Background

Colorectal cancer is the second most common cause of cancer related death in the developed world [1], in consequence advances in our understanding and treatment of colorectal cancers can potentially have a huge impact on cancer morbidity and mortality. Currently much of our understanding of cancer behaviour, including the prediction of likely patient outcomes, is based on histopathological parameters, and from this treatment is tailored to individual patients. At present TNM stage, tumour type and resection margin status are the most widely used parameters in planning adjuvant treatment. Tumour grade of differentiation, vascular invasion and more recently perineural invasion and tumour border configuration have also been used to assist the clinician in predicting colorectal tumour behaviour and hence subsequent patient management [2].

It is well recognised that clinical response and recurrence rates vary within the conventionally staged groups and that this reflects variation in the genetic and molecular make-up of these tumours. Molecular changes occur within cancer cells during tumour progression; these changes provide a potential insight into tumour development and metastasis.

Refining prognostic markers allow treatment to be more accurately tailored to individual patients, as well as suggesting potential mechanisms through which tumour progression occurs which in turn could provide targets for novel therapies.

MUC1 is a membrane bound glycoprotein which has been demonstrated to be predictive of tumour progression and worsening prognosis in both gastric [3–5] and colorectal cancer [6, 7] including those related to HNPCC [8]. This increased expression has been seen more predominantly at the invasive tumour front [9].

MUC3 is also a trans-membrane glycoprotein which is seen in both colorectal cancers and normal colon [10]. Studies have shown an association between MUC3 expression and poor prognosis in a number of cancers including pancreatic [11], breast [12], gastric [13] and renal [14]. There is some evidence suggesting that MUC3 expression is reduced in colorectal cancers and that this varies between histological types [15]. The cellular distribution is also seen to be affected; apolar distribution is thought to reflect abnormal transport systems [6, 28]. For MUC3 tumours displaying moderate or high intensity staining were considered positive, with the remainder considered negative

Statistical analysis

Statistical analysis of the study data was performed using the SPSS package (version 14 for Windows, SPSS Inc., Chicago, IL). Pearson χ2 chi-square tests were used to determine the significance of associations between categorical variables. Disease-specific survival calculations included all patients whose death related to colorectal cancer. In contrast, patients whose deaths resulted from non-colorectal cancer related causes were censored at the time of death. Kaplan-Meier curves were used to assess factors which influenced survival. The statistical significance of differences in disease-specific survival between groups with differing MUC1 and MUC3 expression was estimated using the log-rank test. The Cox proportional-hazards model was used for multivariate analysis in order to determine the relative risk and independent significance of individual factors. In all cases p-values < 0.05 were considered as statistically significant.

Results

Patient and histopathological variables and prognosis

Univariate relationships between known patient/tumour characteristics and DSS were evaluated using the log-rank test (see Table 2). There appeared to be no significant differences in DSS between patients of either gender. Similarly when patient age was considered in three groups (patients 64 years or younger at the time of surgery, patients 65–79 years, and those 80 years and over), no significant differences in DSS were noted. The site of tumour i.e. colon or rectum had no influence on DSS.

Table 2 Univariate survival analysis of patient/tumour characteristics
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Tumour grade showed a trend towards reduced survival with increasing dedifferentiation, with colorectal cancer related deaths occurring in 34.5% (10/29), 48.4% (171/353) and 56.3% (40/71) of patients with well, moderately and poorly differentiated tumours respectively, although this did not reach statistical significance in our cohort of patients. The majority of tumours were adenocarcinomas, however non-adenocarcinoma tumours, did not have a statistically significantly poorer prognosis in this series. Extramural vascular invasion had a strong correlation with survival, 72% (92/128) of patients with evidence of vascular invasion died from colorectal cancer related causes, compared with only 39% (87/224) in patients without. In cases where the vascular invasion status was unknown an intermediate mean DSS was noted. The association between vascular invasion and DSS was highly significant and log-rank testing (log-rank = 44.30, p < 0.0001).

The strongest association of clinicopathological variables with DSS was seen with TNM staging (log-rank = 211.37, p < 0.0001), showing a progressive reduction in DSS with increasing tumour stage.

