Expression of CDX 2 and Villin in Gastric Cardiac Intestinal Metaplasia and the Relation with Gastric Cardiac Carcinogenesis

The gastric cardia is the most proximal part of the stomach adjoining the oesophagus. Interestly, over the past 50 years, adenocarcinoma at this region has increased remarkably due to the recognition that it is epidemiologically distinct from adenocarcinoma of the rest of the stomach (Botterweck et al., 2000; McColl, 2006). Adenocarcinoma of gastric cardia (GCA) tract is associated with several different underlying risk factors and the presence of intestinal metaplasia (IM) is an important one. Studies in the IM in gastric cardia are very important to identify the relationship between IM and GCA. Also very important in finding useful markers to help the earlier diagnosis of GCA (Palli et al., 1992). The caudal relate homologues genes 2 (CDX2) are important during the early differentiation and maintenance of intestinal epithelium but also necessary for the expression of numbers of intestine-specific genes. The CDX2 expression is physiological throughout the small and large intestine, with the proximal limit occurring at the gastroduodenal


Introduction
The gastric cardia is the most proximal part of the stomach adjoining the oesophagus.Interestly, over the past 50 years, adenocarcinoma at this region has increased remarkably due to the recognition that it is epidemiologically distinct from adenocarcinoma of the rest of the stomach (Botterweck et al., 2000;McColl, 2006).Adenocarcinoma of gastric cardia (GCA) tract is associated with several different underlying risk factors and the presence of intestinal metaplasia (IM) is an important one.Studies in the IM in gastric cardia are very important to identify the relationship between IM and GCA.Also very important in finding useful markers to help the earlier diagnosis of GCA (Palli et al., 1992).The caudal relate homologues genes 2 (CDX2) are important during the early differentiation and maintenance of intestinal epithelium but also necessary for the expression of numbers of intestine-specific genes.The CDX2 expression is physiological throughout the small and large intestine, with the proximal limit occurring at the gastroduodenal RESEARCH COMMUNICATION

Expression of CDX2 and Villin in Gastric Cardiac Intestinal Metaplasia and the Relation with Gastric Cardiac Carcinogenesis
Zhong-Yue Xiao 1 , Yi Ru 1 , Jiang-Tao Sun 1 , She-Gan Gao 1 , Yu-Feng Wang 1 , Li-Dong Wang 2 , Xiao-Shan Feng 1 * junction.Ectopic CDX2 expression is considered to involve in the development of intestinal metaplasia and play significant roles in oncogenesis in gastrointestinal tract and its expression has been demonstrated in intestinal metaplasia of the stomach, and Barrett's metaplasia in human beings.So, CDX2 has concerned recently as an early marker for IM epithelium (Eda et al., 2003;Guo et al., 2004;Tsukamoto et al., 2004;Shi et al., 2008).Villin is a tissue-specific actin-binding cytoskeletal protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells.Its expression tends to be correlated with the development of differentiated polarized cells that contain a brush border and microvillus inclusions and can be induced by long-term acid exposure as an end-differentiated marker of intestinal cells, Villin needs to be of a sufficient quantity to result in a mature brush border.Immunohistochemical analyses of gastric intestinal metaplasia and cancer revealed that villin and Cdx2 expression are tightly coupled.Both of them are demonstrated to be useful maker for the diagnosis of Barrett's esophagus and intestinal metastasis in stomach (Dudouet et al., 1987;Fitzgerald et al., 1997;Athman et al., 2002;Niwa et al., 2005;Shi et al., 2008;Yamamichi et al., 2009).
The objectives of this study were to determine whether CDX2 and Villin expression is associated with intestinal differentiation and to evaluate the clinic pathologic patterns of expression in different gastric cardiac lesions before GCA, include cardiac gastritis, dysplasia and adenocarcinoma.

