1476-4598-5-10 1476-4598 Research <p>Expression profile of mucins (MUC2, MUC5AC and MUC6) in <it>Helicobacter pylori </it>infected pre-neoplastic and neoplastic human gastric epithelium</p> Babu Durai Subramani durai_reddy@yahoo.co.in Jayanthi Venkataraman drjayant1@vsnl.com Devaraj Niranjali nirangali@yahoo.com Reis A Celso celso.reis@ipatimup.pt Devaraj Halagowder hdrajum@yahoo.com

Unit of Biochemistry, Department of Zoology, University of Madras, Guindy, Chennai, India

Department of Gastroenerology, Stanley Medical College and Hospital, Chennai, India

Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India

IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Portugal

 Molecular Cancer 1476-4598 2006 5 1 10 http://www.molecular-cancer.com/content/5/1/10 16545139 10.1186/1476-4598-5-10 07 2 2005 19 3 2006 19 3 2006 2006 Babu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Helicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry.

Methods

RUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies.

Results

MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2.

Conclusion

In conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.

Background

Helicobacter pylori colonizes human gastric mucosa and causes gastritis and intestinal metaplasia (IM), which may evolve towards gastric carcinoma 123. H. pylori infection was established as a type I human carcinogen in IARC 4; it was recently shown that the bacteria are also capable of inducing gastritis, IM, and gastric carcinoma in Mongolian gerbils 5678. In humans, H. pylori also colonizes the duodenal mucosa whenever there is gastric metaplasia 9. H. pylori infection in human is therefore dependent on the gastric microenvironment, which is determined to a large extent by the mucin and carbohydrate composition of the gastric mucin layer.

Mucins are heavily glycosylated glycoproteins that are the major components of the mucous viscous gel covering the surface epithelial tissues 10. To date, nine distinct epithelial mucin genes (MUC1, 2, 3, 4, 5AC, 5B, 6, 7, and 8) have been identified 1112131415161718192021. In situ hybridization and immunohistochemical studies have shown that these mucins are differentially expressed in epithelia with cell type specificity 21222324. The normal gastric mucosa shows cell types specific expression of MUC1, MUC5AC, and MUC6, with first two mucins found in the superficial epithelium and MUC6 in the deep glands 222526. MUC6 is mainly expressed in gastric mucosa. The normal gastric mucosa does not express MUC2 2527.

Changes in the expression levels and glycosylation patterns of mucins have been associated with several diseases, including carcinomas 2728293031. In gastric cancer, alterations in mucin polypeptide have been reported: loss of expression of MUC5AC 232632, increased mucin heterogenesity 23. These observations suggest that mucin alterations can be regarded as "molecular" markers of malignant transformation of gastric mucosa. Further more, a recently published investigation strongly suggested MUC5AC as a putative H. pylari receptor 3334.

In this present study, we have characterized the pattern of mucins expression in the progressive stages of H. pylori infected human gastric carcinoma. We have used a comprehensive panel of monoclonal antibodies, with well-characterized specificities directed to MUC2, MUC5AC and MUC6 mucins.

Results

Expression pattern of MUC2, MUC5AC and MUC6 mucins in normal gastric mucosa

MUC5AC was highly expressed in foveolar epithelium and mucous neck cells of antrum (> 75%, Fig. 1b; Fig. 3b; Table 2). Expression of MUC6 was detected in the glands of the antrum (> 75%, Fig. 1c; Fig. 3c; Table 3) and MUC2 was not detected in normal gastric mucosa (Fig. 1a; Fig. 3a; Table 2).

<p>Figure 1</p>

Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients

Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients. a. MUC2 staining of normal gastric mucosa (Original magnification ×100). b. MUC5AC staining of normal gastric mucosa (Original magnification ×100). c. MUC6 staining of normal gastric mucosa (Original magnification ×100). d. MUC2 staining of Atrophic gastritis (Original magnification ×100). e. MUC5AC staining of Atrophic gastritis (Original magnification ×100). f. MUC6 staining of Atrophic gastritis (Original magnification ×100). g. MUC2 staining of Intestinal metaplasia (Original magnification ×100). h. MUC5AC staining of Intestinal metaplasia (Original magnification ×100).

