UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.     

Up-regulated expression of Ezrin and c-Met proteins are related to the metastasis and prognosis of gastric carcinomas

Recent publications demonstrated that abnormal expression of Ezrin and c-Met proteins were related to carcinogenesis, metastasis and prognosis of various sorts of tumors. In this study we detected the expressions of Ezrin and c-Met proteins in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma and analyzed the correlations with metastasis and prognosis of gastric carcinomas. The results demonstrated that both Ezrin and c-Met overexpression were related to the occurrence and progression of gastric carcinoma. Our findings also demonstrated that combined detection of these two tumor-specific biomarkers in gastric carcinomas can provide additional efficacy in predicting the patients' outcomes.     

Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR polymorphism

Aberrant glycosylation of mucins is a common phenomenon associated with oncogenic transformation. We investigated the association between expression of the tumor-associated antigens T, Tn, and sialyl-Tn and polymorphism in the length of the MUC1 mucin tandem repeat in a series of gastric carcinomas. We further evaluated the relevance of MUC1 tandem repeat length on the expression of these tumor-associated carbohydrate antigens (TACAs) using a gastric carcinoma cell line model expressing recombinant MUC1 constructs carrying 0, 3, 9, and 42 repeats. Gastric carcinomas showed a high prevalence of Tn and sialyl-Tn antigens, whereas T antigen was less frequently expressed. The expression of T antigen was significantly higher in gastric carcinomas from patients homozygous for MUC1 large tandem repeat alleles. No significant associations were found for Tn and sialyl-Tn antigens. This novel association was reinforced by the gastric carcinoma cell line model experiments, where de novo expression of T antigen was detected in clones transfected with larger VNTR regions. Our results indicate that polymorphism in the MUC1 tandem repeat influences the expression of TACAs in gastric cancer cells and may therefore allow the identification of subgroups of patients that develop more aggressive tumors expressing T antigen.     

The paraneoplastic phenotype of human gastric glands studied using the carcinoembryonic antigen-specific lectin crustacin

The expression of CEA was studied with the help of rabbit antibody and CEA-specific lectin-krustacin in pepsinogen positive gastric glands near carcinomas and in gastric mucosa with intestinal metaplasia and dysplasia of the epithelium without neoplasia. CEA-krustacin was revealed in 20 out of 41 cases and CEA-antibody in 2 out of 41 cases of gastric glands in mucosa near carcinomas. The results of investigation discover the development in the gastric glands near carcinoma subcellular components. Paraneoplastic phenomenon was found in the majority of the cases.     

Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor lesions

Overexpression of the Multiple Drug Resistance gene (MDR1) has been proposed as a major mechanism related to both intrinsic and acquired resistance to chemotherapeutic agents. The gene product is a membrane protein (P-glycoprotein), that acts as an energydependent drug efflux pump decreasing drug accumulation in resistant tumor cells. We have characterized MDR1 and P-Glycoprotein expression in human gastric adenocarcinoma and in precursor lesions. MDR1 mRNAs, analyzed by dot-blot technique, were detected in 9 of 10 non-tumoral gastric mucosae and in 8 of 10 gastric adenocarcinomas. Immunohistochemical analysis, using the MRK16 monoclonal antibody, revealed heterogeneous expression of P-Glycoprotein in individual cells. The P-Glycoprotein was found on the surface of cells of gastric areas with intestinal metaplasia subtype III. This type of intestinal metaplasia, also called “colonic metaplasia”, has been strongly associated with a high risk for the development of gastric cancer. The fact that the P-Glycoprotein was detected in this precursor lesion is consistent with the intestinal metaplasia dysplasia and carcinoma sequence proposed in the histogenesis of this tumor. The finding that P-Glycoprotein was heterogeneously expressed in malignant cells of some gastric adenocarcinomas also suggests that this transporter system probably contributes to primary and secondary multidrug resistance in this neoplasm.     

