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MUC4 Is a Novel Prognostic Factor of Extrahepatic Bile Duct Carcinoma

Authors' Affiliations: ¹ Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; ² Departments of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska; and ³ Department of Internal Medicine, Sapporo Medical College, Sapporo, Japan

Requests for reprints: Suguru Yonezawa, Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, 890-8544 Kagoshima, Japan. Phone: 81-99-275-5270; Fax: 81-99-265-7235; E-mail: syoneza{at}m2.kufm.kagoshima-u.ac.jp.

	Abstract

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Purpose: Many of the patients with extrahepatic bile duct carcinoma (EHBDC) show a poor outcome. We have reported that MUC4 is a novel prognostic factor of pancreatic adenocarcinoma and intrahepatic cholangiocarcinoma. The aim of this study is to evaluate the prognostic significance of MUC4 expression in EHBDC.

Experimental Design: We examined the expression profile of MUC4 in EHBDC tissues from 70 patients using immunohistochemistry. MUC4 is a membrane mucin like MUC1. In addition, MUC4 is an intramembrane ligand for receptor tyrosine kinase ErbB2 and is related with regulation of p27. We compared the MUC4 expression with MUC1, ErbB2, or p27 expression in EHBDC.

Results: MUC4 was expressed in 36 of the 70 patients with EHBDC. There was no significant correlation between the MUC4 expression and MUC1, ErbB2, or p27 expression. The survival of 19 patients with high MUC4 expression (20% of carcinoma cells stained) was significantly worse than that of the 51 patients with low MUC4 expression (under 20% of carcinoma cells stained; P = 0.0072). The univariate analysis showed that high MUC4 expression (P = 0.0072), high MUC1 expression (P = 0.0092), histologic grading (P = 0.0029), surgical margin involvement (P = 0.0137), and nodal metastasis (P = 0.0036) were statistically significant risk factors. The backward stepwise multivariate analysis showed that high MUC4 expression (P = 0.0195) and surgical margin involvement (P = 0.0358) were statistically significant independent risk factors.

Conclusions: MUC4 expression in EHBDC is a new independent factor for poor prognosis and predicts the outcome of patients with EHBDC.

Mucins are high molecular weight glycoproteins having oligosaccharides attached to serine or threonine residues of the mucin core protein backbone by O-glycosidic linkages. Core proteins for human mucins (MUC1-MUC9, MUC11-13, MUC15-20) have been identified in the past few years (4–7). Our series of immunohistochemical studies for mucin expression in various human tumors have shown that the expression of the MUC1 mucin (membrane mucin) is related to invasive proliferation of tumors and a poor outcome for patients. On the other hand, the expression of the MUC2 mucin (intestinal type secretory mucin) is related to noninvasive proliferation of tumors and a favorable outcome for patients (8–14). In our previous study of EHBDC, we also showed that the patients with high MUC1 expression in their carcinoma cells showed significantly worse survival than those with low MUC1 expression patients. In contrast, the patients with high MUC2 expression in their carcinomas showed significantly better prognosis than those with low MUC2 expression (15).

MUC4 was first reported as a tracheobronchial mucin (16) and it is known that MUC4 is expressed in various normal tissues (17–20). Recently, MUC4 was proven to be a novel intramembrane ligand for receptor tyrosine kinase ErbB2 (21–23), which is a transmembrane glycoprotein encoded by c-ErbB-2 proto-oncogene with a tyrosine kinase domain that is highly homologous with the epidermal growth factor receptor (24, 25). Furthermore, MUC4 was related with the regulation of p27 (26), which is a cyclin-dependent kinase inhibitor used in the control of G₁ and S phases of the cell cycle (27).

MUC4 is a membrane mucin (23), like MUC1, which is a poor prognostic factor in various human tumors (9–15, 28–30). Recently, MUC4 has been reported to be expressed in pancreatic adenocarcinoma, whereas MUC4 was not expressed in the normal pancreatic tissue (31–34). Thus, MUC4 is considered to be a tumor marker for pancreatic adenocarcinoma. In biliary epithelial cells, Vandenhaute et al. (35) reported that MUC4 was not expressed in the normal epithelium. Lee and Liu (36) reported that MUC4 was expressed in 10% of normal controls, whereas it was 67% positive in cholagiocarcinoma. We reported that MUC4 is a new prognostic factor for prognosis in the intrahepatic cholangiocarcinoma mass-forming type (37) and pancreatic adenocarcinoma (38).