 <p>Figure 2</p>

Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients

Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients. a. MUC6 staining of Intestinal metaplasia (Original magnification ×100). b. MUC2 staining of dysplasia (Original magnification ×100). c. MUC5AC staining of dysplasia (Original magnification ×100). d. MUC6 staining of dysplasia (Original magnification ×100). e. MUC2 staining of carcinoma (Original magnification ×100). f. MUC5AC staining of carcinoma (Original magnification ×100). g. MUC6 staining of carcinoma (Original magnification ×100).

 <p>Figure 3</p>

Expression pattern of mucins (MUC2, MUC5AC and MUC6) in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium

Expression pattern of mucins (MUC2, MUC5AC and MUC6) in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. a. MUC2 expression in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. b. MUC5AC expression in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. c. MUC6 expression in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium.

 <p>Table 1</p>

Specificities, origins and dilutions of the monoclonal antibodies.

Monoclonal antibody

Specificity

Reference

Dilution

PMH1

MUC2

Reis et al [11]

Undiluted

CLH2

MUC5AC

Reis et al [12]

1:2

CLH5

MUC6

Reis et al [10]

1:2

 <p>Table 2</p>

Expression of mucins (MUC2, MUCSAC and MUC6) in H. pylori infected pre-neoplastic and neoplastic human gastric epithelium

Stages

H. pylori

MUC2 Expression

MUC5AC Expression

MUC6 Expression

N

P

-

+

++

+++

++++

-

+

++

+++

++++

-

+

++

+++

++++

Normal (n = 7)

7

-

n = 7 (100%)

-

-

-

-

-

-

-

n = 2 (28.6%)

n = 5 (71.4%)

-

-

-

1 (14.3%)

6 (85.7%)

Atrophic gastritis (n = 26)

-

26

n = 22 (84.6%)

n = 4 (15.4%)

-

-

-

-

-

1 (38%)

13 (50%)

12 (46.2%)

-

-

1 (3.8%)

15 (57.7%)

10 (38.5%)

Intestinal metaplasia (n = 21)

-

21

-

-

n = 9 (42.9%)

n = 12 (57.1%)**

-

-

1 (4.8%)

16 (76.2%)**

4 (19%)

-

-

5 (23.8%)

14 (66.7%)**

2 (9.5%)

-

Dysplasia (n = 17)

-

17

-

n = 5 (29.4%)

n = 11 (64.7%)

n = 1 (5.9%)

-

-

3 (17.6%)

10 (58.8%)

4 (23.5%)

-

-

3 (17.6%)

11 (64.7%)

3 (17.3%)

-

Carcinoma (n = 36)

-

36

-

22 (61.1%)

14 (38.9%)

-

-

-

28 (77.8%)

8 (22.2%)

-

-

12 (33.3%)

17 (47.2%)

7 (19.4%)

-

-

N – negative - (> 5%) negative

P – positive + (5–25%) few positive cells

n – No. of cases ++ (25–50%) well-defined area

+++ (50–75%) extensive area stained

++++ (75–100%) most of the cells stained

**p < 0.00, *p < 0.05 (Significant difference in expression)

Immunodetection of mucins in atrophic gastritis

There was expression of MUC5AC in all the cases of columnar cells (Fig. 1e; Fig. 3b; Table 2). The percentage of stained cells varied from case to case, in most cases, there was a trend for higher number of cells expressing MUC5AC (>75%) in the superficial part of the metaplastic glands than in deep part. MUC6 was detected in columnar cells (>50%; Fig. 1f; Fig. 3c; Table 2) and MUC2 was not detected in atrophic gastritis (Fig. 1d).

