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Differences in the expression of mucins in various forms of cystitis glandularis
ARTICLE IN PRESS
Pathology – Research and Practice 203 (2007) 653–658 www.elsevier.de/prp
ORIGINAL ARTICLE
Differences in the expression of mucins in various forms of cystitis glandularis Ljubinka Jankovic Velickovica, Vuka Katica, Takanori Hattorib, Ryoji Kushimab, Goran Marjanovicc, Vladisav Stefanovicd, a
Institute of Pathology, Faculty of Medicine, Nis, Serbia Department of Pathology, Shiga University of Medical Science, Ohtsu, Japan c Clinic of Hematology and Clinical Immunology Faculty of Medicine Nis, Serbia d Institute of Nephrology and Hemodialysis, Faculty of Medicine, Nis, Serbia b
Received 9 March 2007; accepted 7 May 2007
Abstract A wide spectrum of glandular epithelial metaplastic changes may be seen in the bladder. Cystitis glandularis (CG) is a well-known metaplastic lesion occurring in the presence of chronic inflammation, but there are a few data about mucin expression in its two subtypes (typical and intestinal). The purpose of the present study was to determine the expression of mucin core proteins and CD10 in the different types of CG. For this examination, we used a panel of monoclonal-specific antibodies for MUC1, MUC2, MUC5AC, and MUC6. CG of the intestinal type expressed MUC5AC both in goblet and columnar cells, and strongly expressed intestinal mucin MUC2 only in goblet cells in all cases. There was no expression of MUC1, MUC6, and CD10 in the metaplastic cells. CG of the typical type showed an expression of MUC1 similar to normal urotelium, but the CD10 expression was more intensive than in the control. The mucin expression profile in the different types of CG allows the identification of ‘‘gastric mucin’’ (MUC5AC) together with intestinal mucin (MUC2), while typical CG (CGTP) retains MUC1. Different and contrasting immunoprofiles were evident in various forms of CG. The absence of CD 10 in CG of the intestinal type is a finding that points towards an incomplete form of urinary bladder metaplasia. r 2007 Elsevier GmbH. All rights reserved. Keywords: CD10; Cystitis glandularis; Intestinal metaplasia; Mucin
1. Introduction Cystitis glandularis (CG) is a metaplastic alteration of the urothelium in the urinary bladder, which is thought to be induced by chronic inflammation or irritation. In the presence of chronic inflammation, bladder mucosa becomes hyperproliferative, and nests of urothelium Corresponding author. Tel.: +381 18 234092; fax: +381 18 238770.
E-mail addresses: [email protected] (L. Jankovic Velickovic), [email protected] (V. Stefanovic). 0344-0338/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2007.05.003
may appear within the lamina propria (von Brunn’s nests) [18]. With continued inflammation, further differentiation to cystic lesions (cystitis cystica) or glandular lesions (CG) may occur [16,18]. Cystitis cystica is considered benign, whereas CG is of uncertain malignant potential. Several authors have described the progression to bladder adenocarcinoma during longterm follow up [4,6,15,17,27], although others have disputed this finding [9]. In the past, the majority of authors subdivided CG into two subtypes with different morphologic findings. Typical CG (CGTP) is
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characterized by nests of columnar epithelial cells within the lamina propria of the bladder, forming glandular structures, while intestinal CG (CGIT) is characterized by similar glandular structures made of simple columnar epithelial cells that are replaced by mucin-secreting goblet cells. The two subtypes may coexist, and previous studies have specifically described the intestinal subtype as premalignant [4,18,23]. The recent identification of several mucin genes, as well as the production of monoclonal antibodies (mAbs) to mucin protein cores, has led to the immunohistochemical localization of several mucin species. Mucin core proteins comprise an expanded gene family consisting of at least 19 members. Mucins are highmolecular-weight glycoproteins with oligosaccharides attached to the apomucin protein backbone by O-glycosidic linkage. The synthesis and secretion of mucins are common features of glandular epithelial tissue [2,8,13,14,22,24]. Mucins are classified into membrane-associated and secreted types. MUC1 is a transmembrane glycoprotein that was detected in most epithelial tissues, including the breast and pancreas, as well as in urinary, gastrointestinal, and respiratory tracts. It is known that MUC1 has a role in lumen formation, as well as an inhibitory role in the cell to stroma interaction [1,20]. MUC2, a prototype seretory mucin expressed in the colon, small intestine, and airways, differs from MUC1 in its structure [8,14]. CD10 is a neutral endopeptidase, the expression of which represents a useful tool in the classification and diagnosis of malignant leukemia and lymphoma. Immunohistochemical studies have demonstrated that this antigen is expressed in a variety of non-hematopoietic normal and tumor tissues, including utothelial neoplasms and normal urothelium [11,19]. The aim of this study was to evaluate and compare the expression of MUC1, MUC2, MUC5AC, MUC6, and CD10 to determine the immunoprofile of these two types of CG.
