- Email: [email protected]
Expression of trefoil peptides in the subtypes of intestinal metaplasia
Peptides xxx (2004) xxx–xxx
Expression of trefoil peptides in the subtypes of intestinal metaplasia Byung-Wook Kim a,∗ , Kyoung-Mee Kim b , Bo-In Lee a , Lee-So Maeng b , Hwang Choi a , Se-Hyun Cho a , Hiun-Suk Chae a , Jae-Kwang Kim a , Kyu-Yong Choi a , In-Sik Chung a b
a Department of Internal Medicine, The Catholic University of Korea, Seoul, South Korea Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, South Korea
Received 27 August 2003; accepted 8 December 2003 Available online 10 May 2004
Abstract We studied the expression of trefoil peptides in the different types of intestinal metaplasia of the stomach. Endoscopic biopsy was performed in 132 patients with dyspepsia. Intestinal metaplasia subtype was classified according to the pattern of alcian blue/PAS staining and high iron diamine staining. Expression of trefoil peptides was measured by immunohistochemistry. TFF1 and TFF3 were mainly expressed in goblet cells and TFF2 in columnar cells in all the types of intestinal metaplasia. There was a gradual decrease of TFF1 and TFF3, and increase of TFF2, during the progression of intestinal metaplasia from type I to type III via the type II intermediate. © 2004 Elsevier Inc. All rights reserved. Keywords: Trefoil peptide; Metaplasia; Stomach; Immunohistochemistry
1. Introduction Human trefoil peptides are expressed predominantly in the gastrointestinal tract and play key roles in mucosal protection by forming a mucous barrier, and also in mucosal repair by promoting recovery after injury [17]. It has been suggested that changes in their expression are associated with several diseases, including carcinoma of the stomach [11,13]. It also has been proposed that point mutation in the TFF1 gene [14] and alterations of TFF1 structure, including hypermethylation, are associated with the development of gastric cancer [6]. Metaplasia is defined as a potentially reversible change from one fully differentiated cell type to another, and intestinal metaplasia consists of the replacement of the gastric mucosa by an epithelium that resembles histologically the intestinal mucosa, and is a well-established premalignant condition of the stomach [10]. Two main subtypes of intestinal metaplasia have been identified according to their histology: the complete type that is characterized by the presence of absorptive cells, Paneth cells, and goblet cells secreting sialomucins and corresponds to the small intestine pheno∗
Corresponding author. Present address: Division of Gastroenterology, Department of Internal Medicine, Our Lady of Mercy Hospital, The Catholic University of Korea, #665, Pupyong-dong, Pupyong-gu, Incheon, 403-720, South Korea. Tel.: +82-32-510-5500; fax: +82-32-510-5683. E-mail address: [email protected] (B.-W. Kim). 0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.12.021
type; and the incomplete type characterized by the presence of columnar and goblet cells secreting sialo- and/or sulfomucins corresponding to the large intestine phenotype [2]. Intestinal metaplasia can also be subdivided into types I, II and III according to the mucin expression pattern. Type I is the same as the complete type while types II and III both correspond to the incomplete type [5]; type II intestinal metaplasia mainly secrete neutral and acid sialomucin, whereas type III mainly secrete sulfomucin. Intestinal metaplasia type III is often considered an ominous indicator of the future development of the intestinal form of gastric cancer [1,16,18]. Alterations in the expression of trefoil peptides have been reported in intestinal metaplasia [7,19]. However, there have been no reports of the pattern of trefoil peptides in the subtypes of intestinal metaplasia. In the present study, we carried out such a characterization in the hope of obtaining further insight into the process of gastric carcinogenesis.
2. Materials and methods 2.1. Subjects Endoscopic biopsy was performed in patients with non-ulcer dyspepsia after methylene blue chromoscopy. All patients provided written informed consent. Two pieces
2
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
were taken from the corpus and another two pieces from the antrum, and each specimen was fixed in 10% formalin and routinely embedded in paraffin wax. Serial sections were cut and used for histochemistry and immunohistochemistry.
sults and consulted over instances where their classification did not agree.
2.2. Staining for intestinal metaplasia subtypes
Statistical analyses were performed with the χ2 test for trend using SPSS version 10.0 for Windows. P < 0.05 was regarded as statistically significant.