Immunodetection of mucins in intestinal metaplasia

There was decreased level expression of MUC5AC and MUC6 in all the cases (>50%), both in goblet and in columnar cells (Fig. 1h, Fig. 2a; Fig. 3b, Fig. 3c; Table 2). The percentage of stained cells varied from case to case, de novo expression of intestinal mucin MUC2 was observed (>75%), and its expression displayed a diffuse cytoplasmic pattern in goblet cells (Fig. 1g; Fig. 3a; Table 2).

Immunodetection of mucins in dysplasia

There was decreased level expression of MUC5AC and MUC6, than intestinal metaplasia (>50%). MUC5AC and MUC6 displayed a diffuse cytoplasmic expression pattern in columnar cells (Fig. 2c; Fig. 2d; Fig. 3d; Fig. 3; Table 2). MUC2 expression displayed a diffuse cytoplasmic pattern in goblets cells (>50%, Fig. 2b; Fig 3a; Table 2).

Immunodetection of mucins in carcinoma

MUC5AC and MUC6 expression were frequently observed in early carcinoma (>50%) than advanced carcinomas (>25%). In advanced carcinomas, we observed a trend for decreased immunoreactivity in deep areas than in the superficial areas. MUC5AC and MUC6 expressions were decreased in advanced carcinomas (>25%; Fig. 3a; Fig. 3b; Fig. 3c; Fig. 2e, Fig. 2f, Fig. 2g; Table 2).

Discussion

In this present study, we have shown that the pattern of mucins expression in the progressive stages of H. pylori infected human gastric epithelium. Mucins are expressed with a cell- and tissue-specific pattern in normal tissue. Alterations of the expression pattern of mucins have been described in carcinomas as well as in their precursor lesions; in the latter, altered mucin carbohydrate and peptide moieties may constitute molecular markers of increased risk of malignant transformation 273536383940.

In agreement with previous studies reporting the distribution of mucins in normal stomach, we found MUC5AC is highly expressed in foveolar cells of antrum, as reported 262732373839. We also found that MUC6 is expressed in mucous cells of the neck zone in pyloric glands of the antrum with similar pattern to that described previously 222347. The expression of MUC2 was usually not detected in normal gastric mucosa as described in the previous reports 252740.

Intestinal metaplasia is one of the lesions identified in the cascade of event that precedes the development of gastric carcinoma 41. Intestinal metaplasia comprises the replacement of the gastric mucosa by an epithelium that resembles histologically the intestinal mucosa. Altered mucin expression patterns have been observed in H. pylori infected intestinal metaplasia, including under expression of MUC5AC and MUC6 and de novo expression of MUC2 mostly in goblet cells 3048. These results support the assumption that the intestinal metaplasia does represent a differentiation of the mucosa toward a intestinal phenotype 4243.

The decreased expression of MUC5AC and MUC6 mucin in gastric carcinomas were confirmed in the present study. The expression of MUC5AC in early gastric carcinoma of the present series, regardless of the histological type of the tumors, contrasts with the decreased level expression of MUC5AC immunoreactivity in almost half of the advanced carcinomas which suggest that all gastric carcinomas retain at least some cells with a gastric phenotype during the first steps of neoplastic development (26). This assumption is further supported by the decreased immunoreactivity in deep areas compared with superficial areas of the advanced carcinomas. These results are in agreement with previous results showing that gastric cancer progression is accompanied by alteration of the expression of several mucin genes 14. Finally, we observed that the intestinal mucin MUC2 is expressed in every intestinal metaplasia, which is a lesion that appears during the process of gastric carcinogenesis in the context of H. pylori infected individuals.

Conclusion

Although H. pylori has been classified as a class I carcinogen by the IARC 4 the mechanism of gastric carcinogenesis remains poorly understood. In pre-neoplastic gastric mucosa, the expression of all the three mucin antigens did not appear to correlate with the severity of inflammation and H. pylori status. Results from the present study may suggest that H. pylori infection acts as an initiator of this carcinogenesis cascade by inducing inflammation and, in some cases, the subsequent metaplastic change. Intestinal metaplasia is accompanied by the induction of expression MUC2 and decreased expression of MUC5AC and MUC6. These alterations may favor the development of gastric carcinoma. The present study suggests that MUC2 is a marker of intestinal metaplasia and may be used for the early detection of this lesion in H. pylori infected pre-neoplastic human gastric epithelium.

