Mucosal Patterns Under Magnification Chromoendoscopy, Mucin Gene Expression And Methylenetetrahydrofolate Reductase (MTHFR) Gene C677T And A1298C Polymorphisms In Barrett's Esophagus

AIM: (1) To screen the patients with Barrett's Esophagus, analyze their clinical, endoscopic and histopathological characteristics and evaluate the predictive value of Magnification Chromoendoscopy for the detection of Barrett's Esophagus. (2) To investigate the role of Helicobacter pylori in the development of Barrett's esophagus. (3) To assess mucin gene expression in Barrett's esophagus. (4) To study two MTHFR polymorphisms, C677T and A1298C in patients with Barrett's esophagus. METHODS: (1) 2,506 patients with gastroesophageal reflux disease (GERD) and 106 patients with Barrett's esophagus undergoing upper endoscopy in the period from February, 2003 to February , 2004 at the Southwest Hospital in Chongqing were studied. An Olympus magnification endoscope (GIF-Q240Z, 80x) was used to screen the patients with Barrett's esophagus. The distal oesophagus was sprayed with 3% Lugol's solution and 0.5% methylene blue and the esophageal columnar mucosa patterns were noted under high magnification and targeted for biopsy. All biopsy specimens were fixed in 10% buffered formalin and then Hematoxylin-eosin (HE) staining was used for the histopathological diagnosis. Traditional mucin histochemistry using the HID/AB (pH 2.5) and periodic acid Schiff/alcian blue (PAS/AB) methods was performed to identify the types of intestinal metaplasia. Some biopsy specimens from the patients with Barrett's esophagus whose histology showed a specialised intestinal epithelium were fixed in 2.5% glutaraldehyde for subsequent transmission electron microscopy (TEM) study. The remaining specimens were snap-frozen and stored at -80℃until assayed. The clinical manifestations of the patients were analyzed. The relationship between the endoscopic findings and the histopathological epithelial type of the patients with Barrett's esophagus was investigated. (2) The prevalence of Helicobacter pylori was determined in 106 Barrett's esophagus patients compared to a 50 control population of patients with GERD including 11 patients with reflux esophagitis and 50 normal controls. Tissues were taken from the distal esophagus and the gastric antrum of the patients. Helicobacter pylori colonization was determined upon the use of rapid urease test and Warthin-Starry staining of all esophageal and gastric biopsy specimens. (3) Mucin core protein-MUC1, MUC2, MUC3, MUC5AC and MUC6 was detected by immunohistochemistry. (4) DNA was isolated from the frozen tissue taken from the distal esophagus of the patients with Barrett's esophagus, GERD, reflux esophagitis and the controls. MTHFR gene C677T and A1298C polymorphisms were examined using a PCR-RFLP method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: (1) In the period from February of 2003 to February of 2004, a total of 15,377 patients were examined with upper endoscopy in our Institute. 2,506 patients were diagnosed to suffer from gastroesophageal reflux disease (GERD), 106 patients from Barrett's esophagus visible macroscopically and 36 patients from Barrett's esophagus proved specifically intestinal metaplasia(IM) histopathologically. The prevalence of Barrett's esophagus in patients who were examined with upper endoscopy was 0.69%, while the prevalence of IM was 0.23%. The prevalence of Barrett's esophagus was 3.15% in patients with GERD, while the prevalence of IM in GERD was 1.44%. The prevalence of Barrett's esophagus in GERD was higher than in general population (p<0.01). The clinical feature of 106 cases of Barrett's esophagus was gastroesophageal reflux which refers to the reflux of acidic fluid from the stomach into the esophagus, and is associated with heartburn, and pains behind the breastbone and obstructive sensation during swallowing. About 1/3 patients with Barrett's esophagus had no symptoms of GERD. Four patterns of columnar mucosa of the distal esophagus were revealed under magnification chromoendoscopy. They were dot pattern, tubular pattern, ridge or villous pattern and irregular/distorted pattern. The detected rate of IM in above mucosal patterns were 3.77%, 3.28%, 96.77% and 100% respectively. The staining of methylene blue was positive in 31 patients which were all proved IM histopathologically. The detected rate of IM in villous pattern and irregular patterns was significantly higher than that in dot and tubular patterns (p<0.01). AB/PAS and HID/AB stainings showed that all of specialized IM of Barrett's esophagus was typeⅡand some cases was with type Ⅲor with typeⅠtogether. Under transmission electron microscopy, an intermediate cell type between mucous cell and goblet cell was found in the surface of Barrett's specialized IM, and a special cell type