Alcian blue (pH 2.5)/periodic acid-Schiff and the high iron diamine-alcian blue technique were used to identify neutral, sialomucins, and sulfomucins [3]. Intestinal metaplasia was classified according to Filipe as follows: type I, mature absorptive cells and goblet cells, the latter secreting sialomucins; type II, few or no absorptive cells, presence of columnar intermediate cells in various stages of differentiation secreting neutral and acid sialomucins and goblet cells secreting sialomucins or, occasionally, sulfomucins, or both; and type III, columnar intermediate cells secreting predominantly sulfomucins, and goblet cells secreting sialomucins or sulfomucins, or both [2,5]. 2.3. Immunohistochemistry of trefoil peptides Immunohistochemistry was performed with mouse monoclonal antibodies (TFF1, TFF2; kindly provided by Drs. N. Wright and G. Elia, Cancer Research UK, London, UK) and rat polyclonal antibody (TFF3; kindly provided by Dr. A. Giraud, University of Melbourne, Melbourne, Australia). In brief, formalin-fixed 5 m sections were deparaffinized, rehydrated, and washed in tris-buffered saline (TBS). Endogenous peroxidase activity and nonspecific binding were blocked by incubation with 3% hydrogen peroxide (H2 O2 ) for 5 min, and blocking solution (normal immune serum) was added for 10 min followed by TFF1 primary antibody (dilution 1:400) for 60 min. After rinsing with TBS, biotinylated secondary antibody was added for 10 min and the slides again rinsed with TBS. Enzyme conjugation was performed with streptavidin for 10 min. With TFF2 (dilution 1:200), goat antimouse IgM biotinylated secondary antibody was added for 30 min and with TFF3 (dilution 1:200), rabbit anti-rat IgG biotinylated secondary antibody was added for 60 min. The slides were finally incubated with avidin-biotinylated peroxidase complex (ABComplex/HRP, Dako, Glostrup, Denmark). All of the outcomes were visualized with diaminobenzidine (DAB) and counterstained with Meyer’s hematoxylin. TBS alone without primary antibody acted as negative control for the immunostaining. Normal human gastric mucosa obtained from endoscopic biopsy and normal colonic mucosa from surgical specimen were used for positive controls along with TFF1 and TFF2 antibodies, and TFF3 antibody, respectively. The immunoreactivity of the trefoil peptides was graded from (−) to (+++) according to the percentage of positive cells. Immunoreactivity in goblet cells and columnar cells was assessed separately. Two pathologists reviewed the re-
2.4. Statistics
3. Results 3.1. Subtypes of intestinal metaplasia In the 132 patients included in the study, intestinal metaplasia was detected in the corpus in 88 patients and in the antrum in 107 patients. Type I intestinal metaplasia was detected in the corpus in 69 patients (78.4%), and in the antrum in 22 patients (20.6%) while type II was present in the corpus in 17 patients (19.3%), and in the antrum in 46 patients (43.0%). Finally, type III was found in the corpus in two patients (2.3%), and in the antrum in 39 patients (36.4%). Types II and III intestinal metaplasia occurred more commonly in the antrum than in the corpus (P < 0.05). 3.2. Immunohistochemistry of trefoil peptides TFF1 was mainly expressed in goblet cells and rarely in columnar cells in all the types of intestinal metaplasia (Fig. 1A). TFF2, however, was not expressed in goblet cells but only in columnar cells in all three types (Fig. 1B). TFF3 was expressed in both goblet cells and in columnar cells, in every type of intestinal metaplasia, but was more highly expressed in the goblet cells (Fig. 1C). TFF3 showed the strongest immunoreactivity of the trefoil peptides in all the types of intestinal metaplasia. The expression of TFF1 and TFF3 in goblet cells tended to decrease from type I to III via type II (Fig. 2A and C P = 0.045, P = 0.002, respectively). There was no difference between the subtypes in the expression of TFF3 in columnar cells. However, TFF2 expression in columnar cells tended to increase from type I to type III via type II (Fig. 2B, P < 0.001). The immunoreactivity of trefoil peptides in types I and II was similar in antrum and corpus.