Materials and methods

Tissue samples

Fifty nine gastric biopsy specimens and 3 ml of blood were obtained from Stanley Medical College and Hospital, Chennai. All biopsy tissue samples were collected after informed consent. This study was carried out after obtaining clearance from the ethical board of the hospital. All biopsy specimens were fixed in 10% buffered formalin and routinely embedded in paraffin wax. 4 μm thin serial sections were cut and used for immunohistochemistry. H. pylori infection was confirmed by RUT and ELISA 43. Hematoxylin and Eosin stained sections were used to classify gastritis graded according to revised Sydney classification 44, gastric tumors according to the classification of Lauren 45 and WHO classification 46. We studied 7 cases of normal Human gastric epithelium (7 biopsies), 26 cases of Atrophic gastritis (26 biopsies), 21 cases of Intestinal metaplasia (21 biopsies), 17 cases of dysplasia (17 biopsies) and 36 cases of carcinoma in which 21 cases early carcinoma and 15 cases advanced carcinoma (16 biopsies and 20 surgical specimens).

Monoclonal antibodies

A panel of three monoclonal antibodies was used to determine the expression of mucins (MUC2, MUC5AC and MUC6). The specificities, references and dilutions of the antibodies are listed in Table 1.

Immunohistochemistry

Sections from normal, pre-neoplastic and neoplastic gastric tissues were immuno stained according to Reis et al 47 with slight modifications. Sections designed for neuraminidase treatment were washed twice in phosphate buffered saline (PBS), pH 7.4 and incubated with neuraminidase from clostridium perfringens Type VI (Sigma; St Louis, MO) diluted in 0.1 M Na-acetate buffer, pH 5.5 to a final concentration of 0.1 U/ml. The incubation was carried out for 2 h at 37°C and was followed by three washings in ice-cold PBS. Sections were treated with 0.5% H2O2 in methanol for 30 min, followed by 20 min of incubation with rabbit non-immune serum. Sections were rinsed and incubated with primary antibody (Table 1), over night at 4°C. The sections were rinsed and incubated with HRP conjucated rabbit anti-mouse secondary antibody (1:100 dilution) for 1 h followed by rinsing with PBS and developing with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (SRL, Mumbai, India) freshly prepared in PBS containing 0.1% H2O2. Sections were counter stained with hematoxylin, dehydrated, and mounted. All series including positive and negative controls were performed by substitution of the primary mAbs with immunoglobulins of the same class and at the same concentration.

Scoring of mucins expression in H. pylori infected pre-neoplastic and neoplastic human gastric tissues

A semi quantitative approach was used to score the immunostaining of human gastric tissues. The slides were examined under light microscope, every 100 cells were scored in five different fields and the results were expressed as mean percentage. (-), negative; +, few positive cells (<25%); ++ well-defined areas with positive cells (25–50%); +++, extensive areas with positive cells (50–75%); ++++, most cells stained (>75%).

Statistics

Data analysis was performed using statistical software package SPSS 10.0 for windows. The Chi-Square test or Fisher's exact test was used to analyze differences in frequencies. Spearman's correlation test was used to analyze correlation between parameters. P < 0.05 was considered statistically significant.

Abbreviations

DAB – 3,3'-diaminobenzidine tetra hydrochloride; ELISA – Enzyme-linked immunosorbent assay; HRP – Horseradish peroxidase; IARC – International Agency for Research on Cancer; IM – Intestinal metaplasia; mAbs – Monoclonal antibodies; PBS – Phosphate buffered saline; WHO – World Health Organization; RUT – Rapid Urea Test.

Authors' contributions

SDB carried out all experiments along with the designing and coordination of HD. VJ provided patients data and specimens. HD reviewed the immunohistochemical results and ND, CAR provided suggestion for the finalization of this paper. All authors read and approved the final manuscript.