2. Materials and methods Tissues from 10 patients containing CG were obtained from the Surgical Pathology archives of the Institute of Pathology between 1996 and 2005. Tissue samples consisted of biopsy material obtained from noncancer patients. Seven of the 10 patients were male and three were female, with a median age of 52.7716.06 (24–74) years. Although both types of CG were found in every sample, CGIT was a predominant finding. Eight normal bladder urothelia were used as controls. All tissues were fixed in 10% formalin for 24–48 h and embedded in paraffin. We classified CG into CGTP and the CGIT on hematoxylin- and eosin-stained sections.
2.1. Mucin histochemical and immunohistochemical studies Biopsies with both types of CG were stained with PAS and the AB/HID method (pH 2.5) to determine whether they expressed neutralmucins, sialomucins, or sulphomucins [10]. The intensity of mucin content was scored as negative ( ), weak (+), or strong (2+). The histochemical detection of mucins was used to determine the type of mucins secretion in various forms of CG. Immunohistochemical techniques are widely used to identify intestinal and gastric cell differentiation for the classification of intestinal metaplasia. We detected mucin by using the following mAbs from Novocastra, Newcastle, UK: MUC1 (MA695, 1:100), MUC2 (CCP 58, 1:100), MUC5AC (CLH2, 1:100), MUC6 (CLH5, 1:100), and CD10 (56C6; 1:50). Immunohistochemical study using the streptavidin–biotin method (Histofine SAB-PO (M) kit) was performed at the Department of Pathology, Shiga University of Medical Science, Japan. Peroxidase-binding sites were visualized using diaminobensidine, and nuclei were lightly counterstained with hematoxylin. Positive staining for the MUC gene and CD10 was graded as diffuse (2+: over 30% of cells), sporadic (1+: less than 30% of cells), or negative (-: less than 5% of cells) [11].
3. Results 3.1. Microscopic features Most of the analyzed bladder slides showed numerous glands in the lamina propria, showing the features of CG of the typical and intestinal type. The lining epithelial cells in CGTP are columnar and surrounded by several layers of transitional cells. The predominant finding in all cases was CGIT, usually the florid type. It consisted of numerous glands lined by a single layer of goblet cells with basally situated nuclei. Occasionally, we noted abrupt transition from CGTP to CGIT (Fig. 1A). Some glands were lined by tall columnar epithelium with plenty of goblet cells. Extravasation of mucin into the stroma was present only in one case, without desmoplastic response. The coexistence of areas with typical and intestinal type of CG formed the mass that simulated a neoplasm.
3.2. Histochemical features Almost all crypts of CGIT were PAS and AB/HID positive. The mucin production was predominantly intracellular, and was expressed in all 10 cases of frank intestinal metaplasia. Most goblet cells contained acid
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(predominantly sialomucins) product. The goblet cells at the surface of the upper half of the crypt contained both sulpho and sialomucins, while the lower goblet cells contained predominantly sulphated mucins with HID–AB reactivity (Fig. 1B). The extracellular mucin content, which was seen focally in one case of CGIT, consisted of sialo and neutralmucins. The intracellular mucin production was expressed in CGTP with PAS, but its amounts were small. Acid product was not detected in CGTP (Fig. 1B). The histochemical findings in CG are summarized in Table 1. Eight normal urothelium samples showed no PAS and HID–AB reactivity.
3.3. MUC1, MUC2, MUC5AC, MUC6, and CD10 expression In the normal urothelium, MUC1 was limited predominantly to the apical membranes of the umbrella cell layer, while CD10 was found to be expressed weakly (+), or the reaction was negative in the apical portion of the cytoplasm along the luminal surface (Table 2). On the other hand, all samples of CGTP showed very strong CD10 reaction with brown staining of cell membrane, and distribution was superficial or diffuse (Fig. 2A). MUC1 was expressed in all samples of CGTP (Fig. 2B). All samples of both normal urothelium and CGTP were uniformly negative for MUC2, MUC5AC, and MUC6 (Tables 2 and 3). In sharp contrast to this, MUC1 was not detected in CGIT, but on the other hand, MUC2 and MUC5AC were expressed in all 10 cases (100%). We detected MUC2 in goblet cells only (Fig. 3A) and MUC5AC in columnar cells (Fig. 3B). MUC6 (pyloricgland mucin) and CD10 were negative in all CGITs (Table 4).