4. Discussion In the West, the frequency of type III intestinal metaplasia is reported to be about 10% [4]. In this study, however, its frequency was 2.3% in the corpus and 36.4% in the antrum. This suggests that intestinal metaplasia begins in the antrum and progress to the corpus. This can be easily explained if H. pylori migrates from antrum to corpus to cardia. It is therefore plausible that H. pylori makes
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
3
Fig. 2. (A) Expression of TFF1 in goblet cells of intestinal metaplasia subtypes. Note the down regulation of TFF1 from type I via type II to type III. (B) Expression of TFF2 in columnar cells of the intestinal metaplasia subtypes. Note the increase of TFF2 from type I via type II to type III. (C) Expression of TFF3 in goblet cells of the intestinal metaplasia subtypes. Down regulation of TFF3 from type I via type II to type III can be seen. (IM I, type I intestinal metaplasia; IM II, type II intestinal metaplasia; IM III, type III intestinal metaplsia).
Fig. 1. (A) TFF1 is mainly expressed in goblet cells (arrow) and weakly expressed in columnar cells (open arrow). (B) TFF2 is weakly expressed in columnar cells (open arrow) and not expressed in goblet cells (arrow). (C) TFF3 is highly expressed in goblet cells (arrow) and somewhat weakly expressed in columnar cells (open arrow).
an important contribution to the progression of intestinal metaplasia. About 57.8% of adult Korean patients are infected with H. pylori [9], and gastric cancer is still the most common cancer [12]. This difference between Western inhabitants and Koreans may account for the high incidence of intestinal metaplasia type III and the subsequent high incidence of antral gastric cancer. In this study population, H. pylori infection was detected in 121 patients. TFF1 was expressed in each type of intestinal metaplasia, mainly in goblet cells. However, the immunoreactivity was weaker than in the adjacent normal gastric epithelium. This is in agreement with a previous study [19]. TFF1 was more highly expressed in goblet cells than in columnar cells in all the types of intestinal metaplasia. Furthermore, the
4
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
immunoreactivity of TFF1 tended to decrease from type I to type III via type II. These results suggest that the different types of intestinal metaplasia retain a gastric component and lose it progressively from type I to type III via type II. In the light of other reports [8], it is possible that down regulation of TFF1 production may start in type I and progress to type III via type II as an intermediate that is much more susceptible to damage from other carcinogens, and finally progress to gastric cancer. TFF2 was not expressed in goblet cells and only weakly expressed in columnar cells in every type of intestinal metaplasia. TFF2 expression in columnar cells increased from type I via type II to type III. According to other reports, TFF2 begins to be upregulated from the stage of dysplasia [8]. However, our study suggests that its re-expression starts from intestinal metaplasia and progresses from type I to III via type II. It has also been suggested in another study [8] that upregulation of TFF2 is associated with gastric carcinogenesis. TFF3 is at best weakly expressed in normal gastric epithelium. It is noteworthy that its immunoreactivity was higher than that of TFF1 and TFF2 in every types of intestinal metaplasia, especially in goblet cells. This has also been reported in another study [19], and may suggest that intestinal type epithelia, such as intestinal metaplasia, secrete predominantly the intestinal types of trefoil peptides, though they still have gastric components. TFF3 also showed a decreasing incidence from type I to type III via type II and it is therefore uncertain whether TFF3 can play any role in the suppression of gastric cancer. It will be important to clarify whether TFF3 can play any role in an acidic milieu and whether it can act as a cytoprotectant of gastric epithelia with intestinal metaplasia. We had intended to compare the expression pattern of trefoil peptides in the antrum and corpus in intestinal metaplasia, but type III intestinal metaplasia was detected in the corpus in only two cases, and it was therefore difficult to make the comparison. However in types I and II, where the comparison was possible there was no difference in TFF1, TFF2, and TFF3 expression in antrum and corpus. In normal gastric mucosa TFF1 is secreted by the surface cells of the corpus and antrum, and TFF2 by the mucous neck cells. However, we found that in intestinal metaplasia, goblet cells secreted TFF1 and TFF3 and columnar cells secreted all three trefoil peptides, regardless of type, though TFF3 showed the highest expression. Though only TFF1 is known to be a gastric-specific tumor suppressor gene, the other trefoil peptides may participate in the process of gastric cancer formation. This question should be elucidated at the gene level. It is well known that trefoil peptides are co-expressed with mucin, and that peptides and mucin play synergistic roles in maintaining mucosal integrity. The expression pattern of mucin glycoprotein in the intestinal metaplasia subtypes was reported previously [15]. The trefoil peptides alone cannot be used as markers of the intestinal metaplasia subtypes, and
studies of the co-expression patterns with mucin might provide insight into the role of the trefoil peptides in intestinal metaplasia, and clarify whether they can be used as markers for subtyping intestinal metaplasias. In conclusion, there is progressive loss of TFF1 and TFF3, together with induction of TFF2, during the development of intestinal metaplasia from type I to type III via the type II intermediate. Inhibition of these events may block the progression of intestinal metaplasia and prevent the ensuing gastric cancer.
