Japanese Journal of Clinical Oncology 30:89-94 (2000)
© 2000 Foundation for Promotion of Cancer Research
Clinical Significance of MUC1 and MUC2 Mucin and p53 Protein Expression in Colorectal Carcinoma
1Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 2Hitachi Chemical Research Center, Irvine, CA, USA and 3First Department of Pathology, Niigata University, Niigata, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Up-regulation of MUC1, down-regulation of MUC2 and p53 overexpression are seen in colorectal carcinomas. However, there have been few reports about the associations between MUC1, MUC2 and p53 expression and metastatic potential. The aim of this study was to investigate MUC1, MUC2 and p53 expression in colorectal carcinoma with special reference to regional and distant metastasis.
Methods: Eighty-six colorectal carcinomas were collected from patients undergoing tumor resection. Sections were used for MUC1, MUC2 and p53 immunostaining. Cancers were regarded as MUC1 or MUC2 positive when the positive cells were beyond 30% of cancer cells. Cancers with diffuse or nested patterns were regarded as having p53 overexpression.
Results: Of 86 cancers, 37 (43%) were MUC1 positive, 28 (33%) were MUC2 positive and 59 (69%) showed p53 overexpression. A difference was observed only in the frequency of MUC1 positivity with respect to depth of tumor invasion. Neither depth of tumor invasion nor histological differentiation had a positive correlation with MUC1, MUC2 and p53 overexpression. The frequency of MUC1 positive cells in Dukes’ C and D tumors was significantly higher than that in Dukes’ A and B tumors. The frequency of MUC1 positivity in tumors with hepatic involvement was significantly higher than that in tumors without hepatic involvement (100 vs 39%; p < 0.01). There was no difference in the frequency of MUC2 or p53 positivity in Dukes’ stage or hepatic metastasis. MUC1 immunoreactivity of the surface was identical with that of the whole tumor in 81% (70/86) of carcinomas, MUC 2 in 87% and p53 in 100%.
Conclusions: The results suggest that up-regulation of MUC1 is involved in the progression from the non-metastatic to the metastatic stage and that p53 abnormality is not directly involved in it. The data also imply that immunostaining of preoperative biopsy samples is useful for evaluating the immunoreactivity of the whole tumor.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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MUC1 is the apomucin for epithelial membrane antigen and related structures identified with monoclonal antibodies raised against the membrane component of human milk fat globules (1,2). MUC1 expression is up-regulated in a variety of cancers including colorectal cancer (3). MUC2 is expressed by intestinal goblet cells. Interesting relations between MUC2 expression and the pathogenesis of colorectal neoplasia have been suggested (4,5). MUC2 is expressed by adenomas and mucinous carcinomas (6). Down-regulation of MUC2 is seen in non-mucinous adenocarcinomas arising within adenomas (6). However, there have been few reports about the associations between MUC1 and MUC2 expression and metastatic potential in colorectal carcinomas (7,8).
The p53 mutation is common in human cancers and overexpression of its product is detected in 60–70% of colorectal cancers with immunohistochemistry (9–12). Overexpression of p53 immunoreactive cells is reported as a useful marker for the diagnosis of carcinoma, because there is a high concordance between carcinoma and p53 mutations (9,12–15). However, the relation between p53 expression and metastasis in colorectal carcinoma is still controversial (12,16,17).
The aim of this study was, therefore, to investigate MUC1, MUC2 and p53 expression in colorectal carcinoma with special reference to regional and distant metastasis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Eighty-six colorectal carcinomas were collected from patients undergoing tumor resection at the Division of Surgical Oncology, Department of Surgery, University of Tokyo. Sixty-two were male and the remaining 24 were female. The mean age was 61.9 years with a range of 33–86 years. The tumor location was the cecum in three, ascending colon in 11, transverse colon in five, descending colon in one, sigmoid colon in 25 and rectum in 31 patients. The depth of tumor invasion was sm (pT1) in 19, mp (pT2) in 14, ss (pT3) in 25 and se or si (pT4) in 28.
Four 3 µm thick sections were cut from formalin-fixed paraffin-embedded blocks. The first section was stained with hematoxylin and eosin (H&E) and the remaining sections were used for MUC1, MUC2 and p53 immunostaining.
