ARTICLE |
Correspondence to: Jeremy R. Jass, Dept. of Pathology, Mayne Medical School, Herston QLD 4006, Australia.
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Summary |
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We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum. (J Histochem Cytochem 47:10391047, 1999)
Key Words: mucin, MUC1, MUC2, MUC4, MUC5AC, traditional adenoma, hyperplastic polyp, serrated adenoma, colon, neoplastic potential
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Introduction |
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THE MUCOSAL SURFACE of the gastrointestinal tract is covered with mucus secreted by specialized epithelial cells to lubricate and protect the epithelium against potential pathogens, mutagens, and mechanical and chemical damage (
The MUC1 gene located on chromosome 1q21-24 is fully sequenced and encodes a transmembrane glycoprotein (
Alterations in the synthesis and expression of MUC1, MUC2, MUC3, and MUC4, and the subsequent glycosylation of these protein core molecules, have been described in colorectal polyps, cancer, and inflammatory bowel disease (
Traditionally, colorectal polyps have been classified into two common types: hyperplastic polyps and adenomas. In 1990, Longacre and FenoglioPreiser introduced the concept of mixed epithelial polyps, which can be divided into two subtypes. The first type consists of both adenomatous and hyperplastic glands that can be clearly identified as hyperplastic polyp and adenoma (HP/AD). The second type combines architectural and cytological features of hyperplastic and adenomatous epithelium. These authors proposed the term "serrated adenoma" for the latter, a distinctive subtype of colorectal epithelial polyp. Serrated adenomas show prominent glandular serration similar to hyperplastic polyps and therefore are often misdiagnosed as hyperplastic polyps. However, cytological features such as incomplete mucinous differentiation, nuclear enlargement, pseudostratification, increased mitotic activity, and increased architectural complexity are indicative of the neoplastic nature of serrated adenomas. Intramucosal carcinoma occurs in 10% of serrated adenomas (
It has been suggested that hyperplastic polyps may develop into serrated adenomas as a separate histogenetic pathway of carcinogenesis (
In the present study we used monoclonal antibodies (MAbs) for the mucins MUC1, MUC2, MUC4, and MUC5AC to determine the different patterns of mucin distribution and to clarify the histogenetic relationships in hyperplastic polyps, serrated adenomas and traditional adenomas.
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Materials and Methods |
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Formalin-fixed, paraffin-embedded human tissue specimens of endoscopically resected hyperplastic polyps (n = 12), serrated adenomas (n = 27), tubular adenomas (n = 18), and tubulovillous adenomas (n = 2) were obtained from the files of the Pathology Department, Niigata University Medical School, Japan. Histologically normal mucosa from the margins of the specimens served as control tissue. The size ranges for hyperplastic polyps, serrated adenomas, and traditional adenomas were 4.510 mm (mean 5.5 mm), 330 mm (mean 10.5 mm), and 325 mm (mean 8.0 mm), respectively. All of the colorectal polyps were sessile except for 9/27 serrated adenomas and 5/20 traditional adenomas, which were polypoid. Low-grade dysplasia was observed in all adenomas (
Preparation of Slides
Paraffin sections (34 µm) were affixed to Superfrost Plus adhesive slides (Menzel-Gläser; Braunschweig, Germany) and air-dried overnight at 37C. Sections were dewaxed in xylol and rehydrated through descending graded alcohols to Tris-buffered saline (0.05 M Tris, 0.15 M NaCl; TBS), pH 7.27.4. Sections were incubated in 1% periodic acid in distilled H2O for 30 min (MUC1), or in 0.1% porcine trypsin (ICN Biomedicals Australasia; Sydney Australia) with 0.1% CaCl2 in PBS for 30 min (MUC2), or transferred to 0.01 M citric acid buffer, pH 6, and boiled twice for 5 min each and then transferred to TBS (MUC4, MUC5AC) (
The sections were then incubated in 1.0% H2O2, 0.1% NaN3 in TBS for 10 min to block endogenous peroxidase activity, then washed in three changes of TBS for 5 min each. Nonspecific antibody binding was inhibited by incubating the sections in 4% skim milk powder in TBS for 15 min, followed by a brief wash in TBS. The sections were then placed in a humidified chamber and incubated with 10% normal (nonimmune) goat serum (Zymed; San Francisco, CA) for 20 min. Excess normal serum was decanted from the sections and the primary antibody applied overnight at room temperature, except for BC2 (MUC1), which was applied for 60 min. MAbs used were BC2 (1.5 µg·ml-1) against MUC1 (
Sections were washed in three changes of TBS for 5 min each [the first buffer change contained 0.5% (v/v) Triton X-100] and then incubated with biotinylated goat anti-mouse immunoglobulins (Zymed) for 30 min. Sections were washed again in three changes of TBS for 5 min each [the first wash contained 0.1% (v/v) Triton X-100], incubated with streptavidinhorseradish peroxidase conjugate (Zymed) for 15 min, and washed in three changes of TBS for 5 min each. Color was developed in 3,3'-diaminobenzidine (Sigma Chemical; St Louis, MO) with H2O2 as substrate for 5 min. Then sections were washed in running tapwater, lightly counterstained in Mayer's hematoxylin, dehydrated through ascending graded alcohols, cleared in xylene, and mounted using DePeX (BDH Gurr; Poole, UK).