Acknowledgments We thank Dr. N. Wright and G. Elia of Cancer Research UK, London, UK for providing the TFF1 and TFF2 mouse monoclonal antibodies. We also thank Dr. A. Giraud of the University of Melbourne, Melbourne, Australia for providing the TFF3 rat polyclonal antibody. This study was partially supported by the Research Institute of Clinical Medicine, Our Lady of Mercy Hospital, The Catholic University of Korea. References [1] Craanen ME, Blok P, Dekker W, Ferwerda J, Tytgat GNJ. Prevalence of subtypes of intestinal metaplasia in gastric antral mucosa. Dig Dis Sci 1991;36:1529–36. [2] Filipe MI, Jass JR. Intestinal metaplasia subtypes and cancer risk. In: Filipe MI, Jass JR, editors. Gastric carcinoma. London: Churchill Livingstone; 1986. p. 87–115. [3] Filipe MI, Lake BD. Appendix 3. In: Filipe MI, Lake BD, editors. Histochemistry in pathology. 2nd ed. London: Churchill Livingstone; 1990. p. 449–53. [4] Filipe MI. Gastrointestinal carcinoma and precursor lesions. In: Filipe MI, Lake BD, editors. Histochemistry in pathology. 2nd ed. London: Churchill Livingstone; 1990. p. 175–98. [5] Filipe MI. Borderline lesions of the gastric epithelium: new indicators of gastric risk and clinical implications. In: Fenoglio-Preiser CM, Wolff M, Rilke F, editors. Progress in surgical pathology. Philadelphia: Field & Wood Medical Publishers; 1992. p. 269–90. [6] Fujimoto J, Yasui W, Tahara H, Tahara E, Kudo Y, Yokozaki H, et al. DNA hypermethylation at the pS2 promoter region is associated with early stage of stomach carcinogenesis. Cancer Lett 2000;149:125–34. [7] Hanby AM, Poulsom R, Singh S, Elia G, Jeffery RE, Wright NA. Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach. Gastroenterology 1993;105:1110–6. [8] Hu GY, Yu BP, Dong WG, Li MQ, Yu JP, Luo HS, et al. Expression of TFF2 and Helicobacter pylori infection in carcinogenesis of gastric mucosa. World J Gastroenterol 2003;9:910–4. [9] Kim HS, Lee YC, Lee HW, Yoo HM, Lee CG, Kim JM, et al. Seroepidemiologic study of Helicobacter pylori in Korea. Kor J Gastroenterol 1999;33:170–82. [10] Leung WK, Sung JJ. Review article: intestinal metaplasia and gastric carcinogenesis. Aliment Pharmacol Ther 2002;16:1209–16. [11] Machado JC, Nogueira AM, Carneiro F, Reis CA, Sobrinho-Simoes M. Gastric carcinoma exhibits distinct types of cell differentiation: an immunohistochemical study of trefoil peptides (TFF1 and TFF2) and mucins (MUC1, MUC2, MUC5AC, and MUC6). J Pathol 2000;190:437–43.
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx [12] Ministry of Health and Welfare, Republic of Korea. Annual Report on National Cancer Registry; 2001. [13] Nogueira AM, Machado JC, Carneiro F, Reis CA, Gott P, Sobrinho-Simoes M. Patterns of expression of trefoil peptides and mucins in gastric polyps with and without malignant transformation. J Pathol 1999;187:541–8. [14] Park WS, Oh RR, Park JY, Lee JH, Shin MS, Kim HS, et al. Somatic mutations of the trefoil factor family 1 gene in gastric cancer. Gastroenterology 2000;119:691–8. [15] Reis CA, David L, Correa P, Carneiro F, de Bolos C, Garcia E, et al. Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression. Cancer Res 1999;59:1003–7.
