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10.1245/ASO.2004.03.086
Annals of Surgical Oncology 11:40-44 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Correlation Between MUC5AC Expression and the Prognosis of Patients With Adenocarcinoma of the Uterine Cervix

Akira Mitsuhashi, MD, Kouji Yamazawa, MD, Yuuichiro Nagai, MD, Naotake Tanaka, MD, Hideo Matsui, MD and Souei Sekiya, MD

From the Departments of Reproductive Medicine (AM, KY, NT, HM, SS) and Molecular Pathology (YN), Graduate School of Medicine, Chiba University, Chiba, Japan.

Correspondence: Address correspondence and reprint requests to: Akira Mitsuhashi, MD, Department of Reproductive Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan; Fax: 81-43-226-2122; E-mail: makira-cib{at}umin.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Mucin expression shows some correlation with prognosis in patients with various cancers. However, few studies have been conducted on adenocarcinomas of the uterine cervix.

Methods: An immunohistochemical study with a monoclonal anti-MUC5AC antibody, 45M1, was performed on 47 adenocarcinomas of the uterine cervix and on 40 specimens of normal endocervical epithelium. The correlations between clinicopathologic variables and MUC5AC expression were evaluated in the cervical adenocarcinomas.

Results: A significant reduction of MUC5AC expression was evident in the adenocarcinomas of the cervix in comparison with that in the normal endocervical epithelium (53.2% vs. 100%; P < .001). MUC5AC expression was correlated with paracervical invasion and histological type. Patients with negative MUC5AC expression showed poorer survival than those with positive MUC5AC expression (P = .03). However, multivariate analysis revealed that only the depth of invasion was an independent prognostic variable.

Conclusions: MUC5AC expression was suppressed in adenocarcinoma of the uterine cervix. Absence of MUC5AC expression seems correlated with worse survival.

Key Words: Mucin • MUC5AC • Immunohistochemistry • Cervical adenocarcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucins are glycoproteins that have a high degree of O-glycosylation and that are major components of the viscous mucous gel that covers the surfaces of epithelial tissues.1 At least 14 MUC genes (MUC1–MUC13) have been identified recently.2–9 Under normal physiological conditions, MUCs are known to offer some protection of adjoining epithelial tissues. However, in various instances, changes in the biochemical characteristics of MUCs were shown to occur in both preneoplastic and neoplastic lesions.10,11

Aberrant expression of MUCs in epithelial tissues showing malignant changes is also well known. Several studies have described altered synthesis, secretion, and structure of MUCs in various cancers. In pancreatic cancers, both MUC2 messenger RNA (mRNA) and protein are expressed in noninvasive pancreatic tumors, but not in invasive cancers.12 MUC4 mRNA is expressed in pancreatic adenocarcinomas but not in benign pancreatic tissues.13 MUC1 expression is associated with poor outcome and MUC2 expression with favorable outcome in patients with gastric cancer.14 Expression of MUC7 mRNA was detected in bladder cancer cell lines and invasive transitional cell carcinomas, but neither in superficial, noninvasive bladder tumors nor in normal bladder urothelium by reverse-transcription-polymerase chain reaction.15 These reports suggest that the upregulation of the expression of various MUC genes that is concomitant with abnormal glycosylation patterns may be associated with the onset of malignant transformation, tumor invasion, and metastatic potential.

MUCs play an important role in the defense of the female reproductive tract against invaders.16 Endocervical RNA samples showed positive binding of the probes to MUCs 1, 4, 5AC, 5B, and 6 and confirmed the infrequent presence of MUC2 and the absence of MUC3 and MUC7. The MUC5AC message was confined to the endocervical tissue.17,18 The normal mucinous epithelium of the endocervix consistently expresses MUC5AC, but not MUC2.18,19

Little is known about the expression of MUC in adenocarcinomas of the uterine cervix. In this study, we focused on MUC5AC expression in cervical adenocarcinoma. We evaluated the association between expression of these MUCs and clinicopathologic parameters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
A total of 47 consecutive patients with adenocarcinoma of the uterine cervix treated at the Chiba University Hospital between 1990 and June 1999 were examined. All patients were staged before surgery by following the guidelines of the International Federation of Gynecology and Obstetrics (FIGO). The primary tumors included 47 cases of invasive adenocarcinoma of the uterine cervix, which were classified with the current World Health Organization classification scheme into stages IA (n = 6), IB (n = 25), IIA (n = 4), and IIB (n = 12). The histological types of these tumors were as follows: endocervical type, n = 32; endometrioid type, n = 8; adenosquamous type, n = 4; and other types, n = 3. Sections of 40 samples of normal endocervical epithelium were used as control.