4. Discussion Mucins constitute a large family of glycoproteins expressed by many epithelial cells and their malignant counterparts. MUC1 is a type of mucin encoded by the MUC1 gene, located on chromosome 1 (1q21). MUC1 is known to have a role in lumen formation, and has an inhibitory role in the cell-to-stroma interaction [20]. In normal urothelium, MUC1 expression was consistently strong and diffuse in the apical membranes of the umbrella cell layer. This membrane-associated mucin was also detected in malignant urothelium [7,20,25]. The gel-forming mucins, MUC2 and MUC5AC, are found in a cluster on chromosome 11p15.5. The MUC2 gene codes for a typical secretory mucin that predominates in goblet cells of the small and large intestine [3,8]. MUC5AC was demonstrated in gastric columnar epithelial cells, while MUC6 was present on the cells
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in the glandular compartment of pyloric mucosa. MUC5AC and MUC6 are two major secretory mucins of the stomach [2,3,22]. Our immunohistochemical study demonstrated an intestinal and gastric phenotype in CGIT by the detection of MUC2 and MUC5AC expression. In contrast to normal urinary bladder mucosa, CGIT displayed no expression of MUC1. Normal colorectal mucosa showed positive staining for MUC1, MUC2, and MUC4 [3]. MUC1 expression is most obvious within the proliferative zone in normal mucosa, whereas mature goblet cells secreting MUC2 are present mainly in the maturation zone [12]. In normal adults, MUC5AC is not expressed in the intestinal tract. Buisine et al. have shown that MUC5AC is expressed in emryonic intestine up to 8 weeks of gestation. However, after 12 weeks, expression is significantly reduced, as mRNAs were not detected in any intestinal region from the 13th week of gestation onwards. Thus, MUC5AC is developmentally expressed during intestinal ontogenesis [5,14]. Also, MUC5AC was shown to be expressed in rectosigmoid villous adenomas [11]. Also, CD10 was never detected in CGIT. To our knowledge, the present study is the first report that clearly demonstrates an inverse finding between MUC2 expression and CD10 activity in CGIT. CD10 is the intestinal marker, where it delineates the brush border of the surface epithelium of the small intestinal mucosa [21]. CD10 is a surface glycoprotein metalloenzyme that uses zinc as a cofactor, and modulates cellular responses to peptide hormones by regulating local peptide concentrations. CD10 has many biologic effects, including attenuation of the neutrophil inflammatory response [19]. Incomplete intestinal differentiation implied a combination of intestinal (MUC2) and gastric (MUC5AC) differentiation without CD10 expression [12], which was present in CGIT. In addition, intestinal metaplasia of the urinary bladder, despite its morphologic resemblance to intestinal metaplasia in the other organs, has different immunohistochemical profiles [23]. Incomplete forms of gastric intestinal metaplasia exhibit MUC1 and MUC5AC in both goblet and absorptive cells, MUC2 in goblet cells only, and MUC6 in over 60% of cases [8,22]. Also, moderate to strong MUC1 and CD10 expression was observed in almost all cases of CGTP, an expression pattern that is similar to normal urothelium. CGTP displayed only small amounts of neutralmucin, while CGIT had abundant cytoplasmic sialo, sulpho and neutralmucins. This reaction has been regarded as unique to the epithelial mucins in the normal terminal ileum and large intestine. Cdx2, an intestinal homeobox gene, is an important factor for the development of intestinal metaplasia. Cdx2 mRNAs are widely present in the human
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Fig. 1. Histological and histochemical features in cystitis glandularis: (A) typical cystitis glandularis with tall columnar epithelial cells and its abrupt transition to cystitis glandulari of the intestinal type with goblet cells in the lamina propria (HE staining – original magnification 20), (B) the goblet cells in the upper half of the crypt in cystitis glandularis of the intestinal type contained both sulphomucins (black) and sialomucins (blue), while lower goblet cells contained predominantly sulphated mucins (arrow). Acid product not detected in typical cystitis glandularis (upper right side) (HID-AB, pH 2.5 – original magnification 20).
