Abstract Objective To investigate mucin expression of MUC2, the relationship between the typing of CDX2 in colorectal adenocarcinoma and its clinical pathological factors. The method applying immunohistochemical method in 30 cases of colorectal adenomas, 60 cases of colorectal adenocarcinoma and 30 cases of normal colorectal mucosa mucin MUC2, CDX2 detection. Results in normal colon mucosa, adenoma, adenocarcinoma, MUC2 positive rate were 100%, 93.3%, 58.3%; of CDX2 was 90.0%, 76.7% and 31.7%, respectively. MUC2 and CDX2 expression in colorectal adenocarcinoma the colorectal adenocarcinoma divided into four types: the expression of MUC2 + of CDX2 +, MUC2 + of CDX2-, expression of MUC2-of CDX2 + expression of MUC2-of CDX2 expression of MUC2 + of CDX2 + type and degree of differentiation, depth of invasion, Dukes related to staging and survival; expression of MUC2 + of CDX2-type and lymph node metastasis; MUC2-CDX2 + clinicopathological factors not related; expression of MUC2-of CDX2-type and degree of differentiation, depth of invasion, lymph node metastasis, Dukes stage, and the survival of a clear correlation. Conclusion MUC2, down-regulated expression of CDX2 may be involved in the occurrence of colorectal adenocarcinoma, expression of MUC2 + of CDX2 expression of MUC2-of CDX2-type colorectal adenocarcinoma tumorigenesis, development, invasion and metastasis, larger clinical prognosis .

Key words colorectal tumors; expression of MUC2; of CDX2; immunohistochemistry

Expression and Clinical Significance of MUC2, CDX2 in Colorectal Neoplasm

    YU Xiu  wen, WANG Xian  yan, SUN Yu  rong, XU Guang  you, ZHANG Xiao  jie

    Department of Pathology, Qiqihaer Medical College, Qiqihaer 161042, ChinaAbstract: Objective To investigate the relation between the classification of MUC2 and CDX2 in colorectal adenocarcinoma and its clinical pathological parameters.Methods The expression of MUC2 and CDX2 was detected by immunohistochemistry in 30 specimens of colorectal adenoma, 60 specimens of colorectal adenocarcinoma and 30 specimens of normal colorectal tissues. Results The positive rates of MUC2 in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma were 100%, 93.3%, 58.3% respectively. CDX2 were 90.0%, 76.7%, 31.7%. Colorectal adenocarcinoma could be classified into four phenotypes: MUC2 + CDX2 +, MUC2 + CDX2-, MUC2-CDX2 +, MUC2-CDX2-.MUC2 + CDX2 + showed significant correlation with differentiation, depth of invasion, Dukes staging and survival period. MUC2 + CDX2-showed significant correlation with lymph node metastasis. MUC2-CDX2 + showed no significance in relation to all the clinical pathological parameters. MUC2-CDX2-showed significant correlation with differentiation, depth of invasion, lymph node metastasis, Dukes staging and survival period. Conclusion The dawn  regulation of MUC2 and CDX2 may participate in the pathogenesis of colorectal adenocarcinoma. Classification of colorectal adenocarcinoma is relevant to the genesis, progression, invasion and metastasis of colorectal adenocarcinoma., which can be used to predict the patients’ prognosis.

    Key words: Colorectal neoplasm; MUC2; CDX2; Immunohistochemistry

 Colorectal cancer is the occurrence of malignant tumors from colorectal mucosal epithelial cells after gastric cancer and esophageal cancer, the incidence of gastrointestinal cancer in China and also one of the common malignant tumors. In recent years, the incidence of colorectal cancer presents a rising trend. The incidence of colorectal cancer is colorectal mucosa epithelium by a variety of genetic and environmental factors lead to changes in multiple genes, mucin (Mucin 2, MUC2) MUC2 and CDX2 tail type homeobox transcription factor  2 (Caudal  relatedhememe  odomorin  transcripoin, CDX2) gene expression abnormalities, changes in the quality and quantity of its product MUC2 mucin and CDX2 protein. By immunohistochemical methods we to sticky protein MUC2 and CDX2 expression study of 60 patients with colorectal adenocarcinoma explore it as a tumor marker for colorectal cancer diagnosis, classification, and prognostic value.

