We have shown that CDX2 protein is present in BO containing SIE, as was recently reported by others.26,27
CDX2 expression was also detected in oesophageal adenocarcinoma. In contrast, no CDX2 protein was seen in biopsies containing gastric type epithelium of the distal oesophagus taken from patients with BO, or in the squamous epithelium of patients with reflux oesophagitis without BO. However, low amounts of Cdx2 mRNA were detected in approximately one third of the squamous epithelium samples of patients with BO. The presence of Cdx2 mRNA also correlated with goblet cell specific Muc2 mRNA in BO samples (fig 3).
The homeobox protein CDX2 is involved in the differentiation and maintenance of intestinal epithelium.14
Expression of CDX2 is detected at the time of morphogenesis in the visceral endoderm of mouse intestine,28
and continues to be present throughout adulthood, but then is normally restricted to the intestine.29
It is detectable in the crypts of the intestine and in the villi,15
and is thought to be a key regulator of intestinal differentiation.19
Exogenous expression of CDX2 in IEC6 cells, an undifferentiated rat intestinal cell line that does not express CDX2, causes differentiation of IEC6 cells into goblet cells and absorptive enterocytes.14
Similar observations have been made in an animal model, in which ectopic expression of CDX2 induced the development of metaplastic changes of the gastric antrum, and in Helicobacter pylori
related intestinal metaplasia of the human stomach.30,31
These metaplastic changes of the mouse gastric antrum were also characterised by the development of goblet cells and absorptive enterocytes, and the expression of intestine specific proteins such as MUC2, alkaline phosphatase, villin, guanylyl cyclase C, and trefoil factor 3.21
In contrast, heterozygous CDX2 knockout mice developed polyp-like lesions in their colon during the first 3 months of life, which lacked CDX2 expression.32
These lesions were composed of heterotopic, well differentiated, stratified squamous epithelium, stomach, and small intestinal mucosa.33
It was concluded that CDX2 directs epithelial differentiation towards a caudal phenotype. For these reasons, CDX2 expression is thought to be an early marker of intestinal differentiation, and may therefore play a role in the development of SIE in the lower part of the oesophagus, as seen in BO.
“We hypothesise that inflammation in the oesophagus caused by duodeno–gastro–oesophageal reflux induces CDX2 expression in a subset of patients”
Although all additional biopsies taken from the BO segment for histological evaluation in the group of patients in whom Cdx2 mRNA was determined showed SIE, Cdx2 mRNA was not detected in six of 19 (32%) BO segments. In the biopsies taken from these segments, Muc2 transcription was absent, which suggests that goblet cells were not present in these samples. Because goblet cells are a hallmark of BO, these biopsies may have contained another type of columnar epithelium, probably gastric type epithelium, as was detected in 26 of 79 biopsies of the BO segment in our study. This is in agreement with findings in another study, which reported that goblet cells were only found in 51% of patients with 3–4 cm columnar-like epithelium of the oesophagus on a first endoscopy.34
This increased to 88.9% after three endoscopies.34
This suggests that the absence of SIE in the biopsies taken from the columnar lined segment might be the result of sampling error. Because in our study only one of the six patients negative for Cdx2 mRNA had an oesophageal adenocarcinoma, and no dysplasia was seen in the adjacent biopsies taken for routine screening in the other five patients, it is unlikely that a neoplasm that is associated with a decreased number of goblet cells was present in these biopsies.
Take home messages
- The expression of CDX2 protein and mRNA is strongly associated with the presence of specialised intestinal epithelium in the oesophagus
- Cdx2 mRNA was also present in endoscopically normal appearing squamous epithelium of one third of patients with Barrett’s oesophagus (BO) who have, and may precede the morphological changes seen in BO
- Pathways that induce Cdx2 transcription in squamous epithelial cells may be important in the development of BO
To assess whether CDX2 is an early marker for metaplastic replacement in the oesophagus, Cdx2 mRNA expression was also determined in reflux exposed squamous epithelium of patients with BO. Low amounts of Cdx2 mRNA were indeed seen in six of the 19 squamous epithelium samples tested (fig 3). In addition, transcription of Muc2 was not detected in these samples, which excludes the possibility that SIE was covered by a stratified epithelial layer. This indicates that healthy appearing squamous epithelium 5 cm above the squamo–columnar junction of the oesophagus in a subset of patients with columnar metaplasia of the distal oesophagus may already have undergone molecular changes, which may make them prone to the development of SIE, although this needs to be determined in a longitudinal follow up study of patients with reflux oesophagitis without BO. Patient to patient variation in the extent of reflux, the severity of inflammation, and the effect of the medication used, may explain why not all squamous epithelium samples of patients with BO contained detectable amounts of Cdx2 mRNA.
The development of BO is associated with the pathological reflux of acid35
Taken together with recent reports that CDX2 expression can be induced in keratinocytes by prolonged exposure to acid,37
Cdx2 transcription may be an early step in the metaplastic replacement of oesophageal squamous epithelium by SIE. We hypothesise that inflammation in the oesophagus caused by duodeno–gastro–oesophageal reflux induces CDX2 expression in a subset of patients. Pathways involved in de novo CDX2 expression in oesophageal squamous epithelium may be important for the development of BO. Elucidating these pathways may result in a greater understanding of why only a subset of patients with GORD develop BO.