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[16] Rokkas T, Filipe MI, Sladen GE. Detection of an increased incidence of early gastric cancer in patients with intestinal metaplasia type III who are closely followed up. Gut 1991;32:1110–3. [17] Sands BE, Podolsky DK. The trefoil peptide family. Annu Rev Physiol 1996;58:253–73. [18] Silva S, Filipe MI. Intestinal metaplasia and its variants in the gastric mucosa of Portuguese subjects: a comparative analysis of biopsy and gastrectomy material. Hum Pathol 1986;17:988–95. [19] Taupin D, Pedersen J, Familari M, Cook G, Yeomans N, Giraud AS. Augmented intestinal trefoil factor (TFF3) and loss of pS2 (TFF1) expression precedes metaplastic differentiation of gastric epithelium. Lab Invest 2001;81:397–408.
Expression of trefoil peptides in the subtypes of intestinal metaplasia Byung-Wook Kim a,∗ , Kyoung-Mee Kim b , Bo-In Lee a , Lee-So Maeng b , Hwang Choi a , Se-Hyun Cho a , Hiun-Suk Chae a , Jae-Kwang Kim a , Kyu-Yong Choi a , In-Sik Chung a b
a Department of Internal Medicine, The Catholic University of Korea, Seoul, South Korea Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, South Korea
Received 27 August 2003; accepted 8 December 2003 Available online 10 May 2004
Abstract We studied the expression of trefoil peptides in the different types of intestinal metaplasia of the stomach. Endoscopic biopsy was performed in 132 patients with dyspepsia. Intestinal metaplasia subtype was classified according to the pattern of alcian blue/PAS staining and high iron diamine staining. Expression of trefoil peptides was measured by immunohistochemistry. TFF1 and TFF3 were mainly expressed in goblet cells and TFF2 in columnar cells in all the types of intestinal metaplasia. There was a gradual decrease of TFF1 and TFF3, and increase of TFF2, during the progression of intestinal metaplasia from type I to type III via the type II intermediate. © 2004 Elsevier Inc. All rights reserved. Keywords: Trefoil peptide; Metaplasia; Stomach; Immunohistochemistry
1. Introduction Human trefoil peptides are expressed predominantly in the gastrointestinal tract and play key roles in mucosal protection by forming a mucous barrier, and also in mucosal repair by promoting recovery after injury [17]. It has been suggested that changes in their expression are associated with several diseases, including carcinoma of the stomach [11,13]. It also has been proposed that point mutation in the TFF1 gene [14] and alterations of TFF1 structure, including hypermethylation, are associated with the development of gastric cancer [6]. Metaplasia is defined as a potentially reversible change from one fully differentiated cell type to another, and intestinal metaplasia consists of the replacement of the gastric mucosa by an epithelium that resembles histologically the intestinal mucosa, and is a well-established premalignant condition of the stomach [10]. Two main subtypes of intestinal metaplasia have been identified according to their histology: the complete type that is characterized by the presence of absorptive cells, Paneth cells, and goblet cells secreting sialomucins and corresponds to the small intestine pheno∗
Corresponding author. Present address: Division of Gastroenterology, Department of Internal Medicine, Our Lady of Mercy Hospital, The Catholic University of Korea, #665, Pupyong-dong, Pupyong-gu, Incheon, 403-720, South Korea. Tel.: +82-32-510-5500; fax: +82-32-510-5683. E-mail address: [email protected] (B.-W. Kim). 0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.12.021
type; and the incomplete type characterized by the presence of columnar and goblet cells secreting sialo- and/or sulfomucins corresponding to the large intestine phenotype [2]. Intestinal metaplasia can also be subdivided into types I, II and III according to the mucin expression pattern. Type I is the same as the complete type while types II and III both correspond to the incomplete type [5]; type II intestinal metaplasia mainly secrete neutral and acid sialomucin, whereas type III mainly secrete sulfomucin. Intestinal metaplasia type III is often considered an ominous indicator of the future development of the intestinal form of gastric cancer [1,16,18]. Alterations in the expression of trefoil peptides have been reported in intestinal metaplasia [7,19]. However, there have been no reports of the pattern of trefoil peptides in the subtypes of intestinal metaplasia. In the present study, we carried out such a characterization in the hope of obtaining further insight into the process of gastric carcinogenesis.