    1 Materials and methods

    1.1 specimen source

    60 cases of primary colorectal adenocarcinoma were randomly selected from the Heilongjiang Provincial Tumor Hospital from January 1996 to December 1997 surgical resection and confirmed by pathology, paraffin-embedded tissue specimens, all patients had complete follow-up data. Including 33 males and 27 females; age of onset is 20 to 75 years old, with a median age of 52.88 years; 19 cases which occurred colon and rectum 41 cases; according to the 1989 WHO tumor histological classification: 20 cases of well-differentiated, differentiated, 20 cases poorly differentiated 20 cases; organs other than 26 cases of serosal invasion and myometrial more than 34 cases, invading serosa and (or); lymph node metastasis and 23 cases without lymph node metastasis in 37 cases; Dukes stage A 19 cases, 13 cases of B, C of 23 cases, five cases; survived more than 5 years, 32 cases, less than 5 years in 28 cases. 30 cases in the control group over the same period for the same institution adenoma and colorectal adenocarcinoma far cut end of the normal tissue (from the cancer edge> 5cm, pathologically confirmed non-cancer tissue infiltration) of paraffin-embedded tissue specimens. The specimens were confirmed by HE staining microscope.

    1.2 Reagents

    Mouse anti-human MUC2 monoclonal antibody concentrate (clones Ccp58), mouse anti-human monoclonal antibody concentrate (CDX2 clones CDX2  88) and SP immunohistochemistry kit, DAB chromogenic kit are Beijing Zhongshan Biotechnology The company’s products.

    1.3 Methods

    In this study, using immunohistochemistry SP method (chain avidin – peroxidase). An anti-MUC2, working concentration of CDX2 are 1:50. All specimens were fixed in 10% neutral buffered formalin solution, conventional dehydration, paraffin-embedded, 4μm thick serial sections, HE staining and immunohistochemical staining. High temperature and pressure citrate antigen retrieval, DAB color, the rest of the steps by manual operation. Gastric positive sections as a positive control, PBS instead of primary antibody as negative control.

    1.4 Results decision

    All the slices in a blinded and reading independently by two pathologists expression of MUC2 protein is localized in the cytoplasm, CDX2 is localized in the nucleus, random observation 10 high power field, 100 cells per field count ratio, according to which positive cells and staining depth assessment, the last comprehensive assessment. (1) by positive cell ratio ratings: 0 is divided into the number of positive cells in the total number of cells (≤ 5%; 1 divided into the number of positive cells in the total cell count> 5%). (2) according to the dyeing depth Rating: 0 is divided into cells brown particles is consistent with the background; divided into cells appear brown or brown particles. (3) Total Points = ratio of positive cells ratings + dyed depth ratings: 0 to 1 is divided into negative (); 2 is divided into the positive (+)

    1.5 statistical methods

    Application SPSS13.0 statistical software for statistical analysis, the relationship between MUC2, CDX2 co-expression with clinicopathological factors χ2 test, P <0.05 was statistically significant.

    2 Results

    2.1 MUC2, CDX2 expression in normal colorectal mucosa, adenoma and adenocarcinoma

    MUC2 positive staining in the cytoplasm to the nucleus and nuclear envelope obvious, some intracytoplasmic was diffuse heterogeneity positive nucleus was not stained, as shown in Figure 1. MUC2 expression in normal colorectal mucosa, adenoma, adenocarcinoma rate were 100% (30/30), 93.3% (28/30), 58.3% (35/60) in normal colorectal mucosa gland by the chi-square test 0.05)。">cancer, between adenoma and adenocarcinoma were statistically significant (P <0.01), not statistically significant (P> 0.05) between the normal colorectal mucosa and adenoma. CDX2 positive staining in the nucleus, and the cytoplasm is not colored, as shown in Figure 2. CDX2 expression in normal colorectal mucosa, adenoma, adenocarcinoma rates were 90% (27/30), 76.7% (23/30), 31.7% (19/60) in normal colorectal mucosa gland by the chi-square test 0.05)。">cancer, between adenoma and adenocarcinoma were statistically significant (P <0.01), not statistically significant (P> 0.05) between the normal colorectal mucosa and adenoma. MUC2, CDX2 expression in normal colorectal mucosa, adenoma and adenocarcinoma are shown in Table 1.

