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Keywords:

  • cholangiocarcinoma;
  • mucin;
  • MUC5AC;
  • tumor marker;
  • bile duct;
  • prognosis;
  • survival;
  • serum;
  • blood group;
  • neoplasm

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

The authors recently showed that MUC5AC mucin, which is expressed aberrantly in tumor tissue, is present in significant concentrations in serum from patients with cholangiocarcinoma. Subsequently, determination of serum MUC5AC had high sensitivity and specificity for cholangiocarcinoma. In this study, the possible association between serum MUC5AC mucin and the clinical findings of the patients and their prognostic value were explored.

METHODS

The expression of MUC5AC mucin in serum samples from 179 patients with histologically confirmed cholangiocarcinoma were determined using immunoblotting.

RESULTS

Detection of serum MUC5AC was associated with patients with blood group Type A, larger-sized tumors (> 5 cm), and advanced-stage disease. Patients who had positive serum MUC5AC status had a significantly poorer prognosis (median survival, 127 days; 95% confidence interval [95% CI], 107–180 days) compared with patients who had negative serum MUC5AC status (median survival, 329 days; 95% CI, 199–458 days; P < 0.001). Multivariate analysis with adjustment for all covariates showed that patients who had positive serum MUC5AC status had a 2.5-fold higher risk of death compared with patients who had negative serum MUC5AC status (P < 0.001).

CONCLUSIONS

Serum MUC5AC was associated with tumor burden. The determination of serum MUC5AC may be predictive of poor patient outcome and may be useful in selecting possible treatment options for patients with cholangiocarcinoma. Cancer 2003;98:1438–43. © 2003 American Cancer Society.

DOI 10.1002/cncr.11652

Cholangiocarcinoma (CCA) is a relatively rare tumor, although it is second among primary malignant liver tumors. Its incidence is highest in Laos and Northeast Thailand, where infection with the liver fluke, Opisthorchis viverrini, is endemic. The long-term survival of patients with bile duct carcinoma remains far from satisfactory because of the difficulty of curative resection, even with advances in surgical technology.1–3

Mucins are heavily glycosylated glycoproteins, the major components of the mucous viscous gel covering the surface of epithelial tissues. Sixteen distinct epithelial mucin genes, designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, and MUC17, have been identified4–8 and are divided into secreted (MUC2, MUC5AC, MUC5B, MUC6, and MUC7) and membrane-bound (MUC1, MUC3, MUC4, MUC13, MUC15, MUC16, and MUC17) forms. Secreted (or gel-forming) mucins play an important role in lubrication, protection, and formation of a selective barrier of epithelial surfaces. They are produced throughout the entire gastrointestinal tract, mammary and salivary glands, the pancreas and gallbladder, the respiratory tract, and reproductive organs.9 The membrane-bound mucins apparently serve a protective function, protecting the glycoproteins from cell surface proteolysis and protecting the cells from attack by other cells. However, the exact function of this group of mucins is not understood fully.

In many human carcinomas, the expression profile of mucins is altered; certain mucins are up-regulated, whereas others are down-regulated.4, 10–13 Some mucins are correlated with a poor prognosis and with increased metastatic potential in certain malignancies.4, 5, 9

Biliary epithelial cells in the intrahepatic large bile ducts constantly express MUC3, MUC6, and MUC5B apomucin; whereas MUC5AC, a secretory mucin found in abundance in the gastric mucosa, is expressed rarely.14–16 However, aberrant and altered mucin expressions occur in CCA. Extensively expressed MUC1 apomucin, focally expressed MUC2 apomucin, and frequently expressed MUC5AC have been documented.17–19

We recently showed that MUC5AC apomucin originating from CCA tissues can be detected in patient serum with high sensitivity and specificity.19 In the current study, we analyzed the association of MUC5AC found in serum with the clinical and pathologic findings and with survival in patients with CCA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients

Between 1998 and 1999, we obtained serum samples prospectively from 179 consenting patients who presented to the Department of Surgery, Faculty of Medicine, Khon Kaen University (Khon Kaen, Thailand), for the definitive treatment of CCA. Serum was drawn before any surgical or drug treatment and was stored at − 20 °C until analyzed. Informed consent was obtained from each patient, and the Human Research Ethics Committee at Khon Kaen University approved the research protocol. Blood group typing and concurrent liver function tests (total protein, albumin, total bilirubin, alkaline phosphatase, and transaminases) were performed by the hospital laboratory using standard protocols. The diagnosis of CCA was verified histologically. The International Union Against Cancer TNM classification and staging system20 was used for tumor assessment. Tumor size was calculated using the greatest perpendicular dimensions of each liver lesion and was evaluated only in 65 patients with mass-forming type CCA who underwent hepatectomy. The survival of each patient with CCA was recorded after surgery until May 15, 2001; 151 patients (84.35%) had died by the end of the follow-up period.

