We examined 88 surgical specimens of gallbladder cancer, 45 chronic cholecystitis specimens (including 7 Xanthogranulomatous cholecystities (XGC)) and 5 normal gallbladder specimens obtained at the time of surgical treatment for gastrointestinal disease. Three pathologists, who are the co-authors of this study, independently performed pathologic evaluations of the specimens and disclosed their the findings of their individual determinations.

For the 88 cancer specimens, the primary tumors (T category) have been classified according to the TNM criteria (₈) as follows: T1, Tumor limited lamina propriae or muscle layer; T1a, Tumor limited in the mucosa, T1b, Tumor extended to muscle layer. T2: Tumor invades perimuscular connective tissue, but no extension beyond the serosa or into the liver, T3: Tumor perforates the serosa (visceral peritoneum) or directly invades one adjacent organ, or both (extension 2 cm or less into the liver), T4: Tumor extends more than 2 cm into the liver, and/or into two or more adjacent organs (stomach, duodenum, colon, pancreas, omentum, extrahepatic bile ducts, any involvement of the liver). Out of the 88-gallbladder cancers, 14 tumors were T1, 15 were T2, 45 were T3 and the remaining 14 were T4.

All specimens were fixed in 10% formalin, embedded in paraffin, serially sectioned into 4 µm thick cuts and mounted on polysine coated slides. The slides were immersed for 20 min in 0.3% hydrogen peroxide in methanol to deplete the endogenous peroxidase. After washing, they were incubated with a protein blocking agent for 5 minutes. Primary antibodies against MUC1 (monoclonal antibody, mouse anti-human CA 15-3, clone DF3 (IgG1, DAKO corporation) were used at a dilution of 1:50. For the negative controls, the primary antibody was replaced with PBS. The slides were incubated with primary antibody in a humid chamber for 1 hour, washed with PBS for 15 minutes, and underwent changing of the buffer 3 times. Then, biotinylated secondary antibodies were applied for 10 minutes at room temperature. After washing, streptavidin peroxidase reagent was applied and the samples were incubated for 10 minutes at room temperature. Lastly, the slides were visualized by incubation within solution containing 0.3% hydrogen peroxide and diaminobenzidine tetrahydrochloride in PBS. Counterstaining was perfored with haematoxylin, prior to mounting in crystal.

Three pathologists examined the expression of MUC1 under a light microscope independently. The MUC1 expression was judged as positive when more than 5% of cells were stained.

Statistical analysis was conducted via the Chi-square test and p values of less than p<0.05 were regarded as significant.

MUC1 expression in epithelial cells and cancer cells in the gallbladders was estimated separately in the epithelial layer and the layers deeper than sub-mucosal layer, which consisted of the sub-mucosal layer, the muscle layer, sub-muscular connective tissue and serous layer. Two types of staining patterns were recognized: polarized and depolarized. In the polarized type, MUC1 expression is restricted predominantly in the apical membrane of the cell (Fig 1-A~D). In the depolarized type, the membrane and cytoplasm of the cell, as well as the stroma adjacent to the basal membrane of the malignant glands were stained (Fig 1-E).

In the normal gallbladder tissue, the expression rate of MUC1 was 20% (1/5) in the epithelial layer, with a polarized staining pattern. There was no MUC1 expression in the sub-mucosal layer (Fig 1-A). In the chronic inflammatory gallbladder tissue samples, only 2 of 45 specimens (4.4%) expressed MUC1 in the epithelial layer and the staining pattern was of the polarized type (Fig 1-B&C). There was no statistical difference in the rate and type of MUC1 expression in the epithelial and sub-mucosal layers between normal and chronic inflammation tissues.

On the other hand, in the gallbladder cancer samples, the epithelial layer had a significantly (p<0.0001) higher expression of MUC1 -- 78.4% (69/88) -- than in non-cancerous (normal and chronic inflammation) tissues (Table 1). The staining pattern of MUC1 was exclusively of the polarized type in the epithelial layer of the non-cancerous gallbladder samples. However, in the gallbladder cancer samples, both staining patterns were marked: 34.8% (24) for the polarized type and 65.2% (45) for the depolarized type. In 74 specimens with cancer cell invasion to the layers deeper than sub-mucosal layer, the MUC1 expressed in 89% (66/74) of them and 88% of MUC1 expressed specimens (58/66) showed depolarized type and only 12% (8/66) demonstrated polarised type.

Figure 1

Table 1: MUC1 expression in the epithelium of different gallbladder lesions

According to the TNM classification, 82 specimens were adenocarcinomas and two were adenosquamous carcinomas, while two showed signet ring cell carcinoma features and the remaining two were mucinous carcinomas. Adenosquamous and mucinous carcinomas expressed MUC1 with a depolarized staining pattern, while the signet ring cells did not show MUC1 expression. Out of 88 carcinomas, 51 were classified as well-differentiated, 21 were classified as moderately differentiated and 16 were classified as poorly differentiated carcinomas. The MUC1 expression rates in the cancer cells on the epithelial layer were as follows: 73% in well, 81% in moderate and 94% in poorly differentiated carcinomas, respectively. In the sub-mucosal layer, it was 86%, 90%, 94% in well, moderate and poorly differentiated carcinomas, respectively. These results suggested no correlation in MUC1 expression rate and cancer differentiation.

The MUC1 expression rate of 29% (4/14) in the epithelial layer of T1 tumors was significantly (p<0.0001) less than the rate noted in the advanced T categories; that is, 93% (14/15) in T2, 89% in T3 (40/45) and 79% in T4 tumors (11/14), respectively (Table 2). All of the cells in the MUC1-positive T1 cancers displayed polarized staining. On the other hand, 69 % (45/65) of the MUC1-expressing cancer cells in the advanced T categories -- T2 (14), T3 (40) and T4 (11) -- had a depolarized pattern. In these advanced T categories, cancers penetrating beyond the epithelial layer demonstrated very high MUC1 expression rates: as 100% (15/15), 84.4% (38/45) and 92.8% (13/14) in T2, T3 and T4, respectively. There is no correlation between the positive expression of MUC1 and the staining pattern among T2, T3 and T4 tumors in the both epithelial and sub-mucosal layers.

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T category is a criterion based upon the extent of primary tumor invasion classified by TNM criterion

Venous invasion was assessed in 74 advanced cancers (T2~T4) under the microscope and was noted in 32 specimens of these specimens. In these venous invasion-positive specimens, the mucin expression rates are 84% and 94 %, respectively, in the epithelial layer and in the sub-mucosal layer. In the specimens where there is no venous invasion, the mucin expression rates are 77% and 86%, respectively. Therefore, the mucin expression rate has no relation to the degree or presence of venous invasion.

Figure 1A-E:

MUC1 expression detected by the immunoperoxidase method using DAB as a coloring agent (brownish pigment).

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Less than 5% of epithelial cells (arrow) expressed MUC1 on the apical surface.

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Less than 5% of epithelial cells (arrow) expressed MUC1 in a polarized pattern.

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Less than 5% of the epithelial samples (arrow) expressed MUC1 in a polarized pattern.

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Cancer cells extensively expressed MUC1 in a polarized pattern.

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Muc1 is expressed in the cytoplasm, as well as along the entire surface and within the stroma adjacent to the basal membrane (depolarized pattern).