    Expression of MUC2 2.2 of CDX2 joint expression of various types of colorectal adenocarcinoma and its clinical pathological factors Relations

    MUC2, CDX2 joint expression in colorectal adenocarcinoma, colorectal cancer is divided into four types: the expression of MUC2 + of CDX2 +, MUC2 + of CDX2-, expression of MUC2-of CDX2 + expression of MUC2-of CDX2 various types of colorectal adenocarcinoma and clinicopathological The relationship between the factors are shown in Table 2.

    As can be seen from Table 2, 60 patients with colorectal adenocarcinoma, MUC2 + CDX2 + type accounted for 17 cases, with the degree of differentiation, depth of invasion, Dukes stage has a significant correlation; expression of MUC2 + of CDX2-type accounted for 18 cases, the lymph node metastasis correlation; MUC2  CDX2 + 2 cases, no correlation with clinicopathological factors; expression of MUC2-of CDX2-type 23 cases, this type of colorectal adenocarcinoma and degree of differentiation, depth of invasion, lymph node metastasis, Dukes stage has a clear correlation with statistical significance by χ2 test.

    Expression of MUC2 2.3 of CDX2 joint expression of colorectal adenocarcinoma and its survival of the relational tables 1 expression of MUC2 expression of CDX2 in normal colorectal mucosa, adenoma and adenocarcinoma from Kaplan  Meier survival curve display, as shown in Figure 3, MUC2 + CDX2 +-type colorectal adenocarcinoma survival curves in the expression of MUC2 the + of CDX2-, MUC2-of CDX2-type top, with a statistically significant (Log rank = 9.857, P = 0.002). Description MUC2 + CDX2 +-type expression and prognosis was positively correlated with that expression of MUC2 + of CDX2 + expression rate, survival long period. The MUC2-CDX2-type survival curves are also located in the bottom of the other group, a statistically significant (Log  rank = 11.107, P = 0.001). Description the MUC2-CDX2-type expression and prognosis was negatively correlated, that is, the the MUC2-CDX2-type expression rate, short survival. While the expression of MUC2 + of CDX2-type, MUC2-CDX2 + type, respectively, compared with other types was not statistically significant (Log  rank = 0.215, P = 0.643; Log  rank = 1.242, P = 0.265).

    3 Discussion

    MUC2 intestinal type mucin [1], is the main component of the normal intestinal mucus, mainly in the peripheral part of the cytoplasm of goblet cells in intestinal mucosa, perinuclear and nuclear upper cytoplasmic mucus droplets compared to negative The positive staining of goblet cells in the shape of a “ring", and columnar cells and surface absorption cells were negative [2]. This study shows that, MUC2 expression in the cytoplasm of goblet cells in normal colorectal mucosa, the same as reported in the literature. In colorectal adenomas, MUC2 expression in the cytoplasm of the tumor cells, the perinuclear and nuclear upper obvious cytoplasm, showed diffuse expression in colorectal adenocarcinoma. The results of this study illustrate MUC2 expression site differences from normal colorectal mucosa the  colorectal adenoma the  colon adenocarcinoma development process. The reason for this difference may be due to the lesions of different, MUC2 appeared incomplete glycosylation the excessive glycosylation, so that the full exposure of the epitope of the antigen results. Under normal circumstances, MUC2 expression in intestinal mucosal epithelium, and decreased expression in colorectal adenomas and adenocarcinomas [3]. Ajioka Y et al [4] The results showed that the expression of MUC2 mucin expression in normal mucosa was 100%, lower expression rate in colorectal adenoma and severe atypical hyperplasia than mild dysplasia adenomas express low, but the MUC2 expression adenoma naked eye regardless of the type of expression of MUC2 in primary colorectal cancer positive expression rate was 45.2% [5], Huang Wenbin, etc. [6] reported a positive rate of 36.5%. This study shows that, MUC2 in normal colonic mucosa, colorectal adenomas, colorectal adenocarcinoma positive expression rate were 100%, 93.3%, 58.3%, was down-regulated expression trends between normal mucosa and adenoma no statistically significance. This may be due to the large table expression of MUC2 relationship of CDX2 co-expression of the various types of colorectal adenocarcinoma and clinicopathological factors P <0.01 intestinal adenoma number of cases is too small in cases needs to be further expanded in-depth study. Colorectal cancer in this process but from normal mucosa greatest expression of MUC2 decreased expression of this trend is there. MUC2 may participate in the occurrence of colorectal tumors and malignant transformation.