Tumour marker expression

MUC1

Analysis of MUC1 expression was possible in 403 of the 462 tumours on the TMA (87%), with the remainder being lost during antigen retrieval or not demonstrating viable tumour cells within the core. This level of core loss is within the rates described by previous authors using TMAs [29, 30]. The majority of staining was seen within the cytoplasm and cell membrane, no staining was seen within the nucleus or surrounding stromal tissue. No staining was seen in 188 (47%) tumours, with <5% and 5–30% of cells staining in 47 (12%) and 41 (10%) respectively. There was 30–60% staining in 62 (15%) cores and greater than 60% in 65 (16%). When dividing the tumours according to previous studies [6, 28] 276 (68%) tumours were MUC1 negative and 127 (32%) positive.

MUC3

Analysis of MUC3 expression was possible in 387 cores (84%). The staining was seen mainly within the cytoplasm 354 (91%) but also with the cell membrane 147 (38%), there was no nuclear staining but occasional stromal staining seen. The majority of tumours displayed either moderate 187 (48%) or strong staining 99 (26%), weak or no staining was seen in 68 (18%) and 33 (8%) respectively.

Representative examples of positive and negative staining for each antigen are shown in figure 1.

Figure 1
figure 1

Immunohistochemical staining of tissue microarray cores with MUC1 and MUC3 antibodies. A & B show cores from tumour demonstrating positive (A) and negative (B) MUC1 staining. C & D show cores of tumour demonstrating strong (C) and weak MUC 3 staining. All are at × 100 magnification.

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Relationships between tumour markers and standard clinicopathological variables

MUC1

For the purposes of analysis the tumours were divided into those with positive or negative expression, as described previously [6, 28]. There did not appear to be any relationship between any of the clinicopathological variables, including stage, and MUC1 expression (see table 3).

Table 3 Patient and Tumour characteristics in relation to MUC1 expression (n = 403)
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MUC3

As the majority of tumour cells within each core expressed a uniform staining pattern, the cores were classified according to intensity of staining as opposed to the proportion of cells staining. Cores were deemed positive if moderate or strong staining was seen. Using this system 286 (74%) tumours were positive and 101 (26%) negative. No correlation between MUC3 cytoplasmic expression and any clinicopathological variables, including stage, was seen (see table 4). Equally there was no correlation of membranous staining with any clinicopathological variables (data not shown).

Table 4 Patient and Tumour characteristics in relation to MUC3 expression (n = 387)
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Relationship between tumour markers and patient survival

Correlation between MUC1 and MUC3 expression and DSS was assessed using Kaplan-Meier plots and log rank testing (see table 2, figures 2 and 3). A significant association was seen between tumours with high MUC1 expression and a reduced DSS (mean DSS 54 months vs. 65 months; p = 0.038). In contrast, there was no correlation between MUC3 expression and DSS.

Figure 2
figure 2

Kaplan-Meier plot for disease specific survival, MUC1 (+) vs. MUC1 (-) tumours (n = 403).

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Figure 3
figure 3

Kaplan-Meier plot for disease specific survival, MUC3 (+) vs. MUC3 (-) tumours (n = 387).

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In order to determine the relative influence of MUC1 and other patient and tumour variables known to affect prognosis, a multivariate analysis was performed using the Cox proportional hazards model. We included only those variables which had been shown to be significantly related to DSS on univariate analysis i.e. intramural vascular invasion and TNM stage (see table 5). In this model, vascular invasion (p < 0.001) and TNM staging (p < 0.001) were seen to retain independent prognostic significance. High expression of MUC1 was also seen to be an independent prognostic marker of poor outcome, with a hazard ratio of 1.339 (95%CI 1.002–1.790, p = 0.048), when compared with tumour demonstrating low MUC1 expression.

Table 5 Cox multivariate regression analysis of variables in relation to disease specific survival
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Discussion

This study investigates the role of MUC1 and MUC3 as prognostic markers in colorectal cancer. Previous studies have suggested a link between MUC1 and MUC3 expression and poor prognosis both in colorectal and other tumour types [3–8]. These studies have frequently suffered from small sample sizes and/or heterogeneous methodology and study populations. The current study comprises the largest analysis of MUC1 and MUC3 expression in colorectal cancer to date; including 463 consecutively treated representative patients, who were representative of the colorectal cancer population within the UK. With a comprehensive data set of clinicopathological variables and patient outcome, over a median 3 year postoperative period, a thorough and comprehensive analysis was possible between these variables and disease specific survival.

In our study population 32% of tumours were positive for MUC1. This compares favourably with previous authors work, who also used the same semi-quantitative scoring system and found 32% and 43% MUC1 positivity in colorectal tumours respectively[6, 28].

