for T stage. Therefore, the LI-cadherin expression appears to be a useful biological marker for lymph node metastasis.
Recent molecular analyses have clarified the relationship between lymph node metastasis and the biologic markers for gastric cancer, such as E-cadherin17,18 and the E-cadherin related molecular subjects, including catenins,19,20 p53,21 S100A4,22 and mucin (MUC1).23 Nevertheless, these molecular markers are hardly satisfactory to clinically apply, because one has to evaluate two or more of associated markers, and has to interpret the reciprocal interactions of expression.
In the present study, we have demonstrated that lymph node metastasis was closely associated with several proven clinicopathological features and the LI-cadherin expression on the multivariate analysis, and that the methods used were relatively simple. Furthermore, significantly reduced expressions in the tumor specimens obtained by endoscopic biopsy prior to surgical resection suggest that the LI-cadherin expression may serve as a prognostication for lymph node metastasis. We strongly believe that preoperative LI-cadherin evaluation could help the decision on the extent of lymphadenectomy for early gastric cancer; however, this should be validated for its accuracy through prospective clinical setting in advance, because false-negative information for N stage in preoperative period would rather give harm to patients.
In conclusion, reduced expression of LI-cadherin has been shown to be closely associated with tumor aggressiveness and lymph node metastasis of human gastric carcinoma. Although further studies are in need to verify the impact of LI-cadherin expression on human gastric carcinoma, its expression could help us determine the lymph node status of the patients suffering with this malignancy.
Received for publication May 25, 2006.
Accepted for publication May 25, 2006.
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ABSTRACT
INTRODUCTION