    CDX2 homeobox gene caudal family, is a intestinal type of differentiation markers, mainly expressed in intestinal epithelial cells, cells of the epithelial cells of the pancreas, and stomach and intestinal metaplasia of the esophagus, the normal esophageal, gastric mucosa and other normal the epithelium no expression [7]. Choi BJ [8] using immunohistochemical methods using tissue microarray analysis of 123 patients with sporadic colorectal cancer patients CDX2 expression, CDX2 expression in normal intestinal epithelial cells, colorectal adenocarcinoma, 29 (23.6% ) patients had positive expression, and the degree of differentiation of colorectal adenocarcinoma, clinical stage, lymph node metastasis. Kim GH [9] by real-time quantitative PCR method to detect CDX2 mRNA in normal colorectal mucosa, adenocarcinoma and its relationship with clinicopathological factors, CDX2 mRNA expression in normal colorectal mucosa in colorectal adenocarcinoma phase than statistically significant, and has nothing to do with various clinicopathological factors. The results of this study show that of CDX2 positive staining in the nucleus in normal colorectal mucosa, adenoma, adenocarcinoma expression rates were 90.0%, 76.7%, 31.7%, showing a trend of down-regulated expression, but in normal colorectal mucosa, adenoma Expression is not statistically significant, indicating that CDX2 involved in the occurrence of colorectal cancer, identify colorectal tumors benign and malignant very good one of the markers of Molecular Biology.

    MUC2 and CDX2 has obvious relevance [10], Yamamoto H, etc. [11] In order to study the relationship of MUC2 and CDX2, CDX2 gene into the intestinal epithelial cell lines by RT  PCR and EMSA method detected CDX2 could specifically MUC2 gene promoter binding, significantly increased the level of the expression of MUC2. In this paper, 60 cases of colorectal adenocarcinoma MUC2 and CDX2 joint detection, results show that the expression of MUC2 + of CDX2 + colorectal degree of differentiation, depth of invasion, the Dukes staging as well as the survival of a significant correlation, that is well differentiated, not invasion serosa, Dukes stage for the AB period, MUC2 and CDX2 protein expression levels. Kaplan  Meier survival curves of this type of colorectal cancer patients with favorable prognosis. Strong correlation was found between MUC2 + of CDX2-type and lymph node metastasis correlation description expression of MUC2 + of CDX2-may be involved in the metastasis of colorectal cancer. MUC2-CDX2 + type and clinicopathological factors not relevant description expression of MUC2-of CDX2 + behavior has nothing to do with the biology of colorectal cancer.

    MUC2-CDX2-type colorectal adenocarcinoma and degree of differentiation, depth of invasion, lymph node metastasis, Dukes stage has a clear correlation that is poorly differentiated, invading the serosa, Dukes stage is a CD of the survival of <5 years MUC2 and CDX2 protein expression level is low. Colorectal participate in the differentiation, invasion, metastasis, and Kaplan  Meier survival curves show that the disadvantage of this type of colon adenocarcinoma patients indicates a poor prognosis. Therefore, the expression of MUC2 mucin through joint detection of CDX2 expression and colorectal adenocarcinoma parting comprehensive analysis of various clinicopathological factors to estimate the prognosis of patients with a very important value.