Acknowledgements

The authors thank the Lady Tata Memorial Trust, Mumbai, India for the financial support to one of the authors S. Durai Babu. We grateful to Dr. Narasimhan, Professor and Head, Department of Pathology, Sri Ramachandra Medical College and Hospital, India, for reviewing the histological and immunohistochemical results and Dr. R. Ravanan, Reader, Department of Statistics, Presidemcy College, India for helping in statistical analysis. We would like to thank Dr. Celso A. Reis for provision of the anti-MUC2, anti-MUC5AC and anti-MUC6 monoclonal antibodies.

 <p>Human gastric carcinogenesis:A multistep and multifactorial process – First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention</p> Correa P Cancer Res 1992 52 6735 6740 1458460 <p><it>Helicobacter pylori </it>infection and the risk of gastric carcinoma</p> Parsonnet J Friedman GD Vandersteen DP N Engl J Med 1991 325 1127 1131 1891020 <p><it>Helicobacter pylori </it>infection and age on the development of intestinal metaplasia – a multiple logistic regression analysis</p> Wang CC Wu MS Wang HH Hepatogastroenterology 1998 45 2234 2237 9951901 <p>International Agency for Research on Cancer. Infections with Schistosomes, Liver Flukes and <it>Helicobacter pylori</it>. Evaluation of Carcinogenic risks to Humans</p> IARC: Lyon 1994 <p>Development of Helicobacter pylori – induced gastric carcinoma in Mongolian gerbils</p> Honda S Fujioka T Tokieda M Satoh R Nishizono A Nasu M Cancer Res 1998 58 4255 4259 9766647 <p>Gastric ulcer, atrophic gastritis, and intestinal metaplasia caused by <it>Helicobacter pylori </it>infection in Mongolian gerbils</p> Honda S Fujioka T Tokieda M Gotoh T Nishizono A Nasu M Scand J Gastroenterol 1998 33 454 460 10.1080/00365529850171990 9648982 <p><it>Helicobacter pylori </it>infection induces gastric cancer in Mongolian gerbils</p> Watanabe T Tada M Nagai H Sasaki S Nako M Gastroenterology 1998 115 642 648 10.1016/S0016-5085(98)70143-X 9721161 <p><it>Helicobacter pylori</it>-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils</p> Ikeno T Ota H Sugiyama A Am J Pathol 1999 154 951 960 10079274 <p>Relationship between <it>Helicobacter pylori </it>infection and gastric metaplasia in the duodenal bulb in the pathogenesis of duodenal ulcer</p> Futami H Takashima M Furuta T Hanai H Kaneko J Gastroenterol Hepatol 1999 14 114 119 10.1046/j.1440-1746.1999.01824.x 10029290 <p>Mucin glycoproteins in neoplasia</p> Kim YS Gum J Jr Brockhausen Glycoconj 1996 13 693 707 10.1007/BF00702333 <p>Molecular cloning and expression of human tumor-associated polymorphic epithelian mycin</p> Gendler SH Lancaster Ca Taylor-Papadimitriou J Peat T Burchell J Pemberton L Lalani EN Wilson D J Biol Chem 1990 265 15286 15293 1697589 <p>Clonging and sequencing of a human pancreatic tumor mucin cDNA</p> Lan MS Batra SK Qi WN Metzgar RS Hollingsworth MA J BIol Chem 1990 265 15294 15299 2394722 <p>Molecular cloning of human intestinal mucin cDNAs</p> Gum JR BByrd JC Hicks JW Toribara NW Lamport DTA Kim YS J BiolChem 1989 264 6480 6487 <p>Molecular cloning of cDNAs derived from a novel human intestinal mucin gene</p> Gum JR Hicks JW Swallow DM Laglace RL Byrd JC Lamport DTA Siddiki B Kim YS Biochem Biophys Res Commun 1990 171 407 415 10.1016/0006-291X(90)91408-K 2393399 <p>Degenerate 87-base-pair tandem repeats create hydrophilic/hydrophobic alternating domains in human mucin peptides mapped to 11p15</p> Dufosse J Prochet N Audie JP Guyonnet-Duperat V Laine A Van Seuninegen I Marrakchi S Aubert JP Biochem J 1993 293 329 337 1134363 7916618 <p>Characterization of the human mucin gene MUC5AC : a consensus cysteine-rich domain for 11p15 mycin genes?