In our study population MUC1 expression was not related to any of the clinicopathological variables examined. Some previous studies demonstrated increased MUC1 expression was related to increasing TNM or Dukes stage [31–33]; however, a number of other studies are in line with our findings [9, 34]. Variations in the findings of the current and previous studies may relate to differences in immunohistochemical protocols, antibodies used, scoring systems and area of the tumour examined e.g. Hiraga et al and Kimura et al only assessed staining at the invasion front [31, 32]. A large study by Lugli et al examines the prognostic significance of MUC1 and MUC2 in relation to differing mismatch repair status in colorectal cancer, with tumours divided into three subgroups. Significant correlations were found in the "mismatch repair proficient group" between MUC1 positivity and tumour stage and grade [33]. There was no such correlation in our cohort, however our analysis did not involve sub-stratification of the population and hence may explain the dissimilar results.

Univariate and multivariate analysis of our patient population confirmed that TNM staging and vascular invasion are strong independent prognostic markers in colorectal cancer. Of particular interest was the large effect vascular invasion had on survival. Presence of vascular invasion reduced mean DSS significantly (38 vs 75 months p < 0.0001), yet no previous studies investigating the prognostic value of MUC1 have included this obviously strong predictor of survival in their analysis. Our data confirm that high expression of MUC1 in colorectal cancer confers a worse prognosis both on univariate and multivariate analysis, even when taking into account the potentially confounding influence of vascular invasion status.

The association of MUC1 with poor prognosis has been linked to effects on cell adhesion and the potential for metastasis. Regimbald et al [35] showed that MUC1 was a ligand for ICAM-1 in breast cancer and might have a pivotal role in haematogenous spread, and it has been speculated that this mechanism may occur in colorectal cancer [36]. MUC1 is also seen to have effects on the extra cellular matrix components through inhibition of kalinin and laminin [37, 38].

MUC1 has been demonstrated to affect beta-catenin, a nuclear transcription factor, and its intracellular distribution has been shown to influence progression of colorectal cancer [39], it has been suggested that MUC1 exerts some of it's effects through interaction with beta-catenin, with over expression of MUC1 leading to increased levels of nuclear beta-catenin [40]. A recent study has shown that the co-expression of MUC1 and nuclear beta-catenin at the invasion front of colorectal tumours may be correlated with a worse prognosis [9].

MUC3 expression was present in moderate to high levels in 76% of tumours assessed. Some studies have suggested that MUC3 may in fact be down-regulated in colorectal cancer compared with normal colon [10, 15]. We did not see any correlation between the clinicopathological variables and MUC3; in particular there was no correlation with tumour stage as is seen with gastric cancers [13]. Furthermore, MUC3 expression did not appear to correlate with prognosis, as has been reported in other tumour types [11–14]. Rakha et al demonstrated MUC3 expression in 91% of breast cancers which was associated with increased local recurrence and lymph node stage. They argued that membranous expression of MUC3 was a poor prognostic feature, which correlated with higher grade and poorer Nottingham Prognostic Index (NPI) [12]. Wang reported that increased MUC3 expression in gastric cancer worsened prognosis, with no significant differences in expression seen in relation to patient sex, tumour location, grade of differentiation, serosal invasion, or Lauren's type. However MUC3 expression was higher in those with metastasis (p < 0.01) and in clinical stage III–IV disease compared to I–II (p < 0.05). MUC3 were not detected in the normal gastric mucosa [13]. MUC3 showed a progressive increase in expression with pancreatic intraepithelial neoplasia of increasing dysplasia and was also highly expressed in ductal adenocarcinoma [11].

Normal lung tissues exhibited a distinct pattern of mucin gene expression, with high levels of MUC1 and low levels of MUC3 immunoreactivity and mRNA. In contrast, lung adenocarcinomas, especially well-differentiated cancers, exhibited increased MUC1 and MUC3 mRNA levels [41]. Copin et al found that coexpression of MUC3 and MUC1 was constant among lung adenocarcinomas [42].

Conclusion

We have demonstrated that using TMA technology and a large cohort of colorectal cancer patients with robust long term follow up data that biomarkers of prognosis can be reliably assessed. Our data clearly demonstrates a role for MUC1 in the progression of colorectal cancer, probably through its effects on cell adhesion and metastasis. MUC1 expression appears to function as an independent prognostic marker in colorectal cancer even when the conventional variables of tumour stage and vascular invasion status are included in the analysis.