2. Materials and methods 2.1. Subjects Endoscopic biopsy was performed in patients with non-ulcer dyspepsia after methylene blue chromoscopy. All patients provided written informed consent. Two pieces
2
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
were taken from the corpus and another two pieces from the antrum, and each specimen was fixed in 10% formalin and routinely embedded in paraffin wax. Serial sections were cut and used for histochemistry and immunohistochemistry.
sults and consulted over instances where their classification did not agree.
2.2. Staining for intestinal metaplasia subtypes
Statistical analyses were performed with the χ2 test for trend using SPSS version 10.0 for Windows. P < 0.05 was regarded as statistically significant.
Alcian blue (pH 2.5)/periodic acid-Schiff and the high iron diamine-alcian blue technique were used to identify neutral, sialomucins, and sulfomucins [3]. Intestinal metaplasia was classified according to Filipe as follows: type I, mature absorptive cells and goblet cells, the latter secreting sialomucins; type II, few or no absorptive cells, presence of columnar intermediate cells in various stages of differentiation secreting neutral and acid sialomucins and goblet cells secreting sialomucins or, occasionally, sulfomucins, or both; and type III, columnar intermediate cells secreting predominantly sulfomucins, and goblet cells secreting sialomucins or sulfomucins, or both [2,5]. 2.3. Immunohistochemistry of trefoil peptides Immunohistochemistry was performed with mouse monoclonal antibodies (TFF1, TFF2; kindly provided by Drs. N. Wright and G. Elia, Cancer Research UK, London, UK) and rat polyclonal antibody (TFF3; kindly provided by Dr. A. Giraud, University of Melbourne, Melbourne, Australia). In brief, formalin-fixed 5 m sections were deparaffinized, rehydrated, and washed in tris-buffered saline (TBS). Endogenous peroxidase activity and nonspecific binding were blocked by incubation with 3% hydrogen peroxide (H2 O2 ) for 5 min, and blocking solution (normal immune serum) was added for 10 min followed by TFF1 primary antibody (dilution 1:400) for 60 min. After rinsing with TBS, biotinylated secondary antibody was added for 10 min and the slides again rinsed with TBS. Enzyme conjugation was performed with streptavidin for 10 min. With TFF2 (dilution 1:200), goat antimouse IgM biotinylated secondary antibody was added for 30 min and with TFF3 (dilution 1:200), rabbit anti-rat IgG biotinylated secondary antibody was added for 60 min. The slides were finally incubated with avidin-biotinylated peroxidase complex (ABComplex/HRP, Dako, Glostrup, Denmark). All of the outcomes were visualized with diaminobenzidine (DAB) and counterstained with Meyer’s hematoxylin. TBS alone without primary antibody acted as negative control for the immunostaining. Normal human gastric mucosa obtained from endoscopic biopsy and normal colonic mucosa from surgical specimen were used for positive controls along with TFF1 and TFF2 antibodies, and TFF3 antibody, respectively. The immunoreactivity of the trefoil peptides was graded from (−) to (+++) according to the percentage of positive cells. Immunoreactivity in goblet cells and columnar cells was assessed separately. Two pathologists reviewed the re-
2.4. Statistics
3. Results 3.1. Subtypes of intestinal metaplasia In the 132 patients included in the study, intestinal metaplasia was detected in the corpus in 88 patients and in the antrum in 107 patients. Type I intestinal metaplasia was detected in the corpus in 69 patients (78.4%), and in the antrum in 22 patients (20.6%) while type II was present in the corpus in 17 patients (19.3%), and in the antrum in 46 patients (43.0%). Finally, type III was found in the corpus in two patients (2.3%), and in the antrum in 39 patients (36.4%). Types II and III intestinal metaplasia occurred more commonly in the antrum than in the corpus (P < 0.05). 3.2. Immunohistochemistry of trefoil peptides TFF1 was mainly expressed in goblet cells and rarely in columnar cells in all the types of intestinal metaplasia (Fig. 1A). TFF2, however, was not expressed in goblet cells but only in columnar cells in all three types (Fig. 1B). TFF3 was expressed in both goblet cells and in columnar cells, in every type of intestinal metaplasia, but was more highly expressed in the goblet cells (Fig. 1C). TFF3 showed the strongest immunoreactivity of the trefoil peptides in all the types of intestinal metaplasia. The expression of TFF1 and TFF3 in goblet cells tended to decrease from type I to III via type II (Fig. 2A and C P = 0.045, P = 0.002, respectively). There was no difference between the subtypes in the expression of TFF3 in columnar cells. However, TFF2 expression in columnar cells tended to increase from type I to type III via type II (Fig. 2B, P < 0.001). The immunoreactivity of trefoil peptides in types I and II was similar in antrum and corpus.