Fig. 2. Expression of CD10 and MUC1 in cystitis glandularis: (A) very strong CD10 reaction in typical cystitis glandularis; brown staining of cell membrane with superficial and diffuse distribution. The goblet cells in intestinal type (arrow) are negative. (immunohistochemistry – original magnification 20) and (B) expression of MUC1 was predominant on the apical membranes in typical cystitis glandularis (immunohistochemistry – original magnification 10).
Table 1. Histochemical finding of neutral and acid mucins in both types of cystitis glandularis Neutralmucins
Sialomucins
Sulphomucins
Typical 1 2 3 4 5 6 7 8 9 10
cystitis glandularis 1+ 1+ 1+ 1+ – 1+ 1+ – 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
Cystitis 1 2 3 4 5 6 7 8 9 10
glandularis intestinal type 2+ 2+ 2+ 2+ 1+ 1+ 1+ 1+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 2+ 2+
1+ 1+ 1+ 2+ 1+ 1+ 1+ 1+ 1+ 1+
Fig. 3. Expression of MUC2 and MUC5AC in cystitis glandularis: (A) goblet cells in intestinal type cystitis glandularis show strong diffuse expression of MUC2. Typical cystitis (arrow) is negative (immunohistochemistry – original magnification 20) and (B) positive expression of MUC5AC in columnar cells of intestinal type cystitis glandularis, and negative finding in typical cystitis glandularis (arrow) (immunohistochemistry – original magnification 20).
intestinal-differentiated enterocytes [23]. Sung et al. studied Cdx2 and hepatocyte-specific antigen (Hep) in CG and found nuclear staining of Cdx2 in 83% cases of CGIT, but Cdx2 was not observed in any case of CGTP. However, none of the cases of intestinal metaplasia of the urinary bladder expressed Hep in contrast to its expression in the upper gastrointestinal tract. The absence of Hep staining in intestinal metaplasia of
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Table 2. Expression of MUCs and CD10 in normal urothelium Control
MUC1
MUC2
MUC5AC
MUC6
CD10
1 2 3 4 5 6 7 8
2+ 2+ 2+ 2+ 2+ 2+ 1+ 2+
– – – – – – – –
– – – – – – – –
– – – – – – – –
— 1+ 1+ 1+ 1+ — 1+ —
Table 3. Expression of MUCs and CD10 in typical cystitis glandularis Typical cystitis glandularis
MUC1 MUC2 MUC5AC MUC6 CD10
1 2 3 4 5 6 7 8 9 10
– 1+ 2+ 1+ 2+ 1+ 1+ 1+ 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
– – – – – – – – – –
2+ 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 1+
Table 4. Expression of MUCs and CD10 in cystitis glandularis of the intestinal type Cystitis glandularis intestinal type
MUC1 MUC2 MUC5AC MUC6 CD10
1 2 3 4 5 6 7 8 9 10
– – – – – – – – – –
2+ 1+ 1+ 2+ 2+ 1+ 2+ 2+ 1+ 1+
1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
the urinary bladder, despite its morphologic resemblance to normal colonic mucosa and intestinal metaplasia in other organs, may suggest the presence of unique metaplastic pathways in the urinary bladder [23,26]. In conclusion, CGIT and CGTP have different and contrasting immunoprofiles. The intestinal type expresses secreted gel-forming mucin (MUC2 and MUC
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5AC), while positive MUC1 expression and CD10 expression were observed in the typical type.