To date, there has been no research on the relationship between MUC4 expression and patient prognosis in EHBDC. In the present study, we expected MUC4 to also be a candidate for one of the poor prognostic factors in EHBDC. We examined the expression profile of MUC4 in EHBDC tissues, and found that MUC4 expression is a very useful factor for poor prognosis in patients with EHBDC. In addition, ErbB2 and p27 expression were examined to investigate the relationship of their expressions with MUC4 expression in EHBDC.

	Materials and Methods

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Patients and tissue samples
Surgically resected EHBDCs from 70 patients (47 males and 23 females) were studied. All tissue specimens were registered from 1990 to 2000 and were filed in the Kagoshima-shi Medical Association Hospital. The mean age of the patients was 69.2 years (range: 41-88 years). The 70 tumors were histologically classified according to ref. 39. The study was approved by the Kagoshima University Faculty of Medicine Human Investigation Committee.

In histologic grading (39), 37 cases (53%) were well-differentiated type (G₁), 21 cases (30%) were moderately differentiated type (G₂), and 12 cases (17%) were poorly differentiated type (G₃). Surgical procedures were as follows: 31 cases (44%) were treated by conventional pancreatoduodenectomy, 18 cases (26%) were treated by pylorus-preserving pancreatoduodenectomy, and 21 cases (30%) were treated by extrahepatic bile duct resection. Lymph node dissection was done in all cases, and lymph node metastasis was positive in 23 cases (33%). Clinical outcome data were available in these 70 patients with EHBDC. Overall survival was analyzed in the current study. All specimens were fixed in formalin, embedded in paraffin, and cut into 4-µm-thick sections for immunohistochemistry, in addition to the usual H&E staining.

Immunohistochemistry
Antibodies. Immunohistochemistry was done by using the following antibodies. MUC4 was detected by mouse monoclonal antibody, clone 8G7 (generated by one of us, by Surinder K. Batra, Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE; ref. 32). For the comparative study, MUC1 was also examined by monoclonal antibody DF3 (mouse IgG, Toray-Fuji Bionics, Tokyo, Japan). ErbB2 was detected by rabbit polyclonal antibody (DakoCytomation, Glostrup, Denmark). p27 was detected by monoclonal antibody NCL-p27 (Novocastra, Newcastle, United Kingdom).

Biotinylated affinity-purified horse anti-mouse IgG, goat anti-rabbit IgG, and avidin-biotinylated horseradish peroxidase complex (ABC) were purchased from Vector Laboratories (Burlingame, CA) as the Vectastain Elite ABC kit.

Staining procedure. Immunohistochemical stainings were done by an immunoperoxidase method using the ABC complex as described previously (37, 38). Each section was deparaffinized with xylene. Endogenous peroxidase was blocked by incubating the sections in 0.3% hydrogen peroxidase in absolute methanol at room temperature for 30 minutes. After hydration in decreasing concentrations of ethanol in water, the sections were washed in 0.01 mol/L PBS (pH 7.4).

For the epitope retrieval of MUC4, a water bath pretreatment at 80°C for 20 minutes in 0.01 mol/L citrate buffer (pH 6.0) was done. Also, for that of ErbB2, a water bath pretreatment at 98°C for 40 minutes was done. For that of p27, an autoclave pretreatment at 120°C for 5 minutes was done.

The sections were washed twice with PBS. Then, 2% horse or goat serum in PBS was applied for 30 minutes at room temperature to prevent nonspecific staining. In the staining using each antibody, the sections were incubated with dilutions of the primary antibodies (MUC4, 1:3,000; MUC1, 1:10; ErbB2, 1:100; p27, 1:40) in PBS with 1% bovine serum albumin for 16 hours at 4°C. The sections were washed thrice with PBS, incubated with the biotinylated secondary antibodies, and washed thrice with PBS. All sections then received ABC for 30 minutes. After washing with PBS thrice, the sections were finally reacted with diaminobenzidine substrate for 10 minutes for visualization, rinsed with tap water, counterstained with hematoxylin, and mounted. Reaction products were not present when nonimmune serum or PBS was used instead of the primary antibodies.

Evaluation of the results by scoring
Three blinded investigators (S.T., H.S., and S. Y.) evaluated the immunostaining independently. When the evaluation was different among three, the final decision was made by the consensus of the three.

