Expression of Lamininγ2 in Extrahepatic Cholangiocarcinoma Tissues and its Influence on Tumor Invasion and Metastasis

Extrahepatic cholangiocarcinoma (EHCC), which is a kind of malignant tumor derived from the extrahepatic biliary epithelial cells with high malignant severity and recessive attack, is generally treated with surgeries (Pattanathien et al., 2013; Huai et al., 2014). However, the recurrence is extremely high due to the invasion and metastasis in the early stage of EHCC, which severely threatens patients’ prognosis (Yang et al., 2014). Laminin332, as a macromolecular glycoprotein, is widely distributed in epithelial cells. It plays an important biological function in the metastasis, proliferation, growth and differentiation of cells by combing with receptors to mediate the adhesion between cells and basilar membrane (Kamoshida, et al., 2014). During the developmental process of tumors, the interaction of tumor cells and basilar


Introduction
Extrahepatic cholangiocarcinoma (EHCC), which is a kind of malignant tumor derived from the extrahepatic biliary epithelial cells with high malignant severity and recessive attack, is generally treated with surgeries (Pattanathien et al., 2013;Huai et al., 2014). However, the recurrence is extremely high due to the invasion and metastasis in the early stage of EHCC, which severely threatens patients' prognosis (Yang et al., 2014).
Laminin332, as a macromolecular glycoprotein, is widely distributed in epithelial cells. It plays an important biological function in the metastasis, proliferation, growth and differentiation of cells by combing with receptors to mediate the adhesion between cells and basilar membrane (Kamoshida, et al., 2014). During the developmental process of tumors, the interaction of tumor cells and basilar membrane is the basis of the invasion and metastasis of tumors. Studies demonstrated that the abnormal distribution of Laminin332 might be in close association with the malignant transformation of epithelial cells as well as the invasion and metastasis of tumors (Kariya et al., 2012;Kim et al., 2012). Lamininγ2 chain, as a special part of Laminin332, is secreted in monomeric form, which has no expression in normal intrahepatic biliary epithelial cells and the basilar membrane of biliary epithelial tissues, but expresses highly in the cytoplasm of cholangiocarcinoma cells and extracellular matrix (ECM) (Liu et al., 2014).
To further definite the effect of Lamininγ2 in the development and progression of EHCC, this research analyzed the expression of Lamininγ2 and its relationship with clinical pathological characteristics and prognosis of EHCC patients, hoping to provide a theoretical basis for the predication of the invasion and metastasis of EHCC.

Methods
The expression of Lamininγ2 in the cancer tissues, para-cancer tissues, lymph nose metastatic tissues and hepatic metastatic tissues of EHCC was detected by immunohistochemical method. The detailed processes were as follows: the paraffin embedding samples were collected and cut into 4 μm slices, followed by routine dimethylbenzene deparaffinage, gradient alcohol hydration, sealing of endogenous peroxidase (EPOD) by 3% hydrogen peroxide solution and antibody repair with 10 mmol/L pH 6.0 citric acid buffer solution. After antibody repair, primary antibodies (mice anti-human Lamininγ2 monoclonal antibodies were purchased from American Novus company) were added and put into wet boxes to be incubated at 4℃overnight, after which they were brought out to be re-warmed for 30 min at room temperature, soaked for 3 times by phosphate buffer solution (PBS), 5 min/time. Horseradish peroxidase (HRP)-marked secondary antibody working solution (goat anti-mice HRP-marked secondary antibodies were bought from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) 30 μL was added, incubated in 37 ℃ dry box for 30 min, soaked by PBS for 3 times (5 min/time), colored with diaminobenzidine (DAB) (DAB staining kits were obtained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), washed by flow water, restained by hematoxylin, dehydrated, naturally dried and sealed with neutral resins. The known positive slices served as positive control and PBS as negative control instead of primary antibodies.

Observational indexes
The positive expression rates of Lamininγ2 in cancer tissues, para-cancer tissues, lymph nose metastatic tissues and hepatic metastatic tissues were observed and their relationships with clinical pathological characteristics and prognosis were analyzed.

Evaluation criteria
Two physicians in Department of Pathology, without being informed of the patients' clinical data, were invited to observe and diagnose all slices independently. The positive cells were defined as the brown granules in ECM and cytoplasm. Ten high-fold fields (×400) were randomly selected from each slice to calculate the total number of tumor cells and the positive cell count. The slice was considered to be positive if the positive expression of cells>5%, or be negative.
Statistical data analysis SAS 9.3 software package was applied for all data statistics. The measurement data were expressed by mean±standard deviation (X±S) and analyzed with independent-sample t test. Enumeration data were expressed by percentage (%), and comparison between two samples were analyzed by four-table χ 2 test while that between multiple samples by line-table χ 2 test. Kaplan-Meier survival curve was adopted for the analysis of survival time and Log-rank test was conducted. Cox risk regression model was applied for the multivariate analysis of factors influencing patients' prognosis. Two-sided test was used for all statistical analysis with α=0.05 as the inspection level.