4. Discussion In the West, the frequency of type III intestinal metaplasia is reported to be about 10% [4]. In this study, however, its frequency was 2.3% in the corpus and 36.4% in the antrum. This suggests that intestinal metaplasia begins in the antrum and progress to the corpus. This can be easily explained if H. pylori migrates from antrum to corpus to cardia. It is therefore plausible that H. pylori makes
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
3
Fig. 2. (A) Expression of TFF1 in goblet cells of intestinal metaplasia subtypes. Note the down regulation of TFF1 from type I via type II to type III. (B) Expression of TFF2 in columnar cells of the intestinal metaplasia subtypes. Note the increase of TFF2 from type I via type II to type III. (C) Expression of TFF3 in goblet cells of the intestinal metaplasia subtypes. Down regulation of TFF3 from type I via type II to type III can be seen. (IM I, type I intestinal metaplasia; IM II, type II intestinal metaplasia; IM III, type III intestinal metaplsia).
Fig. 1. (A) TFF1 is mainly expressed in goblet cells (arrow) and weakly expressed in columnar cells (open arrow). (B) TFF2 is weakly expressed in columnar cells (open arrow) and not expressed in goblet cells (arrow). (C) TFF3 is highly expressed in goblet cells (arrow) and somewhat weakly expressed in columnar cells (open arrow).
an important contribution to the progression of intestinal metaplasia. About 57.8% of adult Korean patients are infected with H. pylori [9], and gastric cancer is still the most common cancer [12]. This difference between Western inhabitants and Koreans may account for the high incidence of intestinal metaplasia type III and the subsequent high incidence of antral gastric cancer. In this study population, H. pylori infection was detected in 121 patients. TFF1 was expressed in each type of intestinal metaplasia, mainly in goblet cells. However, the immunoreactivity was weaker than in the adjacent normal gastric epithelium. This is in agreement with a previous study [19]. TFF1 was more highly expressed in goblet cells than in columnar cells in all the types of intestinal metaplasia. Furthermore, the
4
B.-W. Kim et al. / Peptides xxx (2004) xxx–xxx
immunoreactivity of TFF1 tended to decrease from type I to type III via type II. These results suggest that the different types of intestinal metaplasia retain a gastric component and lose it progressively from type I to type III via type II. In the light of other reports [8], it is possible that down regulation of TFF1 production may start in type I and progress to type III via type II as an intermediate that is much more susceptible to damage from other carcinogens, and finally progress to gastric cancer. TFF2 was not expressed in goblet cells and only weakly expressed in columnar cells in every type of intestinal metaplasia. TFF2 expression in columnar cells increased from type I via type II to type III. According to other reports, TFF2 begins to be upregulated from the stage of dysplasia [8]. However, our study suggests that its re-expression starts from intestinal metaplasia and progresses from type I to III via type II. It has also been suggested in another study [8] that upregulation of TFF2 is associated with gastric carcinogenesis. TFF3 is at best weakly expressed in normal gastric epithelium. It is noteworthy that its immunoreactivity was higher than that of TFF1 and TFF2 in every types of intestinal metaplasia, especially in goblet cells. This has also been reported in another study [19], and may suggest that intestinal type epithelia, such as intestinal metaplasia, secrete predominantly the intestinal types of trefoil peptides, though they still have gastric components. TFF3 also showed a decreasing incidence from type I to type III via type II and it is therefore uncertain whether TFF3 can play any role in the suppression of gastric cancer. It will be important to clarify whether TFF3 can play any role in an acidic milieu and whether it can act as a cytoprotectant of gastric epithelia with intestinal metaplasia. We had intended to compare the expression pattern of trefoil peptides in the antrum and corpus in intestinal metaplasia, but type III intestinal metaplasia was detected in the corpus in only two cases, and it was therefore difficult to make the comparison. However in types I and II, where the comparison was possible there was no difference in TFF1, TFF2, and TFF3 expression in antrum and corpus. In normal gastric mucosa TFF1 is secreted by the surface cells of the corpus and antrum, and TFF2 by the mucous neck cells. However, we found that in intestinal metaplasia, goblet cells secreted TFF1 and TFF3 and columnar cells secreted all three trefoil peptides, regardless of type, though TFF3 showed the highest expression. Though only TFF1 is known to be a gastric-specific tumor suppressor gene, the other trefoil peptides may participate in the process of gastric cancer formation. This question should be elucidated at the gene level. It is well known that trefoil peptides are co-expressed with mucin, and that peptides and mucin play synergistic roles in maintaining mucosal integrity. The expression pattern of mucin glycoprotein in the intestinal metaplasia subtypes was reported previously [15]. The trefoil peptides alone cannot be used as markers of the intestinal metaplasia subtypes, and
studies of the co-expression patterns with mucin might provide insight into the role of the trefoil peptides in intestinal metaplasia, and clarify whether they can be used as markers for subtyping intestinal metaplasias. In conclusion, there is progressive loss of TFF1 and TFF3, together with induction of TFF2, during the development of intestinal metaplasia from type I to type III via the type II intermediate. Inhibition of these events may block the progression of intestinal metaplasia and prevent the ensuing gastric cancer.