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[21] T. Niwa, Y. Ikehara, H. Nakanishi, H. Tanaka, K.I. Inada, T. Tsukamoto, M. Ichinose, M. Tatematsu, Mixed gastric- and intestinal-type metaplasia is formed by cells with dual intestinal and gastric differentiation, J. Histochem. 53 (2005) 75–82. [22] C.A. Reis, L. David, P. Correa, F. Carneiro, C. de Bolos, E. Garcia, U. Mandel, H. Clausen, M. Sobrinho-Simoes, Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression, Cancer Res. 59 (1999) 1003–1007. [23] M.T. Sung, L. Lopez-Beltran, J.N. Eble, G.T. MacLennan, P.H. Tan, R. Montironi, T.D. Jones, T.M. Ulbright, J.E. Blair, L. Cheng, Divergent pathway of intestinal metaplasia and cystitis glandularis of the urinary bladder, Mod. Pathol. 19 (2006) 1395–1401. [24] S. Tsukashita, R. Kushima, M. Bamba, H. Sugihara, T. Hattori, MUC gene expression and histogenesis of adenocarcinoma of the stomach, Int. J. Cancer 94 (2001) 166–170. [25] M.D. Walsh, B.G. Hohn, W. Thong, P.L. Devine, R.A. Gardiner, M.L. Samaratunqa, M.A. McGuckin, Mucin expression by transitional cell carcinomas of the bladder, Br. J. Urol. 73 (1994) 256–262. [26] R. Werling, H. Yaziji, C. Bacchi, A. Gown, Cdx2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas,, Am. J. Surg. Pathol. 27 (2003) 303–310. [27] R.H. Young, D.G. Bostwick, Florid cystitis glandularis of intestinal type with mucin extravasation: a mimic of adenocarcinoma, Am. J. Surg. Pathol. 20 (1996) 1462–1468.
Pathology – Research and Practice 203 (2007) 653–658 www.elsevier.de/prp
ORIGINAL ARTICLE
Differences in the expression of mucins in various forms of cystitis glandularis Ljubinka Jankovic Velickovica, Vuka Katica, Takanori Hattorib, Ryoji Kushimab, Goran Marjanovicc, Vladisav Stefanovicd, a
Institute of Pathology, Faculty of Medicine, Nis, Serbia Department of Pathology, Shiga University of Medical Science, Ohtsu, Japan c Clinic of Hematology and Clinical Immunology Faculty of Medicine Nis, Serbia d Institute of Nephrology and Hemodialysis, Faculty of Medicine, Nis, Serbia b
Received 9 March 2007; accepted 7 May 2007
Abstract A wide spectrum of glandular epithelial metaplastic changes may be seen in the bladder. Cystitis glandularis (CG) is a well-known metaplastic lesion occurring in the presence of chronic inflammation, but there are a few data about mucin expression in its two subtypes (typical and intestinal). The purpose of the present study was to determine the expression of mucin core proteins and CD10 in the different types of CG. For this examination, we used a panel of monoclonal-specific antibodies for MUC1, MUC2, MUC5AC, and MUC6. CG of the intestinal type expressed MUC5AC both in goblet and columnar cells, and strongly expressed intestinal mucin MUC2 only in goblet cells in all cases. There was no expression of MUC1, MUC6, and CD10 in the metaplastic cells. CG of the typical type showed an expression of MUC1 similar to normal urotelium, but the CD10 expression was more intensive than in the control. The mucin expression profile in the different types of CG allows the identification of ‘‘gastric mucin’’ (MUC5AC) together with intestinal mucin (MUC2), while typical CG (CGTP) retains MUC1. Different and contrasting immunoprofiles were evident in various forms of CG. The absence of CD 10 in CG of the intestinal type is a finding that points towards an incomplete form of urinary bladder metaplasia. r 2007 Elsevier GmbH. All rights reserved. Keywords: CD10; Cystitis glandularis; Intestinal metaplasia; Mucin
1. Introduction Cystitis glandularis (CG) is a metaplastic alteration of the urothelium in the urinary bladder, which is thought to be induced by chronic inflammation or irritation. In the presence of chronic inflammation, bladder mucosa becomes hyperproliferative, and nests of urothelium Corresponding author. Tel.: +381 18 234092; fax: +381 18 238770.