The results of the immunohistochemical stainings were evaluated by the percentage of positively stained carcinoma cells. Expression of MUC4 in the cytoplasm of the cells was evaluated. According to our previous study of EHBDC (15), the percentage of positively stained carcinoma cells were graded as follows: –, <5% of carcinoma cells stained; +, 5% to <20% of carcinoma cells stained; ++, 20% to <50% of carcinoma cells stained; and +++, 50% of carcinoma cells stained. Additionally, for statistical analysis, the examined cases are divided into two groups: the low-expression group (LEG), composed of the – and + cases (<20% of carcinoma cells stained), and the high-expression group (HEG), composed of the ++ and +++ cases (20% of the carcinoma cells stained).

Statistical analysis
Statistical analysis was done using the ² test, Fisher's exact test, or Spearman rank-correlation test, where appropriate. Survival of the patients was compared between the HEG of MUC4, MUC1 or ErbB2, or p27 expression and the LEG of those expressions according the Kaplan-Meier method. Differences between the survival curves were tested using the log-rank test. Univariate and multivariate survival analyses were done using the Cox proportional hazards regression model. For the multivariate model, we used 0.10 as the cutoff P value to select the analyzed factors from the univariate analysis data. Backward stepwise multivariate analysis was also used to find independent prognostic factors. A probability of P < 0.05 was considered statistically significant.

	Results

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MUC4, MUC1, ErbB2, or p27 expression in normal epithelium of the extrahepatic bile duct. MUC4, MUC1, and ErbB2 were not expressed in normal epithelium of the extrahepatic bile duct. p27 was expressed in the nuclei in most of the normal epithelium.

MUC4, MUC1, ErbB2, or p27 expression in EHBDC tissue. Figure 1 shows representative expression patterns of MUC4, MUC1, ErbB2, and p27 in EHBDC tissue. Positive staining (5% of carcinoma cells stained) of MUC4 was seen in the cytoplasm of the carcinoma cells in 36 (51%) cases of 70 EHBDCs (Fig. 1A). MUC1 was expressed in the cytoplasm and/or membrane in 61 (87%) cases (Fig. 1B), ErbB2 was expressed in membrane and/or cytoplasm in 9 (13%) cases (Fig. 1C), and p27 was expressed in the nuclei of the carcinoma cells in 55 (79%) cases (Fig. 1D).

View larger version (96K): [in this window] [in a new window]   Fig. 1. Immunohistochemical staining of MUC4 (A), MUC1 (B), ErbB2 (C), and p27 (D) in extrahepatic bile duct carcinoma. MUC4 was expressed in the cytoplasm of the carcinoma cells (A, original magnification, x160). MUC1 was expressed in the membrane and/or cytoplasm (B, original magnification, x160). ErbB2 was expressed in the membrane and/or cytoplasm (C, original magnification, x160). The expression of p27 was seen in the nuclei (D, original magnification, x160).

View larger version (111K): [in this window] [in a new window]   Fig. 2. A sample of the four expression levels of MUC4: – (A), + (B), ++ (C), and +++ (D). Original magnification, x25.

The patients were divided into two groups based on the length of survival: short-term survivors (survival period <12 months) and long-term survivors (survival period 12 months). High MUC4 expression was seen in 58.3% of the short-term survivors, and in 22.0% of the long-term survivors. This difference was statistically significant (P = 0.0128; Table 2). High MUC1 expression did not show such a significant difference (Table 2).

Neither expression of ErbB2 nor p27 was related to the clinicopathologic features listed in Table 2.

The relationship between MUC4, ErbB2, or p27 expression and cumulative survival rate. Among the 70 patients examined, 33 patients died during the follow-up period. Median and mean length of survival for patients with EHBDC who underwent operation was 39 and 47.7 months, respectively. The 1-, 3-, and 5-year survival rates of patients with EHBDC were 79.8%, 51.6%, and 35.4%, respectively. The survival of patients with high MUC4 expression (19 patients) was significantly worse than that with low MUC4 expression (51 patients; P = 0.0072, log-rank test; Fig. 3 ). The survival of patients with high MUC1 expression (18 patients) was also significantly worse than those with low MUC1 expression (52 patients; P = 0.0092; Fig. 4 ). A high or low expression of ErbB2 and p27 expression was not related to the survival of 70 patients with EHBDC (P = 0.1198 and P = 0.6004).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Correlation between the MUC4 expression and cumulative survival rate in 70 patients with extrahepatic bile duct carcinoma, as determined by the Kaplan-Meier method. The survival of patients with a high expression of MUC4 was worse than with low MUC4 expression (P = 0.0072). Closed boxes, patients who were alive at the last follow-up.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Correlation between MUC1 expression and cumulative survival rate in 70 patients with extrahepatic bile duct carcinoma, as determined by the Kaplan-Meier method. The survival of patients with a high expression of MUC1 was worse than that with low MUC1 expression (P = 0.0092). Closed boxes, patients who were alive at the last follow-up.