The sections were scored by two independent observers (A-E, B-H, JRJ). Apomucin staining was scored as negative (-), trace (+/-), weak (+), or strong (++). Scores were estimated by comparing intensity with the adjacent normal mucosa of the same specimen. Note was made of both cellular and subcellular localization with regard to goblet cells (theca vs perinuclear cytoplasm) and columnar cells (cytoplasm vs apical membrane).
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Results |
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Normal Mucosa
Normal mucosa was present in 59 cases, and of these 29 (49%) showed positive staining for MUC1 (BC2) in the cytoplasm of goblet and columnar cells of the lower third of crypts, as well as along the apical membrane, whereas the remainder reacted very weakly or not at all. MUC2 (4F1) was expressed in the perinuclear cytoplasm of the goblet cells, albeit focally (Figure 1B, arrow). The cytoplasm of both columnar and goblet cells was stained for MUC4 (M4.275), staining being more intense in the lower two thirds of crypts (Figure 1C). Columnar cells were consistently negative for MUC5AC (M1), whereas in 12 of the 59 cases (20%) focal staining of the cytoplasm and the mucous droplets of goblet cells was observed (Figure 1D).
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Serrated Adenomas
In the serrated adenomas, 17 of the 27 cases showed an increase in MUC1 (BC2) staining, the remaining 10 showing only very focal or negative reactivity. MUC2 (4F1) staining was strongly increased in the cytoplasm of both columnar and goblet cells (Figure 1B). Complete or near-complete loss of MUC4 (M4.275) was seen in all serrated adenomas (Figure 1C). MUC5AC (M1) staining was similar to that of MUC2, being present within the thecae of the entire mucus-secreting population, but in some cases was restricted to goblet cells (Figure 1D).
Hyperplastic Polyps
In hyperplastic polyps, MUC1 (BC2) expression was decreased compared with the adjacent normal mucosa, with only four of the 12 polyps showing positive and mainly focal staining in goblet and columnar cells. MUC2 (4F1) staining was generally increased, with MUC2 present in the cytoplasm of columnar cells as well as goblet cells (Figure 2B). MUC4 (M4.275) was negative in six cases and reduced in four, with only two cases expressing MUC4 (M4.275) at the level seen in normal mucosa (Figure 2C). MUC5AC (M1) presented a staining pattern similar to that of MUC2, with an increase in reactivity in the goblet cell theca (Figure 2D).
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Traditional Tubular and Tubulovillous Adenomas
The 20 traditional adenomas included 15 flat or sessile adenomas and five polypoid adenomas. Two of the polypoid adenomas were tubulovillous and the remainder were tubular. In 12 adenomas the apical membrane and apical cytoplasm were stained for MUC1 (BC2), a weak reaction was observed in five cases, and the remaining three were negative. Of the 20 adenomas, 19 showed a normal pattern of MUC2 (4F1) expression in goblet cells (Figure 3B). One tubular adenoma showed increased MUC2 (4F1) expression in the perinuclear cytoplasm, and no change in either the intensity, intracellular localization, or crypt distribution of MUC4 (M4.275) expression was seen (Figure 3C). MUC5AC (M1) expression was increased in 12 adenomas, with the remaining eight showing only focal positivity. MUC5AC staining was mainly in the theca of goblet cells (Figure 3D).
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Patterns of apomucin immunoreactivity in each category of polyp were assessed with regard to polyp size, and no discernible differences were observed with increasing lesion size. The immunohistochemical results for normal colon and colorectal polyps are summarized in Table 1.
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Discussion |
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The majority of epithelial polyps of the colorectum can be classified as either hyperplastic polyps or adenomas. Traditionally, these have been regarded as fundamentally different lesions, with only the adenomas being neoplastic and having potential for progression to malignancy (
In normal human colon mucosa, MUC2 and MUC4 are abundantly expressed, whereas the expression or nonexpression of MUC1 has been a matter of some controversy (
MUC5AC is strongly expressed in gastric mucosa but not in normal colon mucosa (
The expression of apomucins in adenomas is associated with polyp size, villosity, and degree of dysplasia. Investigations of mucin expression in colorectal tubular adenomas revealed an increase of MUC1 in foci with high-grade atypia and extension throughout the crypt length with increasing atypia (
Hyperplastic polyps and serrated adenomas showed essentially similar changes, but these were very different from the pattern observed in tubular adenomas. There was increased expression of MUC2 and MUC5AC and reduced expression of MUC4, the latter being more consistently seen in serrated adenomas than in hyperplastic polyps. The main difference between these polyps was with regard to MUC1, which showed increased expression in 63% of serrated adenomas but reduced expression in 67% of hyperplastic polyps. MUC1 expression is associated with neoplastic progression and tumor aggressiveness of colorectal adenocarcinoma (
The loss of MUC4 is interesting because this apomucin is expressed by goblet as well as columnar cells in normal colorectal mucosa, albeit at higher levels in columnar cells (
The shared phenotypes, both structural and functional, observed in hyperplastic polyps and serrated adenomas suggest that the lesions are histogenetically related. This is further supported by the finding of low levels of DNA microsatellite instability (MSI-L) in these lesions (
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Acknowledgments |
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We wish to thank Ms Estelle Schoch and Mr Nigel Misso for invaluable technical support, and Ms Lynne Reid and Mr Clay Winterford for contributing their photographic skills.
Received for publication December 7, 1998; accepted March 23, 1999.
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