E-mail addresses: [email protected] (L. Jankovic Velickovic), [email protected] (V. Stefanovic). 0344-0338/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2007.05.003
may appear within the lamina propria (von Brunn’s nests) [18]. With continued inflammation, further differentiation to cystic lesions (cystitis cystica) or glandular lesions (CG) may occur [16,18]. Cystitis cystica is considered benign, whereas CG is of uncertain malignant potential. Several authors have described the progression to bladder adenocarcinoma during longterm follow up [4,6,15,17,27], although others have disputed this finding [9]. In the past, the majority of authors subdivided CG into two subtypes with different morphologic findings. Typical CG (CGTP) is
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characterized by nests of columnar epithelial cells within the lamina propria of the bladder, forming glandular structures, while intestinal CG (CGIT) is characterized by similar glandular structures made of simple columnar epithelial cells that are replaced by mucin-secreting goblet cells. The two subtypes may coexist, and previous studies have specifically described the intestinal subtype as premalignant [4,18,23]. The recent identification of several mucin genes, as well as the production of monoclonal antibodies (mAbs) to mucin protein cores, has led to the immunohistochemical localization of several mucin species. Mucin core proteins comprise an expanded gene family consisting of at least 19 members. Mucins are highmolecular-weight glycoproteins with oligosaccharides attached to the apomucin protein backbone by O-glycosidic linkage. The synthesis and secretion of mucins are common features of glandular epithelial tissue [2,8,13,14,22,24]. Mucins are classified into membrane-associated and secreted types. MUC1 is a transmembrane glycoprotein that was detected in most epithelial tissues, including the breast and pancreas, as well as in urinary, gastrointestinal, and respiratory tracts. It is known that MUC1 has a role in lumen formation, as well as an inhibitory role in the cell to stroma interaction [1,20]. MUC2, a prototype seretory mucin expressed in the colon, small intestine, and airways, differs from MUC1 in its structure [8,14]. CD10 is a neutral endopeptidase, the expression of which represents a useful tool in the classification and diagnosis of malignant leukemia and lymphoma. Immunohistochemical studies have demonstrated that this antigen is expressed in a variety of non-hematopoietic normal and tumor tissues, including utothelial neoplasms and normal urothelium [11,19]. The aim of this study was to evaluate and compare the expression of MUC1, MUC2, MUC5AC, MUC6, and CD10 to determine the immunoprofile of these two types of CG.
2. Materials and methods Tissues from 10 patients containing CG were obtained from the Surgical Pathology archives of the Institute of Pathology between 1996 and 2005. Tissue samples consisted of biopsy material obtained from noncancer patients. Seven of the 10 patients were male and three were female, with a median age of 52.7716.06 (24–74) years. Although both types of CG were found in every sample, CGIT was a predominant finding. Eight normal bladder urothelia were used as controls. All tissues were fixed in 10% formalin for 24–48 h and embedded in paraffin. We classified CG into CGTP and the CGIT on hematoxylin- and eosin-stained sections.
2.1. Mucin histochemical and immunohistochemical studies Biopsies with both types of CG were stained with PAS and the AB/HID method (pH 2.5) to determine whether they expressed neutralmucins, sialomucins, or sulphomucins [10]. The intensity of mucin content was scored as negative ( ), weak (+), or strong (2+). The histochemical detection of mucins was used to determine the type of mucins secretion in various forms of CG. Immunohistochemical techniques are widely used to identify intestinal and gastric cell differentiation for the classification of intestinal metaplasia. We detected mucin by using the following mAbs from Novocastra, Newcastle, UK: MUC1 (MA695, 1:100), MUC2 (CCP 58, 1:100), MUC5AC (CLH2, 1:100), MUC6 (CLH5, 1:100), and CD10 (56C6; 1:50). Immunohistochemical study using the streptavidin–biotin method (Histofine SAB-PO (M) kit) was performed at the Department of Pathology, Shiga University of Medical Science, Japan. Peroxidase-binding sites were visualized using diaminobensidine, and nuclei were lightly counterstained with hematoxylin. Positive staining for the MUC gene and CD10 was graded as diffuse (2+: over 30% of cells), sporadic (1+: less than 30% of cells), or negative (-: less than 5% of cells) [11].
3. Results 3.1. Microscopic features Most of the analyzed bladder slides showed numerous glands in the lamina propria, showing the features of CG of the typical and intestinal type. The lining epithelial cells in CGTP are columnar and surrounded by several layers of transitional cells. The predominant finding in all cases was CGIT, usually the florid type. It consisted of numerous glands lined by a single layer of goblet cells with basally situated nuclei. Occasionally, we noted abrupt transition from CGTP to CGIT (Fig. 1A). Some glands were lined by tall columnar epithelium with plenty of goblet cells. Extravasation of mucin into the stroma was present only in one case, without desmoplastic response. The coexistence of areas with typical and intestinal type of CG formed the mass that simulated a neoplasm.
3.2. Histochemical features Almost all crypts of CGIT were PAS and AB/HID positive. The mucin production was predominantly intracellular, and was expressed in all 10 cases of frank intestinal metaplasia. Most goblet cells contained acid
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(predominantly sialomucins) product. The goblet cells at the surface of the upper half of the crypt contained both sulpho and sialomucins, while the lower goblet cells contained predominantly sulphated mucins with HID–AB reactivity (Fig. 1B). The extracellular mucin content, which was seen focally in one case of CGIT, consisted of sialo and neutralmucins. The intracellular mucin production was expressed in CGTP with PAS, but its amounts were small. Acid product was not detected in CGTP (Fig. 1B). The histochemical findings in CG are summarized in Table 1. Eight normal urothelium samples showed no PAS and HID–AB reactivity.