Immunohistochemical Staining
For MUC1, MUC2 and p53 immunostaining, paraffin-embedded sections were placed on poly-L-lysine-coated glass slides and air-dried at room temperature. Deparaffinized and rehydrated sections were heated in a microwave oven for seven 3 min cycles in citrate buffer to retrieve antigenic activity and cooled for 60 min at room temperature. Endogeneous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxidase in methanol for 20 min at room temperature. After blocking non-specific reactions with 10% normal rabbit serum, the sections were first incubated with MUC1 antibody (mouse monoclonal antibody Ma695; Novocastra Laboratories, Newcastle, UK) overnight at a dilution of 1:100, MUC2 antibody (mouse monoclonal antibody Ccp58; Novocastra) overnight at a dilution of 1:100 or p53 antibody (mouse monoclonal antibody DO 7; Novocastra) overnight at a dilution of 1:100. The sections were then incubated with biotinylated rabbit anti-mouse immunoglobulin for 30 min and next with streptavidin–peroxidase complex (Histofine SAB-PO Kit, Biogenex Laboratories) for 15 min. The sections were carefully rinsed with several changes of phosphate-buffered saline (PBS) between each step of the procedure. The color was developed with diaminobenzidine. The sections were lightly counterstained with H&E and mounted. Negative controls were obtained by replacing the primary antibody with PBS.
Immunohistochemical Analysis
The expression of MUC1 and of MUC2 was scored semiquantitatively as follows: 0, 1 = <5% of cells; 2 = 5–30% of cells; 3 = 30–60% of cells; 4 = >60% of cells. Cancers were regarded as MUC1 or MUC2 positive when the score was 
3, according to previous reports (5,18).
p53-positive cells were defined as cells with brown staining on the nucleus, regardless of staining intensity, but cells with very weak equivocal staining were considered negative. Staining patterns were classified into the following four patterns based upon previous reports of colorectal and gallbladder cancers (12,13): (1) diffuse pattern – nuclear positive cells with positive nuclear staining present diffusely in most areas of the tumor; (2) nested pattern – >20 positive cells aggregated in a part of the tumor; (3) scattered pattern – a small number of isolated positive cells scattered in the tumor; (4) negative pattern – no positive cells in the tumor. Tumors with diffuse or nested patterns were regarded as having p53 protein overexpression according to our previous report (19).
The immunoreactivity of MUC1 mucin, MUC2 mucin and p53 protein was evaluated and compared in three different areas, the surface, the invasive margin and the whole tumor, in order to examine the utility of preoperative biopsy in predicting regional or distant metastasis (Fig. 1).
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Statistical Analysis
The chi-squared test was used to analyze the contingency table. Values of p < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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In normal mucosa, MUC1 was never observed (0/3202 tubules). MUC1 was expressed intraluminally within the glycocalyx and in intracytoplasmic lumina in colorectal carcinomas (Fig. 2). The intraluminal material was dense, eosinophilic and admixed with necrotic debris. Of 86 cancers, 37 (43%) had a MUC1 score of
3. MUC1 positivity was observed in 21% (4/19) of pT1, 50% (7/14) of pT2, 40% (10/25) of pT3 and 57% of pT4 cancers (Fig. 3). There was a significant difference between pT1 cancers and pT4 cancers (p < 0.05, 
2 test). In contrast, no difference was observed in the frequency of MUC1 positivity with respect to histological differentiation (Table 1).
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MUC2 was demonstrated in the perinuclear cytoplasm of normal and malignant goblet cells (Fig. 4). In non-neoplastic areas, every tubule was MUC2 positive (total 3183 tubules). Of 86 cancers, 28 (33%) had a MUC2 score of
3. MUC2 was not evident in either the goblet cell theca or extracellular mucin. In cancers, MUC2 expression was associated with the presence of luminal and interstitial material having the features of secretory mucin: lightly basophilic on staining with H&E and foamy or wispy (19). No difference was observed in the frequency of MUC2 positivity with respect to depth of tumor invasion (Fig. 3). Although signet ring cell carcinomas and mucinous carcinomas tend to be positive for MUC 2, no significant difference was observed with respect to histological differentiation (Table 1).
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p53 was expressed only in the nuclei (Fig. 5). In non-neoplastic mucosa, p53 protein overexpression was never observed (0/3275 tubules). Of 86 cancers, 59 (69%) showed p53 protein overexpression. Neither depth of tumor invasion nor histological differentiation had a positive correlation with p53 overexpression (Fig. 3, Table 1).
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The frequency of MUC1 positive cells in Dukes’ C and D tumors was significantly higher than that in Dukes’ A and B tumors (48 vs 34%; p < 0.05,
2 test) (Fig. 6). On the other hand, MUC2- or p53-positive cells were observed at a similar level between Dukes’ C and D tumors and Dukes’ A and B tumors. As shown in Fig. 7, the frequency of MUC1 positivity in tumors with hepatic involvement was significantly higher than that in tumors without hepatic involvement (100 vs 39%; p < 0.01, 2 test). However, the frequency of MUC2 or p53 positivity in tumors with hepatic involvement was similar to that in tumors without hepatic involvement.
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Next, we examined the heterogeneity in staining pattern of these factors. In 81% (70/86) of carcinomas, MUC1 immunoreactivity of the surface was identical with that of the whole tumor. In tumors that showed MUC1 positivity at the upper surface area, 100% (21/21) showed MUC1 positivity in the whole tumor and the invasive margin (Table 2). When the surface of a cancer showed MUC2 negativity, 96% (49/51) showed MUC2 negativity in the whole tumor or the invasive margin (Table 2). In 87% (75/86) of carcinomas, MUC2 immunoreactivity of the surface was identical with that of the whole tumor. p53 immunoreactivity was identical in these three areas without any exceptions (Table 2).
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In 86 cancers, nine contained an adenomatous component. They consisted of seven pT1, one pT2 and one pT4 cancers. As shown in Table 3, MUC1 was negative in adenomatous areas in the nine cases examined; however, in two of nine cases (22%), MUC1 was positive in carcinomatous areas. On the other hand, MUC2 was positive in adenomatous areas in all cases, but positive in carcinomatous areas in six of nine cases (67%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Mucins are highly glycosylated, high molecular weight glycoproteins with unique core polypeptides rich in threonine and serine (20,21,22). We examined two types of mucins, MUC1 and MUC2, in this study (5,6,8,18,23,24). In the normal mucosa, all tubules were positive for MUC2, but negative for MUC1. In colorectal carcinomas, the frequency of MUC1 positivity was higher than that of MUC2. In carcinomas containing an adenomatous component, MUC1 positivity was not observed in adenomatous areas, but in carcinomatous areas. In contrast, the frequency of MUC2 positivity was higher in adenomatous areas than in carcinomatous areas. These results suggest that up-regulation of MUC1 and down-regulation of MUC2 are involved in tumor progression through the adenoma–carcinoma sequence (5). Furthermore, in this study, the frequency of MUC1 positivity in carcinomas with metastasis (Dukes’ C and D) was significantly higher than that in carcinomas without metastasis (Dukes’ A and B). This indicates that up-regulation of MUC1 is also involved in the progression of carcinoma from the non-metastatic to the metastatic stage. Nakamori et al. (25) also reported that colorectal carcinomas with metastasis expressed MUC1 more strongly than those without metastasis. It remains to be determined how MUC1 mucin is involved in the metastatic process in colorectal carcinomas. As for breast carcinomas, Regimbald et al. (26) showed that MUC1 was a ligand for ICAM-1 and that the interaction between MUC1 and ICAM-1 might be essential in the process of hematogeneous metastasis. Ajioka et al. (18) have speculated that the same mechanism might occur in the process of colorectal carcinoma metastasis.
Whether p53 overexpression is a prognostic indicator or not remains controversial (11,15,16). However, this study showed no difference in the frequency of p53 positivity between carcinomas with metastases (Dukes’ C and D) and those without (Dukes’ A and B), suggesting that p53 abnormality is not directly involved in the progression of carcinoma from the non-metastatic to the metastatic stage.
This is the first study to investigate the site-to-site difference of MUC1, MUC2 and p53 immunoreactivity in colorectal carcinoma and showed that MUC1, MUC2 and p53 immunoreactivity of the surface were identical with those in the whole tumor in 81% (70/86), 87% (75/86) and 100% (86/86) of carcinomas, respectively. These results imply that immunostaining of preoperative biopsy samples is useful for evaluating the immunoreactivity of the whole tumor. Our study suggested that greater attention should be paid in the search for metastatic lesions when the biopsy specimen of the carcinoma shows MUC1 positivity.
The aim of this study was to find a new, useful marker that could predict metastasis in addition to several markers such as metalloproteinases (27,28) and adhesion molecules (29–31). Masaki et al. (32) reported that dedifferentiated histology at the invasive margin of colorectal carcinomas might be a clinical predictor of lymph node metastasis. Ono et al. (33) reported that focal dedifferentiation and expression of sialyl-Lex antigen were good markers for assessing metastatic potential.
In conclusion, this study revealed that MUC1 could be a new, useful marker for synchronous metastasis in colorectal carcinomas. Further prospective studies should be performed to evaluate the predictive power of MUC1 for metachronous metastasis.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This work was presented at the 52nd Japanese Gastroenterological Surgical Academic Meeting, Tokyo, Japan, on July 17, 1998.
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FOOTNOTES |
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+ For reprints and all correspondence: Keiji Matsuda, Hitachi Chemical Research Center, 1003 Health Sciences Road West, Irvine, CA 92612, USA. E-mail: matsudak@bigplanet.com
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received August 9, 1999; accepted November 4, 1999.
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