Acknowledgments We thank Dr. N. Wright and G. Elia of Cancer Research UK, London, UK for providing the TFF1 and TFF2 mouse monoclonal antibodies. We also thank Dr. A. Giraud of the University of Melbourne, Melbourne, Australia for providing the TFF3 rat polyclonal antibody. This study was partially supported by the Research Institute of Clinical Medicine, Our Lady of Mercy Hospital, The Catholic University of Korea. References [1] Craanen ME, Blok P, Dekker W, Ferwerda J, Tytgat GNJ. Prevalence of subtypes of intestinal metaplasia in gastric antral mucosa. Dig Dis Sci 1991;36:1529–36. [2] Filipe MI, Jass JR. Intestinal metaplasia subtypes and cancer risk. In: Filipe MI, Jass JR, editors. Gastric carcinoma. London: Churchill Livingstone; 1986. p. 87–115. [3] Filipe MI, Lake BD. Appendix 3. In: Filipe MI, Lake BD, editors. Histochemistry in pathology. 2nd ed. London: Churchill Livingstone; 1990. p. 449–53. [4] Filipe MI. Gastrointestinal carcinoma and precursor lesions. In: Filipe MI, Lake BD, editors. Histochemistry in pathology. 2nd ed. London: Churchill Livingstone; 1990. p. 175–98. [5] Filipe MI. Borderline lesions of the gastric epithelium: new indicators of gastric risk and clinical implications. In: Fenoglio-Preiser CM, Wolff M, Rilke F, editors. Progress in surgical pathology. Philadelphia: Field & Wood Medical Publishers; 1992. p. 269–90. [6] Fujimoto J, Yasui W, Tahara H, Tahara E, Kudo Y, Yokozaki H, et al. DNA hypermethylation at the pS2 promoter region is associated with early stage of stomach carcinogenesis. Cancer Lett 2000;149:125–34. [7] Hanby AM, Poulsom R, Singh S, Elia G, Jeffery RE, Wright NA. Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach. Gastroenterology 1993;105:1110–6. [8] Hu GY, Yu BP, Dong WG, Li MQ, Yu JP, Luo HS, et al. Expression of TFF2 and Helicobacter pylori infection in carcinogenesis of gastric mucosa. World J Gastroenterol 2003;9:910–4. [9] Kim HS, Lee YC, Lee HW, Yoo HM, Lee CG, Kim JM, et al. Seroepidemiologic study of Helicobacter pylori in Korea. Kor J Gastroenterol 1999;33:170–82. [10] Leung WK, Sung JJ. Review article: intestinal metaplasia and gastric carcinogenesis. Aliment Pharmacol Ther 2002;16:1209–16. [11] Machado JC, Nogueira AM, Carneiro F, Reis CA, Sobrinho-Simoes M. Gastric carcinoma exhibits distinct types of cell differentiation: an immunohistochemical study of trefoil peptides (TFF1 and TFF2) and mucins (MUC1, MUC2, MUC5AC, and MUC6). J Pathol 2000;190:437–43.
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