Relevance between expression of Lamininγ2 and the clinical pathological characteristics of EHCC patients
Of the 79 EHCC patients, Lamininγ2 expression was in obvious association with lymph node metastasis (P<0.01), but had no relevance with ages, genders, tumor locations, tumor stages, differentiation severity and distant metastasis in ECM (P>0.05), whereas it was in marked connection with lymph node and distant metastasis in cytoplasm (P<0.05 or P<0.01), but had no relationship with ages, genders, tumor locations, tumor stages and differentiation (P>0.05) ( Table 1).

Influence of Lamininγ2 on prognosis of EHCC patients
The median survival time (MST) of patients with positive expression of Lamininγ2 in both cytoplasm and ECM of cancer cells, only in ECM and only in cytoplasm was 8.5, 9.0 and 8.7 months respectively, and the differences were significant when compared with that (22.0 months) of patients with negative expression in ECM and cytoplasm (P=0.0018). The median recurrent period of patients with positive Lamininγ2 expression in both cytoplasm and ECM of cancer cells, only in ECM and only in cytoplasm was 6.0, 7.0 and 4.0 months, and there were significant differences when compared with that (16.0 months) in patients with negative expression in ECM and cytoplasm (P=0.0001).
To further exclude other influencing factors, Cox regression analysis (involving ages, genders, tumor locations, etc.) was performed and it showed that the positive expression of Lamininγ2 and tumor differentiation severity were the independent risk factors influencing the prognosis of EHCC patients (Table 2).

Discussion
Laminin332, as one of the Laminin (LN) family, is the primary ingredient of basilar membrane, which mainly mediates the adhesion between epithelial cells and basilar membrane. A study indicated that Laminin332 did not express in normal mucous cells, but over-expressed in the  cytoplasm of primary andeocarcinoma cells (Imura et al., 2012), whose expression increase was in close correlation with the metastasis and proliferation of multiple tumor cells (Kinoshita et al., 2012;Fukuchi et al., 2013). Laminin332 is composed of three chains termed by α3, β3 and γ2, in which Lamininγ2, as the only polypeptide chain, is secreted in monomeric form. Some scholars believed that Lamininγ2 could trigger the functional loss of endothelial barrier by extracting vascular endothelial cells and increasing vascular permeability so as to increase the metastasis of tumor cells and improve their invasive growth (Sato et al., 2014). The research results of Degen et al illustrated that during the process of tumor developing into invasive squamouscell carcinoma, the expression of Lamininγ2 increased significantly (Degen et al., 2013). In this study, Lamininγ2, which expressed abnormally high in cancer tissues, had no expression in para-cancer tissues, and the further analysis of the relevance between the expression of Lamininγ2 and the clinical pathological characteristics demonstrated that the expression of Lamininγ2 was significantly connected with lymph node metastasis and distant metastasis in cytoplasm, and with lymph node metastasis in ECM, suggesting that Lamininγ2 expressed abnormally in cancer tissues, which was in obvious association with the invasion and metastasis of tumor cells.
The invasion and metastasis of tumors can directly influence the prognosis of patients and the over-expression of Lamininγ2 may lead to poorer prognosis of tumor patients. It has been reported that in patients with bladder carcinoma, the expression of Lamininγ2 is closely related with the tumor stages and differentiation severity, becoming an independent risk factor for the tumor progression (Kang et al., 2013); that in normal esophageal tissues, Lamininγ2 expresses in basilar membrane, which shows high expression in the oncocyte cytoplasm of esophageal squamous cell carcinoma tissues, and the expression condition is in close correlation with the survival time of patients with esophageal carcinoma in phase Ⅱ, and the higher the expression, the poorer the prognosis (Xue et al., 2011); that the survival time of patients with oral squamous cell carcinoma (OSCC) who are with positive expression of Lamininγ2 is significantly shorter than in patients with negative expression, indicating that Lamininγ2 may be a reliable predicative index for patients with OSCC (Gasparoni et al., 2007). This study, through Kaplan-Meier models, found that in EHCC patients, the survival time and recurrent period in patients with positive expression of Lamininγ2 were evidently lower than in those with negative expression, and further Cox model analysis showed that the positive expression of Lamininγ2 was an independent risk factor impacting the survival time and recurrence of EHCC patients, demonstrating that the positive expression of Lamininγ2 was a potential molecular biological marker for the prognostic diagnosis of cancer patients.
To sum, the invasion and metastasis of tumors as well as postoperative recurrence can all affect EHCC patients' prognosis. The results of this study suggested that Lamininγ2 could lead to the poor prognosis by participating the invasion and metastasis of tumor and influencing the tumor recurrence. However, the mechanism has not been deeply explored in this study, and how the Lamininγ2 improves the invasion and metastasis of tumors and impacts the postoperative recurrence of EHCC patients still need to be further studied.