Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium

MUC5AC was highly expressed in foveolar epithelium and mucous neck cells of antrum (> 75%, Fig. 1b; Fig. 3b; Table 2). Expression of MUC6 was detected in the glands of the antrum (> 75%, Fig. 1c; Fig. 3c; Table 3) and MUC2 was not detected in normal gastric mucosa (Fig. 1a; Fig. 3a; Table 2).

There was expression of MUC5AC in all the cases of columnar cells (Fig. 1e; Fig. 3b; Table 2). The percentage of stained cells varied from case to case, in most cases, there was a trend for higher number of cells expressing MUC5AC (>75%) in the superficial part of the metaplastic glands than in deep part. MUC6 was detected in columnar cells (>50%; Fig. 1f; Fig. 3c; Table 2) and MUC2 was not detected in atrophic gastritis (Fig. 1d).

There was decreased level expression of MUC5AC and MUC6 in all the cases (>50%), both in goblet and in columnar cells (Fig. 1h, Fig. 2a; Fig. 3b, Fig. 3c; Table 2). The percentage of stained cells varied from case to case, de novo expression of intestinal mucin MUC2 was observed (>75%), and its expression displayed a diffuse cytoplasmic pattern in goblet cells (Fig. 1g; Fig. 3a; Table 2).

There was decreased level expression of MUC5AC and MUC6, than intestinal metaplasia (>50%). MUC5AC and MUC6 displayed a diffuse cytoplasmic expression pattern in columnar cells (Fig. 2c; Fig. 2d; Fig. 3d; Fig. 3; Table 2). MUC2 expression displayed a diffuse cytoplasmic pattern in goblets cells (>50%, Fig. 2b; Fig 3a; Table 2).

MUC5AC and MUC6 expression were frequently observed in early carcinoma (>50%) than advanced carcinomas (>25%). In advanced carcinomas, we observed a trend for decreased immunoreactivity in deep areas than in the superficial areas. MUC5AC and MUC6 expressions were decreased in advanced carcinomas (>25%; Fig. 3a; Fig. 3b; Fig. 3c; Fig. 2e, Fig. 2f, Fig. 2g; Table 2).

Fifty nine gastric biopsy specimens and 3 ml of blood were obtained from Stanley Medical College and Hospital, Chennai. All biopsy tissue samples were collected after informed consent. This study was carried out after obtaining clearance from the ethical board of the hospital. All biopsy specimens were fixed in 10% buffered formalin and routinely embedded in paraffin wax. 4 μm thin serial sections were cut and used for immunohistochemistry. H. pylori infection was confirmed by RUT and ELISA [43]. Hematoxylin and Eosin stained sections were used to classify gastritis graded according to revised Sydney classification [44], gastric tumors according to the classification of Lauren [45] and WHO classification [46]. We studied 7 cases of normal Human gastric epithelium (7 biopsies), 26 cases of Atrophic gastritis (26 biopsies), 21 cases of Intestinal metaplasia (21 biopsies), 17 cases of dysplasia (17 biopsies) and 36 cases of carcinoma in which 21 cases early carcinoma and 15 cases advanced carcinoma (16 biopsies and 20 surgical specimens).

A panel of three monoclonal antibodies was used to determine the expression of mucins (MUC2, MUC5AC and MUC6). The specificities, references and dilutions of the antibodies are listed in Table 1.

Sections from normal, pre-neoplastic and neoplastic gastric tissues were immuno stained according to Reis et al [47] with slight modifications. Sections designed for neuraminidase treatment were washed twice in phosphate buffered saline (PBS), pH 7.4 and incubated with neuraminidase from clostridium perfringens Type VI (Sigma; St Louis, MO) diluted in 0.1 M Na-acetate buffer, pH 5.5 to a final concentration of 0.1 U/ml. The incubation was carried out for 2 h at 37°C and was followed by three washings in ice-cold PBS. Sections were treated with 0.5% H₂O₂ in methanol for 30 min, followed by 20 min of incubation with rabbit non-immune serum. Sections were rinsed and incubated with primary antibody (Table 1), over night at 4°C. The sections were rinsed and incubated with HRP conjucated rabbit anti-mouse secondary antibody (1:100 dilution) for 1 h followed by rinsing with PBS and developing with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (SRL, Mumbai, India) freshly prepared in PBS containing 0.1% H₂O₂. Sections were counter stained with hematoxylin, dehydrated, and mounted. All series including positive and negative controls were performed by substitution of the primary mAbs with immunoglobulins of the same class and at the same concentration.

A semi quantitative approach was used to score the immunostaining of human gastric tissues. The slides were examined under light microscope, every 100 cells were scored in five different fields and the results were expressed as mean percentage. (-), negative; +, few positive cells (<25%); ++ well-defined areas with positive cells (25–50%); +++, extensive areas with positive cells (50–75%); ++++, most cells stained (>75%).

Data analysis was performed using statistical software package SPSS 10.0 for windows. The Chi-Square test or Fisher's exact test was used to analyze differences in frequencies. Spearman's correlation test was used to analyze correlation between parameters. P < 0.05 was considered statistically significant.