3.3. MUC1, MUC2, MUC5AC, MUC6, and CD10 expression In the normal urothelium, MUC1 was limited predominantly to the apical membranes of the umbrella cell layer, while CD10 was found to be expressed weakly (+), or the reaction was negative in the apical portion of the cytoplasm along the luminal surface (Table 2). On the other hand, all samples of CGTP showed very strong CD10 reaction with brown staining of cell membrane, and distribution was superficial or diffuse (Fig. 2A). MUC1 was expressed in all samples of CGTP (Fig. 2B). All samples of both normal urothelium and CGTP were uniformly negative for MUC2, MUC5AC, and MUC6 (Tables 2 and 3). In sharp contrast to this, MUC1 was not detected in CGIT, but on the other hand, MUC2 and MUC5AC were expressed in all 10 cases (100%). We detected MUC2 in goblet cells only (Fig. 3A) and MUC5AC in columnar cells (Fig. 3B). MUC6 (pyloricgland mucin) and CD10 were negative in all CGITs (Table 4).
4. Discussion Mucins constitute a large family of glycoproteins expressed by many epithelial cells and their malignant counterparts. MUC1 is a type of mucin encoded by the MUC1 gene, located on chromosome 1 (1q21). MUC1 is known to have a role in lumen formation, and has an inhibitory role in the cell-to-stroma interaction [20]. In normal urothelium, MUC1 expression was consistently strong and diffuse in the apical membranes of the umbrella cell layer. This membrane-associated mucin was also detected in malignant urothelium [7,20,25]. The gel-forming mucins, MUC2 and MUC5AC, are found in a cluster on chromosome 11p15.5. The MUC2 gene codes for a typical secretory mucin that predominates in goblet cells of the small and large intestine [3,8]. MUC5AC was demonstrated in gastric columnar epithelial cells, while MUC6 was present on the cells
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in the glandular compartment of pyloric mucosa. MUC5AC and MUC6 are two major secretory mucins of the stomach [2,3,22]. Our immunohistochemical study demonstrated an intestinal and gastric phenotype in CGIT by the detection of MUC2 and MUC5AC expression. In contrast to normal urinary bladder mucosa, CGIT displayed no expression of MUC1. Normal colorectal mucosa showed positive staining for MUC1, MUC2, and MUC4 [3]. MUC1 expression is most obvious within the proliferative zone in normal mucosa, whereas mature goblet cells secreting MUC2 are present mainly in the maturation zone [12]. In normal adults, MUC5AC is not expressed in the intestinal tract. Buisine et al. have shown that MUC5AC is expressed in emryonic intestine up to 8 weeks of gestation. However, after 12 weeks, expression is significantly reduced, as mRNAs were not detected in any intestinal region from the 13th week of gestation onwards. Thus, MUC5AC is developmentally expressed during intestinal ontogenesis [5,14]. Also, MUC5AC was shown to be expressed in rectosigmoid villous adenomas [11]. Also, CD10 was never detected in CGIT. To our knowledge, the present study is the first report that clearly demonstrates an inverse finding between MUC2 expression and CD10 activity in CGIT. CD10 is the intestinal marker, where it delineates the brush border of the surface epithelium of the small intestinal mucosa [21]. CD10 is a surface glycoprotein metalloenzyme that uses zinc as a cofactor, and modulates cellular responses to peptide hormones by regulating local peptide concentrations. CD10 has many biologic effects, including attenuation of the neutrophil inflammatory response [19]. Incomplete intestinal differentiation implied a combination of intestinal (MUC2) and gastric (MUC5AC) differentiation without CD10 expression [12], which was present in CGIT. In addition, intestinal metaplasia of the urinary bladder, despite its morphologic resemblance to intestinal metaplasia in the other organs, has different immunohistochemical profiles [23]. Incomplete forms of gastric intestinal metaplasia exhibit MUC1 and MUC5AC in both goblet and absorptive cells, MUC2 in goblet cells only, and MUC6 in over 60% of cases [8,22]. Also, moderate to strong MUC1 and CD10 expression was observed in almost all cases of CGTP, an expression pattern that is similar to normal urothelium. CGTP displayed only small amounts of neutralmucin, while CGIT had abundant cytoplasmic sialo, sulpho and neutralmucins. This reaction has been regarded as unique to the epithelial mucins in the normal terminal ileum and large intestine. Cdx2, an intestinal homeobox gene, is an important factor for the development of intestinal metaplasia. Cdx2 mRNAs are widely present in the human
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Fig. 1. Histological and histochemical features in cystitis glandularis: (A) typical cystitis glandularis with tall columnar epithelial cells and its abrupt transition to cystitis glandulari of the intestinal type with goblet cells in the lamina propria (HE staining – original magnification 20), (B) the goblet cells in the upper half of the crypt in cystitis glandularis of the intestinal type contained both sulphomucins (black) and sialomucins (blue), while lower goblet cells contained predominantly sulphated mucins (arrow). Acid product not detected in typical cystitis glandularis (upper right side) (HID-AB, pH 2.5 – original magnification 20).