</p> Guyonnet-Duperat V Audie JP Debailleul V Laine A Buisine MP Galieque-Zouitina S Pigny P Degand P Aubert JP Porchet N Biochem J 1995 305 211 219 1136451 7826332 <p>Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs</p> Porchet N Van-Cong N Dugosse J Audie JP Guyonne-Duperat V Gross MS Denis C Degand P Bernhein A Aubert JP Biochem Res Commun 1991 175 414 422 10.1016/0006-291X(91)91580-6 <p>Molecualr cloning sequence and specificity of expression of the gene encoding the lower molecular weight human salivary mucin (MUC7)</p> Bobek LA Tsai H Biesbrock AR Levine MJ J Biol Chem 1993 268 20563 20569 7690757 <p>Human gastric mucin. Identification of a unique species by expression cloning</p> Toribara NW Roberton AM Ho SB Kyo WL Gum E Hicks JW Gum JR Byrd JC Siddiki B J Biol Chen 1993 268 5879 5885 <p>A novel human airway mucin cDNA encodes a protein with unique tandem-repeat organization</p> Shankar V Gilmore MS Elkins RC Sachdev GP Biochem J 1994 300 295 298 1138160 8002930 <p>Expression of human mucin genes in respiratory, digestive and reproductive tracts ascertained by <it>in situ </it>hybridization</p> Audie JP Janin A Porchet N Copin MC Gosselin B Aubert JP J Histochem Cytochem 1993 41 1479 1485 8245407 <p>MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach</p> DeBolós C Garrido M Real FX Gastroenterology 1995 109 723 734 10.1016/0016-5085(95)90379-8 7657100 <p>Mucin gene expression in normal, pre-neoplastic, and neoplastic human gastric epithelium</p> Ho SB Shekels LL Toribara NW Kim YS Lyftogt C Cherwitz DL Niehans GA Cancer Res 1995 55 2681 2690 7780985 <p>Differential expression of human high molecular weight salivary mucin (MGI) and low molecular weight salivary mucin (MG2)</p> Nielsen PA Mandel U Therkildsen MH Clausen H J Dent Res 1996 75 1820 1826 9003227 <p>Differential apomucin expression in normal and neoplastic human gastrointestinal tissues</p> Carrato C Balague C DeBolos C Gonzalez E Gambus G Planas J Perini JM Andreu D Real FX Gastroenterology 1994 107 160 172 8020658 <p>Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody</p> Reis CA David L Nielsen P Int J Cancer 1997 74 112 121 10.1002/(SICI)1097-0215(19970220)74:1<112::AID-IJC19>3.0.CO;2-H 9036879 <p>Heterogeneity of mucin gene expression in normal and neoplastic tissues</p> Ho SN Niehans GA Lyftoget C Yan PS Cherwitz DL Gum ET Dahiya R Kim YS Cancer Res 1993 53 64 651 <p>Aberrant expression of a human mucin gene (MUC5AC) in rectosigmoid vilous adenoma</p> Buisine MP Janin A Maunoury V Audie JP Delesaut MP Copin MC Colombel JF Degand P Aubert JP Porchet N Gastroenterology 1996 104 93 102 <p>Glycosylation patterns of mucins in colonic disease</p> Corfield A Myerscough N Gough M Brockhausen I Schauer R Paraskeva C Biochem Soc Trans 1995 23 840 845 8654850 <p>Detection of the MUC2 apomucin tandem repeat with a mouse monoclonal antibody</p> Gambús G de Bolós C Andreu D Francí C Egea G Real FX Gastroenterology 1993 104 93 102 7678241 <p>Aberrant expression of gland-type gastric mucin in the surface epithelium