Chemicals

Polyclonal antiserum (MAN-5ACI) was raised against the synthetic peptides coupled to the keyhole limpet hemocyanin from the specific sequence within the MUC5AC mucin.21 Goat antirabbit immunoglobulin G (IgG)-peroxidase was produced by Zymed Laboratories (San Francisco, CA). The chemiluminescence reagent kit and the Hyperfilm and Hybond nitrocellulose membranes were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Agarose Gel Electrophoresis and Immunoblotting

Serum MUC5AC levels were determined qualitatively using agarose gel electrophoresis and immunoblotting, as described by Sheehan et al.22 Serum was treated with loading buffer to a final concentration of 0.04 Mol/L Tris-acetate, pH 8.0; 0.005 Mol/L ethylenediamine tetraacetic acid; 0.1% sodium dodecyl sulfate; and 0.6 Mol/L urea. The treated sample was subjected to 0.7% agarose gel electrophoresis and vacuum-transferred to a nitrocellulose membrane. The membrane was incubated with 1:10,000 MAN-5ACI antisera and 1:8000 goat antirabbit IgG-peroxidase. MUC5AC was visualized by incubated membrane with Chemiluminescence Reagent Plus and was exposed to the Hyperfilm with an intensifying screen. The sample that yielded immunoreactivity on the film was interpreted as positive for serum MUC5AC. The specificity of the MUC5AC antibody was confirmed when no immunoreactivity was observed, i.e., when the antibody was omitted from the detection system.

Statistical Analysis

Statistical analyses for comparisons between clinicopathologic findings, survival, and MUC5AC expression were performed using STATA software (Stata Corp., College Station, TX). The association of two categoric variables was analyzed using the chi-square test or the Fisher exact probability test. Multivariate analysis for variables that were influenced by the presence of MUC5AC in serum was carried out using a logistic regression model. Potentially predictive variables that were identified at a significance level of P < 0.25 were used for the multivariate logistic regression analysis. The log (likelihood ratio) test was used for backward elimination and to assess the fit of the model.

Patient survival was calculated from the time patients underwent surgical resection to either death or most recent contact. The survival curves were generalized by using the Kaplan–Meier method and were compared using the log-rank test. Several clinicopathologic factors were subjected to univariate and multivariate analyses using the Cox proportional-hazard regression model. The relative risk of death was compared using the assessment of hazard ratio. Differences were considered significant at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Of 179 patients with CCA who were examined, 71.5% were men, and the ratio of males (n = 128) to females (n = 51) was 2.5:1.0. The median patient age was 56 years (range, 33–84 years). Peripheral or intrahepatic CCA was the major tumor type (49.7%), 29.4% of tumors were central or extrahepatic CCA, and 20.9% of tumors were a combination of both types. Most tumors (89%) were advanced-stage lesions (Stage IVA or IVB).

Correlation between Serum MUC5AC and Clinicopathologic Features

Using univariate analysis, the association of serum MUC5AC in patients with CCA and blood analysis was determined. The expression of serum MUC5AC had no association with age, gender, body mass index, blood group, serum markers of liver functions, parameters of complete blood count, or clinicopathologic findings (data not shown). Two variables, tumor stage and tumor size, were identified as statistically significant (Table 1). The proportion of patients with positive serum MUC5AC status was significantly higher in patients who had Stage IVB disease compared with patients who had disease in other stages (P = 0.009). The detection of serum MUC5AC apomucin was more frequent in patients who had larger tumors (> 5 cm; P = 0.01).

Table 1. Correlation between Expression of Serum MUC5AC and Clinicopathologic Features of Patients with Cholangiocarcinoma
VariableNo. of patientssMUC5ACP value
NegativePositive
  1. sMUC5AC: serum MUC5AC (secretory mucin of the gastric mucosa).