A chart review was performed to record the tumor pathology, stage, therapies, and clinical outcome for each patient. The median follow-up time of the surviving patients was 58 months (range, 7–156 months). The tumor size was determined from the clinical record or pathologic slides. Depth of invasion was categorized as less than two thirds or two thirds or more of cervical stromal invasion.20,21 All patients with the exception of three stage IA patients were treated with radical hysterectomy and pelvic lymphadenectomy. Adjuvant radiotherapy was administered to 14 patients with stage I and 14 with stage II tumors. Adjuvant chemotherapy was given to six patients, three of whom were also treated with radiation.

Immunohistochemistry
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections. Three-micrometer-thick sections were stained with the monoclonal anti-MUC antibody by using an avidin-biotin peroxidase complex kit (Vector Laboratories, Burlingame, CA). Anti-MUC5AC (NeoMarkers, Fremont, CA), a mouse monoclonal antibody (clone 45MI) to a peptide core corresponding to a site on the MUC5AC human core protein, was used at a dilution of 1/100. Sections of normal gastric mucosa (MUC5AC) were used as positive controls. As a negative control, phosphate-buffered saline was substituted for the primary mouse antibody for slide incubation.

All sections were examined by three observers (A.M., K.Y., and Y.N.) by using a conference microscope. The sections were scored according to a consensus of the three observers regarding the overall proportion of positively stained tumor cells. The results of the antibody staining were evaluated by the percentage of positively stained neoplastic cells; positive was considered staining of at least 5% of the cells.14,22 Three degrees of intensity of staining were used: 1+, weaker than positive control; 2+, same as positive control; and 3+, strong staining.

Statistical Analysis
Statistical analysis was performed with the SPSS computer program package (version 10; SPSS Inc., Chicago, IL). Fisher’s exact test or the {chi}2 test was used to determine whether there were significant differences in the frequency of MUC5AC expression between cancer tissues and normal endocervix. Kaplan-Meier analysis was used to determine any correlation between MUC5AC expression and patient disease-free survival. The relationship between the clinicopathologic variables and disease-free survival was assessed by log-rank test. Multivariate analysis was performed with the Cox proportional hazards model. Probability values <.05 were regarded as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The expression of MUC5AC in normal endocervical epithelium and adenocarcinoma of the uterine cervix is shown in Table 1. Normal endocervical epithelium revealed a diffuse pattern of immunoreactivity for MUC5AC in all cases. MUC5AC was distributed mainly in the cytoplasm (Fig. 1). There was an appreciable loss of the expression of MUC5AC in the adenocarcinomas (53.2%; P < .001). Cases of the endocervical type also showed a cytoplasmic pattern of expression (Fig. 2). The loss of expression of MUC5AC was more prominent in the nonendocervical types than in the endocervical type (Table 2).


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  		TABLE 1. MUC5AC in normal endocervical epithelium and adenocarcinoma of the uterine cervix
 


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  		FIG. 1. Expression of MUC5AC in normal glandular epithelium.

 


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  		FIG. 2. Endocervical adenocarcinoma with a strong cytoplasmic staining pattern of MUC5AC.

 

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  		TABLE 2. Relationship between MUC5AC expression and various clinical variables in cervical adenocarcinoma patients
 
We evaluated the relationships between clinicopathologic parameters and MUC5AC expression in adenocarcinoma of the uterine cervix. Loss of MUC5AC expression was more likely to occur in cases of paracervical invasion (P = .04). However, the MUC5AC expression did not correlate significantly with tumor size, lymph node metastasis, depth of invasion, or lymph-vascular space invasion (Table 2).

The MUC5AC expression in disease-free survival was examined by using Kaplan-Meier analysis (Fig. 3A). The disease-free 5-year survival rate was 84% for MUC5AC-positive and 50.6% for MUC5AC-negative cases (P = .03). Stratified by stage, for stage I and II disease, absence of MUC5AC expression was associated with worse prognosis (P = .04; Fig. 3B).



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  		FIG. 3. Survival curves with the Kaplan-Meier method. (A) Disease-free survival decreased from 84% to 50.6% in MUC5AC-negative tumors (P = .03). (B) In every stage, MUC5AC negativity correlated with a poor survival probability compared with MUC5AC positivity (P = .04).

 
Factors influencing recurrence-free survival were FIGO stage, lymph node status, depth of invasion, tumor size, paracervical invasion, lymph-vascular space invasion, and MUC5AC expression. However, multivariate analysis identified depth of invasion as the only statistically significant prognostic variable (P = .01; Table 3).


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  		TABLE 3. Cox regression analysis (dependent variable: disease-free survival)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The normal endocervical epithelium consistently expresses MUC5AC proteins intensely and diffusely.18,19 However, the expression of MUC5AC was diminished in most neoplastic glandular lesions. The persistence of MUC5AC expression was sufficient to identify these neoplasms as originating from the endocervical glands.19 The decreased expression of MUC5AC MUC in adenocarcinomas of the uterine cervix was also confirmed in this study.

Mucin glycoproteins function as cell-cell adhesion molecules, thereby mediating the adhesion and migratory capability of cells. Hilkens et al.23 demonstrated that alteration in mucins may decrease the ability of neoplastic cells to aggregate, thereby facilitating dissociation of tumor cells from the primary tumor site. However, little is known about the function of MUC5AC. Alterations in the expression of mucin glycoproteins may offer cancer cells a better chance of survival and enhance their ability to metastasize.24

Although all reproductive tract epithelia express MUC1, MUC5AC cannot be consistently detected in the endometrial epithelia.18 In this study, the loss of expression of MUC5AC was more prominent in the nonendocervical type than in the endocervical type of adenocarcinoma of the uterine cervix. This suggests that as the cervical epithelium loses the endocervical phenotype, its malignant potential is increased.

A relationship between MUC5AC expression and the biological behavior of tumors has previously been shown in several carcinomas. Patients with MUC5AC mRNA expression in pancreatic tumors had significantly better survival than those with no such expression.25 In colorectal carcinomas, the absence of MUC5AC expression in tumors can be a prognostic factor for a more aggressive form.26 MUC5AC is generally reduced in carcinomas compared with normal or reactively altered tissues,22,27,28 and Baldus et al.29 showed that the loss of MUC5AC immunoreactivity is an independent prognostic marker for worse survival of patients with gastric carcinoma.

In cervical carcinomas, the relationship between clinicopathologic parameters and MUC5AC expression has not been examined. In this study, we confirmed the correlation of MUC5AC expression with paracervical invasion. However, our results showed no correlation of MUC5AC expression with any other clinicopathologic prognostic factor. Disease-free survival of MUC5AC-negative patients was decreased in comparison with that in MUC5AC-positive patients in every FIGO stage. Multiple studies have been undertaken to delineate prognostic factors for adenocarcinoma of the uterine cervix. Some clinicopathologic parameters, such as lymph node metastasis, lymph-vascular space invasion, and depth of invasion, were considered independent risk factors of recurrence.21,30 Flowing from this, because other parameters associated with adverse outcome—including node status, lymph-vascular space invasion, and depth of invasion—are unknown until after surgical staging, identification of low expression of MUC5AC may be helpful for determining those cases at increased risk for adverse outcome before or without surgical staging. This is particularly exciting because this could be a useful marker for minimally invasive disease to further distinguish patients who require aggressive therapy from those who could be treated conservatively.

In conclusion, the absence of MUC5AC expression may be associated with poor prognosis in adenocarcinomas of the uterine cervix. Clearly, more cases with longer follow-up need to be evaluated before further conclusions can be made about whether the expression of MUC5AC is a useful prognostic marker of poor outcome.


   FOOTNOTES
 
An immunohistochemical study with a monoclonal anti-MUC5AC antibody was performed on 47 adenocarcinomas of the uterine cervix. MUC5AC expression was suppressed in adenocarcinoma of the uterine cervix. Absence of MUC5AC expression seems correlated with worse survival.

Received for publication March 26, 2003. Accepted for publication August 25, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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