Tumor samples
A total of 143 gastric cardiac biopsy or resection specimens was selected from high risk section of GCA of HeNan province, China.The mean patient age was 54.9 years (minimum, 31; maximum, 75; standard deviation, 7.94 years), 26 were female and 117 were male.Clinical data and pathologic slides were reviewed in all cases.These specimens including 25 cardiac gastritis specimens with IM, 65 dysplasia specimens with IM and 35 gastric cardiac adenocarcinoma specimens and stained them for CDX2 and villin by Immunohistochemical SP method.15 normal gastric cardiac biopsy specimens were also collected as control.diagnoses were based on the histomorphology according to the criteria of the WHO classification of tumors 2000.

Statistical methods
All the data was analysed by Spass 17.0 statistical package.The χ 2 test or a Fisher exact test was used for comparison of the posi¬tive rates of CDX2 and Villin, and P values of less than 0.05 were regarded as significant.The relation between CDX2 and Villin expression and clinic pathology details was tested by χ 2 test or Fisher exact test, with additional use of the Spearman's rank correlation coefficient (rho) to analyse the correlation between CDX2 and Villin.

The Expression of CDX2 and Villin proteins in precancerous lesion with IM and GCA
The antigens were localized as follows: CDX2 was nuclear (Figure 1).Villin stained the brush border of the superficial IM epithelium and luminal borders of the glandular epithelium with or without cytoplasmic staining (Figure 2).There was no staining for CDX2 or Villin in the 15 control samples.Both cardiac gastritis with IM, dysplasia with IM, the positive rates of CDX2 protein were 84.0%(21/25), 66.7% (32/48) respectively.The positive rates are significant higher then that in the adenocarcinoma tissues 36.4% (20/55).While the positive rates of Villin protein In cardiac gastritis with IM and dysplasia with IM were 76.0% (19/25), 70.8% (34/48) respectively, and 45.5% (25/55) in adenocarcinoma tissues.Same as the result of CDX2, the positive rates are significant higher in precancerous lesion with IM then that in the adenocarcinoma tissues (P<0.01)(Table 1).

The relation between CDX2 and Villin expression and clinic pathology details
Either the expression of CDX2 or Villin was correlated with tumor differentiation (both P<0.05), but was not associated with age, sex, invasion and metastasis of lymph node (P>0.05).

Discussion
GCA have shown an increasing incidence in recent years, leading to increased interest in these tumors (Blot et al., 1991).It is recognised that GCA is epidemiologically distinct from adenocarcinoma of the rest of the stomach (Botterweck et al., 2000;McColl, 2006).JR Siewert (Siewert et al., 2006) also believed that GCA is histopathologic different with distal esophagus adenocarcinoma.Kenneth EL McColl consider that GCA probably arise by two different aetiological and pathogenic pathways.One resembles the cancer of the more distal stomach, due to the damage of H. pylori atrophic gastritis or more rarely autoimmune.The other resembles adenocarcinoma of distal esophagus and is likely to be due to the damage of short-segment gastroesophageal reflux.These two chronic damages contributes to the high incidence of IM and inflammation at the cardiac mucosa (Odze, 2005;McColl, 2006).Our previous research showed that the incidence of IM in tissue adjacent to gastric cardiac adenocarcinoma is much higher than gastric cardiac biopsy tissue from normal population (Gao et al., 2005).The result suggest that IM is an important precancerous lesion.Studies in IM at the cardiac mucosa were important to reveal the evolution of GCA and helpful to find useful markers for early diagnose of GCA.
The cadual homeobox genes 2 (CDX2) is a transcription factor involved in the early differentiation and maintenance of the intestinal epithelial cell during gastrointestinal development (Silberg et al., 2000).Ectopic CDX2 expression was believed related with intestinal metaplasia of the stomach, and Barrett's esophagus(BE) in human beings (Eda et al., 2003;Guo et al., 2004;Shi et al., 2008;Tsukamoto et al., 2004).Silberg DG found that the expression of CDX2 in transgenic mice can lead occurrence of IM in gastric mucosa (Silberg et al., 2002).Analogous results have been found in human beings too (Mesquita et al., 2003).Further study by Mutoh showed that ectopic CDX2 expression not only involved in the appearance of IM in gastric but also involved in the early in the progress of gastric cancer (Mutoh et al., 2004).There are also some reports suggested ectopic CDX2 expression was an important originate event for BE and the expression is involved in the evolution of esophagus adenocarcinoma (Villanacci et al., 2007).In this study, normal gastric cardiac mucosa presented no CDX2 expression.The expression of CDX2 was remarkableness in IM in gastric cardiac mucosa.But the positive rates was significant lower in GCA tissues than that in IM tissues.This suggest that ectopic CDX2 expression was involved in the occurrence of IM in gastric cardia and GCA.
Villin is an tissue-specific actin-binding cytoskeletal protein in the microvillus core of the brush border.As an end-differentiated marker of intestinal cells, its expression can be induced by long-term acid exposure and correlated with the development of differentiated polarized cells that contain a brush border and microvillus inclusions (Fitzgerald et al., 1997).The fre¬quency of villin expression was reported varies from 73% (22/30) to 100% (21/21) in BE and 85% (17/20) in gastric IM (Regaledo et al., 1998;Kerkhof et al., 2006;Zhao et al., 2009).We found villin expression to be present 53 cases in all 73 cases of gastric cardiac IM tissues.There are 25 cases present Villin expression in all 55 GCA tissues, but the positive rate is obviously lower than that in gastric cardiac IM.None of the gastric cardiac biopsy specimens from normal population showed labeling with villin.Besides luminal borders of glands containing goblet cells, villin was also expressed in the cytoplasm of cardiac gastritis and dysplasia tissues.This observation suggests that Villin play an important role in the procedure of gastric cardiac IM to GCA, meanwhile cells expressing villin in the cytoplasmic expression alone may indicate an earlier stage of intestinal differentiation.
Villin was thought to be a less specific marker of IM than CDX2 by some investigators.However, in our hands, the villin and CDX2 expression are tightly coupled.Similar result was reported in BE too (Shi et al., 2008).A recent study found that knockdown of CDX2 in SW480 cells caused a clear down regulation of villin.Indicated that villin is regulated by CDX2 (Yamamichi et al., 2009).Therefore, some investigators suggest to use both CDX2 and Villin as markers for the detection of early IM and supplements for the histologic diagnosis of BE.
The expression of CDX2 and Villin were also found to be correlated with tumor differentiation, and showed down positive rates accompany the differentiation becoming poorer.Whereas no correlate were significant with age, sex, invasion and metastasis of lymph node.Functional studies have already shown CDX2 could regulate many intestine-specific gene transcription in vivo.Such like intestinal-type alkaline phosphatase (ALP); the well characterized brushborder enzyme, sucrase-isomaltase (SI), human defensin-5 (HD), and mucus-secreting goblet cell-mucin marker (MUC2) (Eda et al., 2002;Guo et al., 2004;Weimann et al., 2010).These genes control the differentiation of the IM of gastrointestinal tract.And an in vitro study reported the rate of villin expression to be dramatically increased in the course of enterocyte dif¬ferentiation (Dudouet et al., 1987).Summarise these consequences we found that CDX2 and Villin play a key roles in the differentiation of GCA.In conclusion, Our result suggest that ectopic expression of CDX2 and Villin were involved in early-stage IM at gastric cardia and the expression of Villin was regulate by CDX2.The expression of CDX2 and Villin may play an important role in the procedure of GCA.

Figure 1 .Figure 2 .
Figure 1.The Expression of CDX2 Protins in Precancerous Lesion with IM and GCA. A. negative expression of CDX2 in normal cardiac mucosa (SP×200) B. positive expression of CDX2 in cardiac gastritis with IM (SP×400) C. positive expression of CDX2 in dysplasia with IM (SP×200) D. positive expression of CDX2 in GCA (SP×200)