Age (yrs)    
 ≤569940590.360
 >56802753
Gender    
 Male12851770.290
 Female511635
Blood group    
 O5223290.356
 A29722
 B792950
 AB1358
Tumor type    
 Central5225270.110
 Peripheral883157
 Combined371027
Histologic grading    
 Papillary2813150.852
 Well-differentiated783048
 Moderately differentiated1358
 Poorly differentiated1248
TNM stage    
 I–III201280.009
 IVA462224
 IVB1123379
Tumor size (cm)    
 ≤5221480.010
 >5431330

To identify the independent variables that were related to the expression of serum MUC5AC in patients with CCA, logistic regression analysis was performed. Factors that were identified at a significance level of P < 0.25 in the univariate analysis were included in the logistic regression analysis. Table 2 shows that blood group Type A and Stage IVB tumors were related independently to the expression of MUC5AC in serum samples from patients with CCA, with adjusted odds ratios of 3.24 and 3.20, respectively.

Table 2. Multivariate Analysis Using the Logistic Regression Model for the Detection of Serum MUC5AC in Patients with Cholangiocarcinoma
VariableAdjusted OR95% CIP value
  1. OR: odds ratio; 95% CI: 95% confidence interval.

Age (yrs)   
 ≤561.000.135
 >561.680.85–3.34
Gender   
 Male1.000.532
 Female1.270.60–2.65
Blood group   
 O1.000.007
 A3.241.08–9.73
 B1.380.64–2.97
 AB1.080.29–4.05
Tumor type   
 Central1.000.361
 Peripheral1.520.69–3.37
 Combined1.200.74–5.40
TNM stage   
 I–III1.000.039
 IVA1.580.51–4.92
 IVB3.201.12–9.15

Correlation between Serum MUC5AC and Cumulative Survival Rate

Cumulative survival was compared in patients with primary CCA between patients who had positive serum MUC5AC status and patients who had negative serum MUC5AC status. Patients who survived for < 30 days were considered to have died perioperatively and thus were excluded from the analysis. Patients with positive serum MUC5AC status had a significantly poorer prognosis (median survival, 127 days; 95% confidence interval [CI], 107–180 days) compared with patients who had negative serum MUC5AC status (median survival, 329 days; 95% CI, 199–458 days; P < 0.001) (Fig. 1A).

thumbnail image

Figure 1. Survival curves generated using the Kaplan–Meier method. (A) Survival curves showed that patients with cholangiocarcinoma who were positive for serum MUC5AC had a less favorable prognosis compared with patients who were negative for MUC5AC (P < 0.001). (B) In the subgroup of patients with Stage IVB cholangiocarcinoma, positive serum MUC5AC status was correlated with a lower survival probability compared with negative serum MUC5AC status (P = 0.0003). Tick marks indicate censored patients.

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Nonetheless, a bias may have been introduced into the current analysis by considering the different stages of disease together. Comparisons of the cumulative survival curves according to tumor stage demonstrated that patients who had Stage IVB CCA had a significantly poorer prognosis compared with patients who had Stage IVA or lower CCA (P < 0.001). To eliminate such a bias, cumulative survival rates were compared according to serum MUC5AC status in patients with CCA tumors in the same stage. Among patients with Stage IVA tumors, the median survival for patients with positive serum MUC5AC status was 172 days, compared with 336 days for patients with negative serum MUC5AC status. However, no statistically significant difference was demonstrated between the two groups (P = 0.143). Among patients with Stage IVB tumors, patients who had positive serum MUC5AC status had a significantly poorer prognosis compared with patients with negative serum MUC5AC status, with a median survival of 116 days versus 329 days, respectively (P < 0.0003) (Fig. 1B).

Multivariate Analysis of Prognostic Factors

The Cox proportional-hazards regression model was used to assess the effects of different variables on patient survival. The 18 prognostic factors and serum MUC5AC were analyzed. All factors with P < 0.25 were put in the initial Cox model using backward stepwise selection. Finally, six independent variables (i.e., serum MUC5AC, age, gender, histologic grade, tumor stage, and lymph node resection) were identified as significant for the prediction of survival and were included in the final Cox regression analysis (Table 3). Multivariate analysis with adjustment for all covariates showed that patients who had positive serum MUC5AC status had a 2.5-fold higher risk of death compared with patients who had negative serum MUC5AC status (P < 0.001).