	Discussion

Top Abstract Materials and Methods Results Discussion References

MUC4 is expressed in normal tissues of the respiratory tract, stomach, small intestine, colon, and endocervix (17, 18, 20). MUC4 has been recently proven to be a novel intramembrane ligand for receptor tyrosine kinase ErbB2 (21–23). Ascites sialoglycoprotein-2, a trans-membrane subunit of MUC4, showed specific binding to ErbB2, and MUC4 binding to ErbB2 induced tyrosine phosphorylation of the receptor (22, 23). MUC4 is the only ligand that has been characterized for ErbB2, which was stated to be an orphan receptor with no known ligand (21, 23, 40). Furthermore, although MUC4 alone failed to activate mitogen-activated kinase or protein kinase B/Akt of the phosphatidylinositol 3-kinase pathway, MUC4 expression induced up-regulation of p27. A combination of MUC4 and neuregulin down-regulated p27 and activated protein kinase B/Akt (23, 41). In our present immunohistochemical study, MUC4 expression was not significantly related to ErbB2 or p27 expression in EHBDC. From the comparison of the three factors in the present immunohistochemical study, these three proteins may be controlled and regulated under unknown mechanisms in EHBDC. In intrahepatic cholangiocarcinoma, ErbB2 expression has been reported (42–45). ErbB2 is not expressed in normal biliary epithelium (43, 44). Aishima et al. (44) reported that there was no significant difference in survival between ErbB2-positive and ErbB2-negative patients. The results of the present study support their data. The expression of p27 in intrahepatic cholangiocarcinoma has also been reported (41, 46, 47). As shown also in the present study, p27 is expressed in normal biliary epithelium (41, 46, 47). Fiorentino et al. (46) and Taguchi et al. (47) reported that p27 expression was a prognostic factor in intrahepatic cholangiocarcinoma. In the present study, however, p27 expression was not a significant prognostic factor in EHBDC.

In the MUC4, ErbB2, and p27 investigated in the present study, only MUC4 expression was revealed as a significant independent prognostic factor in the patients with EHBDC. In a recent study using a breast cancer cell line (48), MUC4 was expressed at a higher level in a Herceptin (a monoclonal antibody against a membrane-proximal epitope in the extracelular region of erbB2)–resistant cell line compared with Herceptin-sensitive cell line. Knockdown of MUC4 expression by RNA interference increased binding of Herceptin by exposing ErbB2 that was covered with large size MUC4. In the present immunohistochemical analysis study of EHBDC using a large number of samples (70 cases), however, there was no significant relationship between the expression of MUC4 and ErbB2. There may be a difference between the cascade reaction in vitro and the immunohistochemical localization in vivo in tumor tissues. The relationship between the expression of MUC4 and ErbB2 in the immunohistochemical study of human breast cancer tissue would be interesting to study in the near future.

Our previous study showed that MUC1 expression in EHBDC is a poor prognostic factor (15). This was also confirmed in the present study. In our observation, MUC1 was expressed in the luminal surface membrane and/or in the cytoplasm. In contrast, MUC4 expression showed a cytoplasmic pattern, but it did not show a membrane pattern. Also, in the intrahepatic cholangiocarcinoma mass-forming type, the expression pattern of MUC4 showed a cytoplasmic pattern (37). Like MUC1, MUC4 is a membrane mucin; however, MUC4 acts with a different mechanism. For signaling molecules, MUC1 acts as a docking protein, whereas MUC4 acts as a receptor ligand (23). The difference of expression patterns may suggest the possibility of different mechanisms between MUC1 and MUC4, although both MUC1 and MUC4 are membrane mucins possessing cell signaling function.

In conclusion, we first reported that the expression of MUC4 in EHBCD is an independent poor prognostic factor. MUC4 is hence a useful marker for predicting the outcome of patients with EHBDC who had a surgical resection of the tumor. The patients with EHBDC showing a positive MUC4 expression should be followed-up carefully.

	Acknowledgments

 
We thank Yoshiharu Atsuji, Yoshiko Arimura, and Yukari Nishimura for their excellent technical assistance.

	Footnotes

 
Grant support: Ministry of Education, Science, Sports, Culture, and Technology, Japan, grants 17015038 (S. Yonezawa) and 17109008 (K. Imai); and USPHS grant CA 78590 from the NIH (S.K. Batra).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/27/05; revised 3/30/06; accepted 4/19/06.

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