Fig. 2. Expression of CD10 and MUC1 in cystitis glandularis: (A) very strong CD10 reaction in typical cystitis glandularis; brown staining of cell membrane with superficial and diffuse distribution. The goblet cells in intestinal type (arrow) are negative. (immunohistochemistry – original magnification 20) and (B) expression of MUC1 was predominant on the apical membranes in typical cystitis glandularis (immunohistochemistry – original magnification 10).
Table 1. Histochemical finding of neutral and acid mucins in both types of cystitis glandularis Neutralmucins
Sialomucins
Sulphomucins
Typical 1 2 3 4 5 6 7 8 9 10
cystitis glandularis 1+ 1+ 1+ 1+ – 1+ 1+ – 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
Cystitis 1 2 3 4 5 6 7 8 9 10
glandularis intestinal type 2+ 2+ 2+ 2+ 1+ 1+ 1+ 1+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 2+ 2+
1+ 1+ 1+ 2+ 1+ 1+ 1+ 1+ 1+ 1+
Fig. 3. Expression of MUC2 and MUC5AC in cystitis glandularis: (A) goblet cells in intestinal type cystitis glandularis show strong diffuse expression of MUC2. Typical cystitis (arrow) is negative (immunohistochemistry – original magnification 20) and (B) positive expression of MUC5AC in columnar cells of intestinal type cystitis glandularis, and negative finding in typical cystitis glandularis (arrow) (immunohistochemistry – original magnification 20).
intestinal-differentiated enterocytes [23]. Sung et al. studied Cdx2 and hepatocyte-specific antigen (Hep) in CG and found nuclear staining of Cdx2 in 83% cases of CGIT, but Cdx2 was not observed in any case of CGTP. However, none of the cases of intestinal metaplasia of the urinary bladder expressed Hep in contrast to its expression in the upper gastrointestinal tract. The absence of Hep staining in intestinal metaplasia of
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Table 2. Expression of MUCs and CD10 in normal urothelium Control
MUC1
MUC2
MUC5AC
MUC6
CD10
1 2 3 4 5 6 7 8
2+ 2+ 2+ 2+ 2+ 2+ 1+ 2+
– – – – – – – –
– – – – – – – –
– – – – – – – –
— 1+ 1+ 1+ 1+ — 1+ —
Table 3. Expression of MUCs and CD10 in typical cystitis glandularis Typical cystitis glandularis
MUC1 MUC2 MUC5AC MUC6 CD10
1 2 3 4 5 6 7 8 9 10
– 1+ 2+ 1+ 2+ 1+ 1+ 1+ 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
– – – – – – – – – –
2+ 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 1+
Table 4. Expression of MUCs and CD10 in cystitis glandularis of the intestinal type Cystitis glandularis intestinal type
MUC1 MUC2 MUC5AC MUC6 CD10
1 2 3 4 5 6 7 8 9 10
– – – – – – – – – –
2+ 1+ 1+ 2+ 2+ 1+ 2+ 2+ 1+ 1+
1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
– – – – – – – – – –
– – – – – – – – – –
the urinary bladder, despite its morphologic resemblance to normal colonic mucosa and intestinal metaplasia in other organs, may suggest the presence of unique metaplastic pathways in the urinary bladder [23,26]. In conclusion, CGIT and CGTP have different and contrasting immunoprofiles. The intestinal type expresses secreted gel-forming mucin (MUC2 and MUC
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5AC), while positive MUC1 expression and CD10 expression were observed in the typical type.
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