Table 3. Results of Multivariate Analysis Using a Cox Proportional Hazards Regression Model
VariableAdjusted HR95% CIP value
  1. HR: hazard ratio; 95% CI: 95% confidence interval; sMUC5AC: serum MUC5AC (secretory mucin of the gastric mucosa).

sMUC5AC   
 Negative1.00<0.001
 Positive2.501.50–4.16
Age (yrs)   
 ≤560.771
 >561.070.69–1.66
Gender   
 Male0.213
 Female0.740.46–1.20
Histologic grading   
 Papillary<0.001
 Well-differentiated2.031.06–3.87
 Moderately differentiated1.630.70–3.77
 Poorly differentiated2.410.98–5.92
TNM stage   
 I–III0.009
 IVA1.550.66–3.64
 IVB2.661.24–5.74
Lymph node resection   
 Yes<0.001
 No1.630.99–2.68

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

CCA is an uncommon malignancy for which no specific tumor marker has been found to date. Our previous study showed that MUC5AC mucin, an aberrant mucin produced by bile duct tumors, was detected in the serum of patients with CCA with high specificity and sensitivity.19 In the current study, we demonstrated that MUC5AC apomucin was detected in the sera of patients with CCA regardless of age, gender, blood group, tumor location, histologic tumor type, or clinical features of the patients.

In contrast, the relation between serum MUC5AC and tumor burden was characterized. Patients who had positive serum MUC5AC status seemed to harbor larger tumors compared with patients who had negative serum MUC5AC status. In addition, when the proportion of patients with positive serum MUC5AC status was assessed in accordance with the comprehensive staging, it was found that the rates increased gradually with the stage. Among patients with Stage III or lower CCA, the positive rates were 40%, but the rates increased significantly to 52% for patients with Stage IVA CCA and to 70% for patients with Stage IVB CCA (P = 0.009). The univariate and multivariate analyses using logistic regression also revealed a different frequency of expression for MUC5AC in patients with Stage IVB CCA tumors. This observation suggested the possible role of MUC5AC in the development of tumor metastasis.

Blood group isoantigens of the ABH systems are represented by a variety of glycoproteins and glycolipids, the antigenic specificity of which is determined by variation in their constituent carbohydrate chains.23 Multivariate analysis using the logistic regression in the current study showed significant relation of blood group Type A and the detection of MUC5AC in serum compared with other blood groups when blood group Type O was used as the reference group. The association of blood group and MUC5AC mucin expression has not been reported to date. However, differences in the cumulative survival rates of patients with CCA in different blood groups were not observed in this study (P = 0.69). Further investigation will be needed to explain the possible molecular and genetic basis for this correlation.

Mucins are the main components of mucus and were believed to have the unique function of protecting and lubricating the epithelial surfaces. The study of mucin structure and the correlation between structure and function show that mucins have an important role in growth; fetal development; epithelial renewal, differentiation, and integrity; carcinogenesis; and metastasis.

A number of studies have reported the importance of mucin as a prognostic factor for several types of malignant disease.24–28 In the current study, the expression of MUC5AC was an independent prognostic variable for survival in patients with CCA. Patients who had positive serum MUC5AC status had a significantly poorer survival prognosis compared with patients who had negative serum MUC5AC status (P < 0.001). This poor outcome may not have been due to the high proportion of patients with advanced-stage disease in the MUC5AC positive group, because a significant difference in the survival rate between the two groups still was demonstrated in patients with Stage IVB CCA, even when the patients were stratified according to disease stage. The presence of MUC5AC in serum was correlated with tumor size and metastatic features, suggesting that these variables may be responsible for the poor outcome of patients with CCA who have positive serum MUC5AC status.

The link between MUC5AC expression and malignant progression is significant in two ways. First, MUC5AC is negatively-charged, and cells that express high levels may repel each other and enhance cell migration. Second, the highly viscous gel formed by MUC5AC surrounds the tumor emboli and may protect the tumor from proteolysis and limit the escape of immunogenic cells. Results from experimental data may support this possibility.

The high sensitivity and specificity of serum MUC5AC in the determination of CCA19 and the association of serum MUC5AC with tumor burden, regardless of the histologic grade and tumor type, allow the potential use of serum MUC5AC as a serologic marker for all types of CCA. The determination of serum MUC5AC levels in conjunction with a imaging tests, such as ultrasound or computed tomography scanning, or other biochemical tests (i.e., CA19-9 or carcinoembryonic antigen), may be used to enhance the diagnostic accuracy of CCA in the investigation of patients with a known liver mass or cyst and for monitoring after patients undergo resection or receive chemotherapy for CCA. In addition, serum MUC5AC may be useful in predicting patient outcome and in selecting appropriate treatment options.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Mr. Bryan Roderick Hamman for assistance with the English-language presentation of the article.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES