Prognostic value of mucin 4 expression in colorectal adenocarcinomas

Authors

  • Chandrakumar Shanmugam MD,

    1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
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  • Nirag C. Jhala MD,

    1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
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  • Venkat R. Katkoori PhD,

    1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
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  • Wen Wan PhD,

    1. Department of Preventive Medicine, University of Alabama at Birmingham, Birmingham, Alabama
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  • Sreelatha Meleth PhD,

    1. Department of Preventive Medicine, University of Alabama at Birmingham, Birmingham, Alabama
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  • William E. Grizzle MD, PhD,

    1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
    2. Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama
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  • Upender Manne PhD

    Corresponding author
    1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
    2. Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama
    • Department of Pathology, University of Alabama at Birmingham K515B1-Kracke Building, 1922, Seventh Avenue South, Birmingham, AL 35294-7331===

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    • Fax: (205) 934-4418


Abstract

BACKGROUND:

Mucin 4 (MUC4) is aberrantly expressed in colorectal adenocarcinomas (CRCs) but its prognostic value is unknown.

METHODS:

Archival tissue specimens collected from 132 CRC patients who underwent surgical resection without presurgery or postsurgery therapy were evaluated for expression of MUC4 by using a mouse monoclonal antibody and horseradish peroxidase. MUC4 expression levels were correlated with clinicopathologic features and patient survival. Survival was estimated by both univariate Kaplan-Meier and multivariate Cox regression methods.

RESULTS:

In both normal colonic epithelium and CRCs, MUC4 staining was localized primarily in the cytoplasm. The optimal immunostaining cutoff value (≥75% positive cells and an immunostaining score ≥2.0), which was derived by using the bootstrap method, was used to categorize CRCs into groups of high expression (33 of 132 patients; 25%) or low expression (99 of 132 patients; 75%). Patients who had early stage tumors (stages I and II) with high MUC4 expression had a shorter disease-specific survival (log-rank; P = .007) than patients who had with low expression. Patients who had advanced-stage CRCs (stages III and IV) did not demonstrate such a difference (log-rank; P = .108). Multivariate regression models that were generated separately for patients with early stage and advanced-stage CRC confirmed that increased expression of MUC4 was an independent indicator of a poor prognosis only for patients who had early stage CRCs (hazard ratio, 3.77; 95% confidence interval, 1.46-9.73).

CONCLUSIONS:

The current results indicated that increased MUC4 expression is a predictor of poor survival in CRC, specifically for patients who have early stage tumors. Cancer 2010. © 2010 American Cancer Society.

Mucins are complex glycoproteins that are subdivided into 2 structural and functional classes: secreted, gel-forming mucins (mucin 2 [MUC2], MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC11, MUC12, and MUC17). The products of some MUC genes (MUC7, MUC8, MUC9, MUC13, MUC15, and MUC16) do not fit well into either class.1 In addition to being protective at the surface epithelium, altered expression of some of these mucins is associated with neoplastic progression and metastasis of various cancers, including colorectal cancers (CRCs).1-3

MUC4 is a transmembrane mucin located at chromosome locus 3q29. Although originally considered to be of tracheobronchial origin, it is located in normal stomach, ovary, salivary gland, colon, lung, uterus and prostate.1 MUC4 expression in lungs occurs before organogenesis; in the jejunum and colon, it is expressed at 6.5 weeks of gestation.4 In the esophagus, expression correlates with stages of squamous cell differentiation.4 MUC4 has a mucin-like subunit, MUC4α, which has glycosylated tandem repeats, and a MUC4β subunit, which consists of 3 epidermal growth factor (EGF)-like domains and a short cytoplasmic tail.5 MUC4 expression is regulated in part by pathways associated with interferon-γ, retinoic acid, and transforming growth factor-β signaling.4

MUC4 is implicated in the induction of ultrastructural changes in the transformation of normal epithelium and with tumorigenicity.6 It also is implicated in reducing accessibility of the tumor cell surface antigen to cytotoxic immune cells, thus aiding in evasion of the host immune response.7 With its EGF-like domains, MUC4 acts as a modulator of the human epidermal growth factor receptor 2 (HER2/ErbB2) receptor tyrosine kinase and potentiates tumorigenesis and/or tumor growth in pancreatic carcinoma4, 8 and gallbladder carcinoma.9 Furthermore, MUC4 overexpression confers apoptotic resistance to tumor cells.10, 11 These characteristics indicate that MUC4 has multiple roles in tumor development and progression.

Aberrant expression of MUC4 has been reported in malignancies of the gallbladder,9 biliary tract,12 lung,13, 14 salivary gland,15-17 pancreas,18, 19 ovary,18-20 and prostate.21 The prognostic significance of MUC4 expression is tissue-dependent and varies with the type of malignancy; for example, its expression in mucoepidermoid carcinomas of the salivary gland is associated with improved patient survival and late disease recurrence.15, 17 Its expression in lung adenocarcinomas, however, is associated with increased recurrence of disease and poor survival.14 Since the prognostic significance of MUC4 expression had not been studied previously in colorectal adenocarcinomas (CRCs), we determined the phenotypic expression of MUC4 in retrospective CRC tissues and correlated the extent of its expression with clinicopathologic features and with patient survival based on tumor stage.

MATERIALS AND METHODS

Patient Population

The Institutional Review Board of the University of Alabama at Birmingham (UAB) approved this study. In total, 132 patients with CRC who underwent surgical resection for a “first primary” CRC at the UAB Hospital between 1981 and 1993 were identified. The sample size and power were estimated based on a previous study,12 as described below. The retrospective samples were collected from an “unselected” patient population. The use of patients from this period allowed maximized postsurgery follow-up. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks from these patients were obtained from the Anatomic Pathology Division at UAB.

Patients who had surgical margin involvement, unspecified tumor location, multiple primaries within the colorectum, or multiple malignancies; patients who had a family history of hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis; and patients who had family and/or personal histories of CRC were excluded. Based on information in patient charts, because it was difficult to identify the familial versus sporadic nature of CRCs, this retrospective cohort was a consecutive patient population. The patients who were included were those who had undergone surgery alone as a therapeutic intervention across all tumor stages (stages I-IV); and, to control for treatment bias, patients were excluded if they had received presurgical or postsurgical chemotherapy or radiation therapy. Some patients with CRCs at stages III or IV did not receive adjuvant therapy but underwent surgery with palliative intent.

Pathologic Features

The surgical pathology reports were reviewed by 3 pathologists (C.S., N.C.J., and W.E.G.) who individually reviewed slides stained with hematoxylin and eosin for the degree of histologic differentiation and regraded lesions as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated.22, 23 Disagreements regarding pathology were resolved by re-evaluating the slides to reach a consensus. Well differentiated and moderately differentiated tumors were pooled into a low-grade group, and poorly differentiated and undifferentiated tumors were pooled into a high-grade group.24 Pathologic staging was performed according to American Joint Committee on Cancer criteria.25 International Classification of Diseases for Oncology codes were used to specify the anatomic location of the tumor.22 A tumor was considered mucinous if ≥50% demonstrated mucinous histology.22 The anatomic subsites were the proximal colon, the distal colon, and the rectum. Three-dimensional tumor size was determined, and the greatest tumor dimension was used for statistical purposes.

Patient Demographics and Follow-Up Information

Patient demographics, along with clinical and follow-up information, were retrieved retrospectively from medical records, physician charts, and pathology reports and from the UAB tumor registry. Patients were followed either by the patients' physician or by personnel associated with the tumor registry until the date of death or the date of the last documented contact. Through telephone and mail contacts, these personnel ascertained outcome (mortality) information directly from patients (or relatives) and physicians. This information was validated by examination of the state death registry. Demographic data, including patient age at diagnosis, sex, race/ethnicity, date of surgery, date of last follow-up (if alive), date of recurrence (if any), and date of death, were collected. Collection of follow-up information, which was performed every 6 months, ended in April 2009. Laboratory investigators (C.S. and V.R.K.) were blinded to the outcome information until completion of the assays.

Immunohistochemical Analysis

Tissue sections (5 μm) were cut from FFPE blocks representative of normal and tumor tissue from each patient and were mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburg, Pa). These sections were cut 1 or 2 days before they were immunostained to avoid problems in antigen recognition because of storage.26 Immunohistochemical (IHC) staining was performed as described previously.3, 27, 28 In brief, the sections were melted, incubated overnight at 37°C, deparaffinized in xylene, rehydrated in graded alcohols, and transferred to a Tris-buffer bath (0.05 M Tris base, 0.15 M NaCl, and 0.01% Triton X-100, pH 7.6). Antigen retrieval was accomplished by placing the slides in a pressure cooker for 10 minutes with ethylene diamine tetracetic acid (EDTA) buffer (EDTA 2.1 g, H2O 1000 mL, adjusting to pH 9 with NaOH). Each section was treated with 3% H2O2 for 5 minutes to quench the endogenous peroxidase activity and incubated with 3% goat serum at room temperature for 1 hour to reduce nonspecific immunostaining. The sections were incubated with antihuman MUC4 mouse monoclonal antibody (clone 8G7) diluted at a 1:1500 ratio. This dilution was determined after initial standardization. This antibody was kindly provided by Dr. Surinder K. Batra (University of Nebraska at Omaha, Neb) and was characterized for its epitope (tandem repeat region STGDTTPLPVTDTSSV) recognition and specificity.29 Sections on which the primary antibody was not applied served as controls.

Secondary detection was accomplished with a multispecies system (Signet Laboratory Inc., Dedham, Mass). The sections were exposed to biotinylated multispecies antibodies, including antimouse antibodies, for 20 minutes and then incubated with peroxidase-labeled streptavidin for 20 minutes. A diaminobenzidine tetrachloride super-sensitive substrate kit (BioGenex, San Ramon, Calif) was used to observe the antibody-antigen complex. Each section was counterstained with hematoxylin, dehydrated by graded concentrations of alcohol, and soaked in xylene before coverslips were applied.

Slides were evaluated for MUC4 staining independently by 3 pathologists (C.S., N.C.J., and W.E.G.); if there was a discrepancy in individual scores, then all 3 pathologists re-evaluated the slides together to reach a consensus before combining the individual scores. A semiquantitative immunostaining score (ISS) for MUC4 was obtained as described previously.3, 27, 28 In brief, each pathologist estimated the proportion of cells stained and the intensity of staining in the whole section. The intensity of immunostaining of individual cells was scored on a scale from 0 (no staining) to 4 (strongest intensity). Each pathologist estimated the proportion of cells stained at each intensity. The percentage of cells at each intensity was multiplied by the corresponding intensity value to obtain an ISS ranging from 0 to 4. The scores were combined to obtain an overall mean ISS.

Statistical Methods

Sample size and power calculations

The sample size and power analysis were estimated based on a MUC4 immunohistochemistry study with extrahepatic bile duct carcinoma.12 In that study, increased expression of MUC4 was a poor prognostic indicator of survival, and 19 of 70 patients (27%) were positive for MUC4. The hazard ratio observed was 2.87 (P = .0072). Thus, we proposed to evaluate 132 samples from patients with CRC for MUC4. On the basis of the published results,12 there was enough power to detect a hazard ratio ≥2.87. Therefore, the sample size (n = 132) was sufficient to identify a statistically significant prognostic value for MUC4 expression.

Determining the optimal MUC4 immunostaining cutoff point

The bootstrap method, as described below, was used to find an optimal cutoff point of MUC4 staining that could be used to categorize tumors into groups of low and high expression. A Cox regression model was used to evaluate the results of the optimal cutoff point that was determined by this method after adjusting for other covariates as mentioned below (see Statistical Analyses). The optimal cutoff point was the 1 with the highest statistical significance of MUC4 among the Cox models.

A bootstrap resampling procedure was used to assess the internal validity of the log-rank test for cutoff point analysis. Two hundred different bootstrap samples were drawn randomly with replacement from the original dataset (n = 132). The Kaplan-Meier (KM) analysis was repeated in each sample for each cutoff point, and the log-rank test P value was recorded. The most frequent cutoff value (mode) among the optimal cutoff points was considered an optimal cutoff point that resulted in the lowest P value in the subsequent Cox regression models that were generated for each cutoff point. A cutoff point ≥75% and an ISS ≥2.0 were associated with the lowest P value of .017 and was chosen for dichotomizing tumors into those with high MUC4 expression (≥75% and an ISS ≥2.0) and low MUC4 expression (<75% and an ISS <2.0).

Statistical Analyses

Chi-square analyses were used to assess the univariate associations of baseline characteristics with MUC4 expression. The group with low expression included tumors that lacked staining. The baseline characteristics that were included in these analyses were demographic variables (age and sex), pathologic variables (tumor location, size, histologic type, differentiation, and stage), and MUC4 status. The type I error rate of each test was controlled at <0.05. All analyses were performed with the SAS statistical software package (version 9.0; SAS Institute Inc., Cary, NC).30

Survival analysis was used to model time from the date of surgery to the date of death from CRC. Deaths were the outcomes (events) of interest. Those patients who died from causes other than CRC and those who were alive at the end of the study were considered to be censored. Log-rank tests and KM survival curves31 were used to compare low and high MUC4 expression in each group of patients with early or low-stage disease (stages I and II) and advanced or high-stage disease (stages III and IV). The type I error rate of each test was controlled at <0.05.

In addition to the primary analysis described above to determine the effect of MUC4 phenotypic expression, secondary analyses were performed to consider covariates known as potential confounders or independent risk factors for death. These included age, sex, tumor location, tumor stage, tumor size, and tumor differentiation. For these analyses, Cox regression analyses32 were used within each group (early stage and advanced-stage disease), and a final Cox model was constructed that included those covariates that had P values <.05.

RESULTS

Study Cohort Characteristics

Clinicopathologic features are listed in Table 1. The mean age of the study cohort at the time of surgery was 65 years. At the last follow-up, 32 of 132 patients (24%) remained alive, 61 of 132 patients (46%) had died from CRC, and 39 of 132 patients (30%) had died from other causes.

Table 1. Clinicopathologic Features of the Study Cohort (N=132)
VariableNo. of Patients (%)
  1. CRC indicates colorectal carcinoma; MUC4, mucin 4.

Age group, y 
 <6563 (48)
 ≥6569 (52)
Sex 
 Women57 (43)
 Men75 (57)
Ethnicity 
 African American45 (34)
 Non-Hispanic Caucasian87 (66)
Tumor location 
 Proximal colon59 (45)
 Distal colon48 (36)
 Rectum25 (19)
Tumor stage 
 I23 (17)
 II52 (40)
 III40 (30)
 IV17 (13)
Tumor grade 
 Low97 (73)
 High35 (27)
Tumor size, cm 
 <546 (35)
 ≥586 (65)
Tumor type 
 Mucinous37 (28)
 Nonmucinous95 (72)
Vital status at follow-up 
 Alive32 (24)
 Death from CRC61 (46)
 Death from unknown cause or other than CRC39 (30)
Expression of MUC4 
 Low99 (75)
 High33 (25)

MUC4 Expression

The phenotypic expression patterns of MUC4 are illustrated in Figure 1. In all patients, the normal colonic epithelium exhibited moderate cytoplasmic MUC4 expression with accentuated staining in the lower two-thirds of the normal crypts (Fig. 1A,B). In CRC samples, expression was observed predominantly in the cytoplasm. However, a small proportion of CRC samples (17 of 132 patients; 13%) exhibited membrane staining along with cytoplasmic staining (Fig. 1D, thin arrows). On the basis of the cutoff point, CRCs were categorized into groups of low MUC4 expression (99 of 132 patients; 75%) and high MUC4 expression (33 of 132 patients; 25%) (Table 1). MUC4 staining was absent in 8 CRCs (6%), but the corresponding normal tissues exhibited moderate staining (Fig. 1F). The correlations between MUC4 expression status and clinicopathologic features are shown in Table 2. Most patients in the older age groups (67%) demonstrated increased expression of MUC4 in their CRCs. Higher expression was observed in men (61%) compared with women (39%). Among the patients who had high MUC4 expression, 64% died from CRC compared with 41% of those with decreased MUC4 expression (Table 2).

Figure 1.

Immunohistochemical expression patterns of mucin 4 (MUC4) are shown. (A) Normal colonic epithelium demonstrates cytoplasmic MUC4 staining (note the accentuation of staining in the lower two-thirds of the normal crypts; original magnification, ×200 μm). (B) Colorectal adenocarcinoma exhibits stronger cytoplasmic immunostaining (thick arrows) than the adjacent normal colonic epithelium (thin arrows; original magnification, ×400 μm; in B, 600 μm in inset). (C) This colorectal carcinoma (CRC) has strong cytoplasmic staining (thick arrow), and the adjacent normal epithelium has moderate staining (thin arrow). Inset: The same area from another section is shown in which primary antibody was not applied (original magnification, ×200 μm in C and inset). (D) This CRC with strong cytoplasmic staining (thick arrow) also exhibits mild-to-moderate membrane staining (thin arrows; original magnification, ×600 μm). (E) This CRC demonstrates weak cytoplasmic staining (thick arrow) compared with strong staining in the adjacent normal colonic epithelium (thin arrows; original magnification, ×200 μm). (F) This CRC exhibits the absence of immunostaining (thick arrows), and the adjacent normal colonic crypts exhibit strong cytoplasmic staining (thin arrow). Inset: Another CRC exhibits a lack of immunostaining (original magnification, ×400 μm in F and inset).

Table 2. Association Between Mucin 4 Expression and Clinicopathologic Characteristics (N=132)
 No. of Patients (%)
VariableLow MUC4 ExpressionHigh MUC4 ExpressionChi-square P
  1. MUC4 indicates mucin 4; CRC, colorectal carcinoma.

Total no. of patients99 (75)33 (25) 
Age group, y   
 <6552 (53)11 (33).055
 ≥6547 (47)22 (67) 
Sex   
 Women44 (44)13 (39).612
 Men55 (56)20 (61) 
Ethnicity   
 African American30 (30)15 (45).111
 Non-Hispanic Caucasian69 (70)18 (55) 
Tumor location   
 Proximal colon42 (42)17 (52).454
 Distal colon39 (39)9 (27) 
 Rectum18 (19)7 (21) 
Tumor stage   
 I17 (17)6 (18).965
 II38 (38)14 (42) 
 III31 (32)9 (27) 
 IV13 (13)4 (13) 
Tumor grade   
 Low74 (75)23 (70).569
 High25 (25)10 (30) 
Tumor size, cm   
 <531 (31)15 (45).139
 ≥568 (69)18 (55) 
Tumor type   
 Mucinous25 (25)12 (36).264
 Nonmucinous74 (75)21 (64) 
Vital status (at follow-up)   
 Alive26 (26)6 (18).064
 Death from CRC40 (41)21 (64) 
 Death from unknown cause or cause other than CRC33 (33)6 (18) 

Survival Analyses

Univariate KM survival analyses of the complete study cohort (n = 132) based on MUC4 expression demonstrated that CRCs that had increased expression were associated significantly with shortened disease-specific survival compared with CRCs that had low expression (log-rank P = .023) (Fig. 2A). Survival analyses to assess the significance of MUC4 expression based on tumor stage (early and advanced) demonstrated that patients who had early stage CRCs (stages I and II) with high MUC4 expression had a significantly shorter disease-specific survival than those who had CRCs with low expression (log-rank P = .007) (Fig. 2B). Although patients with advanced-stage CRC (stages III and IV) demonstrated a similar pattern in the survival curves, the survival difference was not statistically significant (log-rank P = .108) (Fig. 2C). Univariate survival analyses based on MUC4 expression status and other patient parameters (age at diagnosis, sex, and ethnicity/race) or tumor variables (location, size, and grade) were not statistically significant (data not shown).

Figure 2.

These Kaplan-Meier survival curves illustrate the significance of mucin 4 (MUC4) expression status in the survival of patients with early and advanced-stage colorectal carcinoma (CRC). (A) Overall, patients with CRC who had increased (high) MUC4 expression had short survival compared with patients who had low MUC4 expression (log-rank; P = .023). (B) In early stage CRC, high MUC4 expression was associated significantly with poor survival (log-rank; P = .007) relative to low MUC4 expression. (C) There was no difference in survival between low and high MUC4 expression among patients who had advanced-stage CRC (log-rank; P = .108).

The independent prognostic significance of MUC4 expression on CRC-specific survival was evaluated with a Cox regression model. These multivariate models confirmed the independent effect of MUC4 on CRC-specific survival (Table 3). A multivariate model that was developed for the complete study cohort (n = 132) demonstrated that patients with increased expression of MUC4 were 2.07 times more likely to die of CRC than patients who had low MUC4 expression (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.14-3.75) (Table 3). These analyses indicated that MUC4 expression and tumor stage were independent prognostic indicators of CRC (Table 3).

Table 3. Cox Regression Analyses Assessing the Prognostic Significance of Mucin 4 Expression
Prognostic VariableIndicator of Poor PrognosisHR (95% CI)P
  1. HR indicates hazard ratio; CI, confidence interval; MUC4, mucin 4.

Overall   
 MUC4 expression: Low vs high expressionHigh expression2.07 (1.14-3.75).017
 Tumor stage   
  I vs IIII1.34 (0.46-3.85).582
  I vs IIIIII4.95 (1.84-13.27).001
  I vs IVIV14.15 (4.64-43.10)<.0001
 Tumor grade: High vs lowHigh grade1.47 (0.77-2.78).243
 Tumor location: Proximal vs distal colonProximal colon0.92 (0.64-1.31).644
 Tumor size: >5 cm vs ≤5 cm>5 cm1.12 (0.63-1.97).693
 Tumor type: Mucinous vs nonmucinousMucinous1.37 (0.73-2.59).321
Early stage: Stages I and II   
 MUC4: Low vs high expressionHigh expression3.77 (1.46-9.73).006
 Tumor grade: High vs lowHigh grade2.91 (1.00-8.44).049
Advanced stage: Stages III and IV   
 MUC4: Low vs high expressionHigh expression1.74 (0.87-3.45).113

The multivariate regression models that were generated separately for early and advanced tumor stages demonstrated that increased expression of MUC4 is an independent indicator of a poor prognosis only in patients with early stage CRCs (Table 3). The HR for early stage tumors was 3.77 (95% CI, 1.46-9.73). For early stage CRCs, tumor grade also was associated significantly with patient survival (HR, 2.91; 95%CI, 1.00-8.44). The prognostic significance of MUC4 in advanced-stage CRCs, however, was not statistically significant (HR, 1.74; 95%CI, 0.87-3.45).

DISCUSSION

To our knowledge, the current study is the first to report the prognostic value of phenotypic expression of MUC4 in primary CRCs. In general, MUC4 expression was observed in the cytoplasm of both normal colonic epithelium and in CRCs. Some CRCs demonstrated membrane staining in addition to cytoplasmic localization. High expression of MUC4 was noted in 25% of CRCs. Survival analyses based on tumor stage indicated that MUC4 expression was associated with shortened survival for patients with early stage CRC, but not for patients with advanced-stage CRC. These findings were confirmed in multivariate Cox regression analyses, suggesting that high MUC4 expression is an independent indicator of a poor prognosis for patients with CRCs.

Similar to what we observed in the current study, earlier investigations reported cytoplasmic staining of MUC4 in normal colonic epithelium with accentuated staining in the lower two-thirds of the normal crypts.33 In our study, increased expression of cytoplasmic MUC4 relative to normal epithelium was observed in 25% of CRCs. Similar increased expression of MUC4 at the messenger RNA level was reported in CRCs compared with normal tissue.34 In the current study, in addition to the cytoplasmic localization of MUC4, we observed membrane staining in 13% of patients. A previous study in lung cancer13 demonstrated staining in the cytoplasm (33%), the membrane (9%), or both (58%). In pancreatic and extrahepatic bile-duct cancers, MUC4 staining was observed primarily in the cytoplasm.12, 35 In our study, CRCs demonstrated either decreased MUC4 expression (69%) or the complete lack of MUC4 expression (6%). This decreased or lack of expression can be attributed to epigenetic mechanisms (DNA hypermethylation and histone modifications), as demonstrated in earlier studies of pancreas and gastric cancer cells.36 However, such a relation (hypermethylation) was demonstrated in only some colon cancer cells.37 These findings suggest that the regulation of MUC4 expression is not organ-specific but varies in individual cancer cells.

Aberrant expression of MUC4 in lung and pancreatic cancers has been evaluated previously as a diagnostic marker.35, 38 Overexpression of MUC4 was observed in pancreatic adenocarcinomas, but its expression was undetectable in normal pancreas and in chronic pancreatitis.18, 19, 35, 39, 40 Thus, MUC4 could be used as an early diagnostic marker of pancreatic adenocarcinomas.35 In addition, MUC4 has been used to differentiate lung adenocarcinomas from malignant mesothelioma in pleural effusions.38 In our current study, because MUC4 expression was observed in the normal colonic epithelium and in CRCs, MUC4 cannot be used as a diagnostic marker.

The expression of MUC4 and its prognostic significance have been demonstrated in cancers of the biliary tract,12 lungs,13, 14 salivary glands,15-17 pancreas,18, 19 ovaries,18-20 and prostate.21 However, to our knowledge, its prognostic value has not been assessed in CRCs. In our study, we observed a significant association between increased MUC4 expression and poor patient survival in both univariate and multivariate survival analyses. Similarly, increased expression of MUC4 was associated with poor survival in patients with extrahepatic bile duct carcinoma12 and intrahepatic cholangiocarcinoma.41 In patients with ovarian carcinoma,20 increased MUC4 expression was associated with a trend toward poor survival. In patients with mucoepidermoid carcinoma of the salivary glands, increased expression of MUC4 was not associated with prognosis.16 In contrast, high MUC4 expression was associated with longer patient survival and late disease recurrence in mucoepidermoid carcinomas of the salivary gland.15, 17 Similarly, in squamous cell carcinomas of the upper aerodigestive tract, MUC4 expression was associated with longer patient survival and a decreased rate of recurrence.42 However, in adenocarcinomas of the lung14 and pancreas,43 MUC4 expression was associated with shortened disease-free and overall patient survival. These conflicting results on the prognostic value of MUC4 in different malignances indicate that its prognostic significance is tissue-dependent and varies with the type of malignancy.

In the current study, stage-based survival analyses demonstrated that only early stage (stages I and II) CRCs with increased expression of MUC4 were associated significantly with shortened patient survival. Similarly, increased expression of MUC4 was associated with poor survival in patients with early stage (stage IA) lung cancer14 and ovarian cancer.20 Furthermore, in lung adenocarcinomas, the corresponding normal epithelium consistently expressed MUC4,14, 38 which we also observed. A statistically significant survival difference was not observed for patients with advanced-stage (stages III and IV) CRCs, although a similar association with survival was observed with MUC4 expression. The multivariate regression models that we generated separately for early and advanced tumor stages confirmed that increased expression of MUC4 is an independent indicator of a poor prognosis only in patients with early stage CRC. However, additional larger studies are needed to assess the prognostic importance of MUC4 expression in patients with advance stage CRC. Although the basis for the prognostic value of MUC4 in early stage CRC, but not in advanced-stage CRC, is not known, MUC4 expression may have distinct roles in different phases of CRC progression, as observed a gradual increase of expression of nuclear factor κB and loss of Bcl-2 expression from adenoma to adenocarcinomas.44 Similarly, MUC4 may be involved in the progression of early stage tumors more than in advanced-stage CRCs. Although our results demonstrated prognostic implications for MUC4 expression in a cohort of patients with CRC, they did not elucidate the role of MUC4 expression in CRC development.

Our current results indicated that tumor grade also is an important prognostic indicator in early stage CRC, but not in advanced-stage CRC. Despite the subjective nature of the histologic grading and considerable interobserver variability, tumor grade has been identified as an independent prognostic factor in CRC.45, 46 In general, patients with advanced-stage CRC (stages III and IV) have a poorer prognosis than patients with early stages (stages I and II); however, a subset of patients with stage II CRCs have an increased risk of early recurrence and death. The underlying molecular mechanisms for these aggressive, early stage CRCs are complex; however, identification of this high-risk subset would be important in the selection of patients for appropriate treatment. In such instances, determination of the MUC4 expression status should aid in identifying patients with aggressive forms of CRCs and help in designing individualized therapies.

In summary, moderate levels of MUC4 expression were observed in uninvolved colonic epithelium; however, in CRCs, MUC4 expression levels varied from high to none. Univariate and multivariate survival analyses indicated that increased MUC4 expression was associated with poor patient survival, specifically for those with early stage CRC. After validating these findings in larger retrospective and prospective studies, a stage-based analyses could establish the utility of MUC4 as a prognostic molecular marker of early stage CRCs. Furthermore, the use of MUC4 as a marker should aid clinicians in identifying patients who have aggressive forms of early stage CRC.

Acknowledgements

We thank Donald L. Hill, PhD, Department of Preventive Medicine, University of Alabama at Birmingham, for his critical review of this article.

CONFLICT OF INTEREST DISCLOSURES

Supported by funds from the National Institute of Health/National Cancer Institute to Dr. Manne (RO1-CA98932-01, R03-CA139629-01, and 2U54-CA118948-03) and to Dr. Grizzle (U24-CA086359).

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