of <it>Helicobacter pylori</it>-infected patients</p> Byrd JC Yan P Sternberg L Yunker CK Scheiman JM Bresailer RS Gasteroenterology 1997 113 455 464 10.1053/gast.1997.v113.pm9247464 <p>Expression of fully andused glycosylated ofrms of MUC1 mucin in gastric carcinoma</p> Reis CA David L Seixas M Burchell J Sobrinho-Sinoes M Int J Cancer 1998 79 402 410 10.1002/(SICI)1097-0215(19980821)79:4<402::AID-IJC16>3.0.CO;2-6 9699534 <p><it>H. pylori </it>colonolizes with MUC5AC in the human stomach</p> Brink GR Tytgat Kmal Hulst LWM Gut 2000 46 601 607 10.1136/gut.46.5.601 10764701 <p>A confirmatory report for the close interaction of Helicobacter pylori with gastric epithelial MUC5AC expression</p> Belma Kover MD Murat Vlas MD Yucel Ustundag MI Sibel Erodogan MD Melih Karabeyoglu MD Osman Yildin MD Bulent oral MD Omer Lengiz MD Atilla Soran MO J Clin Gastroentrol 2004 38 486 502 <p>Aberrant glycosylation in tumours and tumor-associated carbohydrate antigens</p> Hakomori S Adv Cancer Res 1989 52 257 331 2662714 <p>T/Tn pancarcinoma autoantigens: fundamental, diagnostic and prognostic aspects</p> Springer GF Desai PR Ghazizadeh M Tegtmeyer H Cancer Detect Prev 1995 19 173 183 7750105 <p>A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas</p> Girling A Bartkova J Gendler S Gillett C Taylor-Papadimitriou J Int J Cancer 1989 43 1072 1076 2471698 <p>Frequent alteration of the DF3 tumour-associated antigen gene in primary human breast carcinomas</p> Melro GR Siddiqui J Cropp CS Liscia DS Lidereau R Callahan R Kufe DW Cancer Res 1989 49 6966 6971 2582438 <p>Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin</p> Burchell J Gendler S Taylor-Papadimitriou J Girling A Lewis A Millis R Lamport D Cancer Res 1987 47 5476 5482 2443241 <p>Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide</p> Reis CA Sorensen T Mandel U Glycoconj J 1998 15 51 62 10.1023/A:1006939432665 9530956 <p>Human model of gastric carcinogenesis</p> Correa PA Cancer Res 1988 48 3554 3560 3288329 <p>Intestinal metaplasia subtypes and cancer risk</p> Filipe MI Jass JR Gastric Carcinoma, London: Churchill Livingstone Filipe MI, Jass JR 1986 87 115 <p><it>Helicobacter pylori </it>infection and the development of Gastric Cancer</p> Naomi Uemura Shiro Okamoto Sorchiro Yamamoto N Engl J Med 2001 345 32 41 <p>Classification and grading of gastritis : the updated sydney system. International workshop on the histopathology of gastritics, Houton 1994</p> Dixon MF Genta RM Yardley H Lorea P Am J surg Pathol 1996 20 1161 1181 10.1097/00000478-199610000-00001 8827022 <p>The two main histological types of gastric carcinoma: diffuse and so-called intestinal type carcinoma. An attempt at a histo-clinical classification</p> Lauren P Acta Pathol Microbiol Scand 1965 64 31 49 14320675 <p>Histological typing of esophageal and gastric tumors</p> Watanabe H Jass JR Sobin LH WHO International Histological Classification of Tumours Springer, Berlin 2 1990 <p>Immunohistochemical study of the expression of MUC6 mucin and coexpression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas</p> Reis CA David L Carvalho F J Histochem Cytochem 2000 48 377 388 10681391 <p>Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression</p> Reis CA David L Correa P Carneiro F de Bolos C Garcia E Mandel U Clausen H Sobrinho-Simoes M Cancer Res 1999 59 1003 1007 10070955