of both squamous and columnarcell fetures was found under the surface of Barrett's specialized IM. (2) The prevalence of H. pylori colonization in the distal esophagus of controls, GERD, reflux esophagitis, Barrett's esophagus and Barrett's specialized IM were 10.00%, 20.00%, 18.18%, 15.09% and 16.67% respectively. The prevalence of H. pylori colonization in the gastric antrum of the above groups were 60.00%, 32.00%, 72.73%, 31.13% and 30.56% respectively. The positive rate of H. pylori colonization in the gastric antrum of GERD, Barrett's esophagus and Barrett's specialized IM was much lower than that of controls(P<0.01). The positive rate of H. pylori colonization in the gastric antrum with reflux esophagitis was significantly higher than that of the other three groups(P<0.05 or P<0.01). (3) Mild expression of MUC1 was found predominantly in the superficial epithelium of both gastric and specialised intestinal metaplasia. In a small number of specimens mild labelling of MUC1 was also noted in glands. Strong MUC2 labelling was noted only in the goblet cells in Barrett's oesophagus. MUC3 was expressed in the superficial columnar cells of specialised intestinal metaplasia with or without globlet cells but not in gastric metaplasia of the oesophagus. In some specimens MUC3 was expressed in the vacuolus of the globlet cells and the lumen of gland. Strong staining of MUC5AC was noted in the columnar epithelium of both gastric metaplasia and specialised IM in Barrett's oesophagus, and was expressed in the cytoplasm and vacuolus of the globlet cells in some specimens. Expression of MUC6 protein was detected at the base of the crypts in gastric metaplasia and specialised Barrett's glands. (4) We have explored a possible association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and Barrett's oesophagus and found that all Barrett's oesophagus patients had statistically significantly greater percentage of TT genotype than controls and Barrett's specialised IM had statistically significantly greater percentage of TT genotype than gastric metaplasia. Compared with MTHFR 677CC homozygotes, the TT genotype was associated with a 4.49-fold increased risk for Barrett's specialised IM. Analysis of A1298C polymorphism of MTHFR did not show any statistically significant differences in a distribution of MTHFR A1298C genotypes. CONCLUSIONS: (1) There are two criterions for the diagnosis of Barrett's esophagus. The first criterion is the finding at endoscopy of a pink lining mucosa in the esophagus where it normally is not seen. This abnormal lining can have the appearance of circumferential involvement or of tongues and/or islands of pink Barrett's-type mucosa. Thesecond criterion is that biopsies from the pink mucosa reveal the characteristic intestinalized mucosa with the typical goblet cells. (2) The general trend is for Barrett's patients to have more severe GERD. However, not all Barrett's have marked symptoms of GERD, and some patients are detected almost accidentally with minimal symptoms of GERD. Therefore, we have to pay attention to the endoscopic study of common population to screen the cases of Barrett's esophagus. (3) The combination of mythelene blue staining and magnification chromendoscopy is helpful to detect the IM in Barrett's esophagus and plays an important role in the diagnosis and surveillance of the cases of Barrett's esophagus. (4) The special cell type of both squamous and columnar cell fetures seen by TEM study may be the result of transformation of multipotential stem cells of squamous epithelial origin and the intermediate cell may represent an intermediate stage in the development of Barrett's specialized IM. (5) The role of Helicobacter pylori on GERD and Barrett's esophagus is unclear. On one hand, Helicobacter pylori may be protective against the formation of Barrett's esophagus and GERD. On the other hand, it may be a damage facter to esophagus. Therefore, further studies in this field are needed. (6) The MUC gene expression pattern in Barrett's esophagus is similar to that in incomplete intestinal metaplasia of the stomach, suggesting that both Barrett's oesophagus and incomplete intestinal metaplasia reacts to the repeated injury by changing the type of cell in their lining from esophagal squamous and gstric columnar to intestinal-type cells. The expression of MUC3 may prefigure the developing of Barrett's specialized IM. The gastric metaplasia may be a precursor of Barrett's specialized IM. The MUC gene expression pattern in Barrett's esophagus also indicates the cells in Barrett's esophagus have the potential of differentiation, foreboding the perspective of curing Barrett's specialized IM. (7) The MTHFR polymorphisms is one of the inherited factors of Barrett's oesophagus. The MTHFR 677TT genotype increase the risk of Barrett's specialized IM.