The detection, surveillance and treatment of premalignant gastric lesions related to Helicobacter pylori infection

Gastric cancer is an important worldwide health problem and causes considerable morbidity and mortality. It represents the second leading cause of cancer-related death worldwide. A cascade of recognizable precursor lesions precedes most distal gastric carcinomas. In this multistep model of gastric carcinogenesis, Helicobacter pylori causes chronic active inflammation of the gastric mucosa, which slowly progresses through the premalignant stages of atrophic gastritis, intestinal metaplasia and dysplasia to gastric carcinoma. Detection and treatment of premalignant lesions may thus provide a basis for gastric cancer prevention. However, at present, premalignant changes of the gastric mucosa are frequently disregarded in clinical practice or result in widely varying follow-up frequency or treatment. This review provides an overview of current knowledge on detection, surveillance and treatment of patients with premalignant gastric lesions, and identifies the uncertainties that require further research.     

Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor lesions.

Overexpression of the Multiple Drug Resistance gene (MDR1) has been proposed as a major mechanism related to both intrinsic and acquired resistance to chemotherapeutic agents. The gene product is a membrane protein (P-glycoprotein), that acts as an energy-dependent drug efflux pump decreasing drug accumulation in resistant tumor cells. We have characterized MDR1 and P-Glycoprotein expression in human gastric adenocarcinoma and in precursor lesions. MDR1 mRNAs, analyzed by dot-blot technique, were detected in 9 of 10 non-tumoral gastric mucosae and in 8 of 10 gastric adenocarcinomas. Immunohistochemical analysis, using the MRK16 monoclonal antibody, revealed heterogeneous expression of P-Glycoprotein in individual cells. The P-Glycoprotein was found on the surface of cells of gastric areas with intestinal metaplasia subtype III. This type of intestinal metaplasia, also called "colonic metaplasia", has been strongly associated with a high risk for the development of gastric cancer. The fact that the P-Glycoprotein was detected in this precursor lesion is consistent with the intestinal metaplasia-dysplasia and carcinoma sequence proposed in the histogenesis of this tumour. The finding that P-Glycoprotein was heterogeneously expressed in malignant cells of some gastric adenocarcinomas also suggests that this transporter system probably contributes to primary and secondary multidrug resistance in this neoplasm.     

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.     

Statistical model of the interactions between Helicobacter pylori infection and gastric cancer development

Background. The bacterium Helicobacter pylori is associated with a number of gastrointestinal diseases, such as gastric ulcer, duodenal ulcer and gastric cancer. Several histological changes may be observed during the course of infection; some may influence the progression towards cancer. The aim of this study was to build a statistical model to discover direct interactions between H. pylori and different precancerous changes of the gastric mucosa, and in what order and to what degree those may influence the development of the intestinal type of gastric cancer. Methods. To find direct and indirect interactions between H. pylori and different histological variables, log‐linear analyses were used on a case–control study. To generate mathematically and biologically relevant statistical models, a designed algorithm and observed frequency tables were used. Results. The results show that patients with H. pylori infection need to present with proliferation and intestinal metaplasia to develop gastric cancer of the intestinal type. Proliferation and intestinal metaplasia interacted with the variables atrophy and foveolar hyperplasia. Intestinal metaplasia was the only variable with direct interaction with gastric cancer. Gender had no effect on the variables examined. Conclusion. The direct interactions observed in the final statistical model between H. pylori, changes of the mucosa and gastric cancer strengthens and supports previous theories about the progression towards gastric cancer. The results suggest that gastric cancer of the intestinal type may develop from H. pylori infection, proliferation and intestinal metaplasia, while atrophy and foveolar hyperplasia interplay with the other histological variables in the disease process.     

Scanning electron microscopy in gastric adenocarcinoma and intestinal metaplasia

In recent years scanning electron microscopy has been used in gastric biopsy studies, contributing to better recognition of intestinal metaplasia and carcinoma, as a complement to light and transmission electron microscopy. During the second half of 1983, 53 cases of gastric carcinoma were diagnosed at the Department of Pathology of Hospital Mexico, of which six were studied ultrastructurally. A pattern similar to that of intestinal epithelium was found in cases of intestinal metaplasia. Well differentiated adenocarcinomas showed marked tumor cell proliferation with irregular "projections". In poorly differentiated carcinomas, changes were limited to areas where tumor cells invaded the epithelial surface. In summary, scanning electron microscopy is of great help in research and diagnosis of pathologic changes occurring in mucosal surfaces.     

Tetranectin expression in gastric adenocarcinomas

AIMS: The aim was to analyze the immunohistochemical localization of tetranectin in gastric adenocarcinomas and the adjacent tissues of the wall of the stomach. METHODS AND RESULTS: Forty cases of gastric adenocarcinomas were stained by the indirect immunoperoxidase method. Of the ten cases of mucinous signet ring cell carcinomas 5 showed high, 3 moderate and 2 low tetranectin expression. Of the ten cases of well-differentiated intestinal type adenocarcinomas (ITA) 4 showed moderate regional, 3 low regional and 3 negative tetranectin expression. Of the ten cases of moderately-differentiated ITA 3 showed moderate regional, 4 low regional and 3 negative tetranectin expression. Of the ten cases of poorly-differentiated ITA 4 showed focal low and 6 negative tetranectin expression. Overall, the mucinous signet ring carcinomas showed significantly higher tetranectin expression compared to ITA (chi2 = 3.95, p<0.05). In contrast, no significant relationship was found between tetranectin expression and the degree of differentiation in ITA (chi2 = 2.5, p>0.05). In all cases, the perineoplastic desmoplastic reactive stroma showed high expression of tetranectin intra- and extracellularly. The mast cells and goblet cells in the areas of intestinal metaplasia showed high tetranectin expression. CONCLUSIONS: This study shows that: a) tetranectin is produced and deposited extracellularly in the desmoplastic peritumoral stroma of infiltrating gastric adenocarcinomas; b) tetranectin is more highly expressed by the mucinous signet ring cell carcinomas compared to ITA; and c) the amount of tetranectin produced by the ITA is unrelated with the degree of tumor differentiation.     

Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

Endocrine profile in gastric carcinomas. An immunohistochemical study.

22 gastric carcinomas (13 intestinal type and nine diffuse type) were immunostained for neuron specific enolase, chromogranin, Leu-7 and a panel of fifteen different peptide hormones. Five out of the 13 tumours of intestinal type and four out of the nine diffuse carcinomas expressed immunoreactivity for one or more of the pan endocrine markers. Seven out of the 13 tumours of intestinal type and five out of the nine diffuse carcinomas also expressed immunoreactivity for gastrin (3), ACTH (3), serotonin (7) and calcitonin (7). Immunoreactivity for somatostatin (1) and substance P (1) were also seen in two tumours of intestinal type. Seven out of 18 cases with benign mucosa adjacent to the tumours expressed a focal immunoreactivity for chromogranin (6), serotonin (6), gastrin (5) and calcitonin (1). All hormone-producing tumours also expressed immunoreactivity for carcino-embryonic antigen. Our results confirm that a high proportion of gastric carcinomas are hormone producing.     

Identification of CEACAM5 as a Biomarker for Prewarning and Prognosis in Gastric Cancer

MGd1, a monoclonal antibody raised against gastric cancer cells, possesses a high degree of specificity for gastric cancer (GC). Here we identified that the antigen of MGd1 is CEACAM5, and used MGd1 to investigate the expression of CEACAM5 in non-GC and GC tissues (N=643), as a biomarker for prewarning and prognosis. The expression of CEACAM5 was detected by immunohistochemistry in numerous tissues; its clinicopathological correlation was statistically analyzed. CEACAM5 expression was increased progressively from normal gastric mucosa to chronic atrophic gastritis, intestinal metaplasia, dysplasia and finally to GC (p<0.05). In gastric precancerous lesions (intestinal metaplasia and dysplasia), CEACAM5-positive patients had a higher risk of developing GC as compared with CEACAM5-negative patients (OR = 12.68, p<0.001). Besides, CEACAM5 was found positively correlated with invasion depth of gastric adenocarcinoma (p<0.001). In survival analysis, CEACAM5 was demonstrated to be an independent prognostic predictor for patients with GC of clinical stage IIIA/IV (p=0.033). Our results demonstrate that CEACAM5 is a promising biomarker for GC prewarning and prognostic evaluation.     

The roles of REIC gene and its encoding product in gastric carcinoma

REIC is downregulated in immortalized cell lines compared with the parental normal counterparts. It may inhibit colony formation, tumor growth and induce apoptosis. Here, gastric carcinoma or epithelial cells transfected with REIC-expressing plasmid, its siRNA or treated with recombinant REIC were subjected to the phenotypes’ measurement or related molecules’ detection. REIC expression was examined in gastric carcinomas by RT-PCR, western blot and immunohistochemistry. REIC overexpression or treatment resulted in a low karyoplasmic ratio and proliferation, G₁ arrest, high apoptosis, low migration, invasion or lamellipodia formation in AGS cells. REIC knockdown caused the opposite in GES-1 cells. Anti-REIC antibody blocked the effects of REIC overexpression on proliferation, G₁/S progression and apoptosis. Ectopic REIC expression downregulated the expression of β-catenin, phosphorylated S6K (Thr389), phosphorylated Akt1/2/3 (Ser473), cyclin D2 and E, WAVE2 and upregulated phosphorylated mTOR (Ser2448) expression and the mRNA level of Akt1, Akt2, mTOR, Raptor and Rictor in AGS cells. REIC expression was negatively associated with tumor size, lymph node metastasis, dedifferentiation or poor prognosis of carcinoma. The serum REIC level was significantly higher in healthy individuals than the carcinoma patients and inversely linked to tumor size by ELISA. The possible mechanisms underlying the forced REIC overexpression or recombinant REIC mediated the reversal of the aggressive phenotypes of gastric carcinoma cells are to downregulate β-catenin and WAVE2 expression and to alter other related target proteins. Downregulated REIC expression was closely linked to aggressive behaviors and poor prognosis of gastric carcinoma.     

Expression of Mina53 and its significance in gastric carcinoma

AIM: To study the expression of Mina53 and its relationships with clinicopathological characteristics, antioncogene inactivation and tumor proliferation in human gastric carcinoma, and to explore the role of Mina53 in carcinogenesis and tumor progression. METHODS: Expression of Mina53 and proliferating cell nuclear antigen (PCNA) was determined in gastric carcinoma (n=79), gastric dysplasia (n=21) and normal gastric tissues (n=20), while p53 was measured in gastric carcinoma tissues by immunohistochemistry. RESULTS: Mina53 was negatively expressed in all normal mucosa tissues. Dysplasia specimens showed weakly positive staining for Mina53 in 3 of 21 cases. Elevated expression of Mina53 was observed in 72 (91.1%) of the gastric carcinomas. No significant associations were found between Mina53 and clinicopathological characteristics such as sex, age, histological differentiation, distant metastasis and lymph node metastasis (p>0.05). There was a significant association with depth of invasion (X2=5.385, p<0.05) and TMN stage (X2=6.255, p<0.05). In gastric carcinoma, positive staining for p53 was detected in 53 of 79 cases (67.1%), showing a significant association with Mina53 (X2=5.161, p<0.05). The mean (+/- SD) PCNA labeling index for gastric carcinoma was 39.47+/-16.92%. Mina53 expression was positively associated with PCNA level (r=0.756, p<0.01). CONCLUSION: Mina53 was overexpressed in gastric carcinoma and associated with tumor proliferation and antioncogene inactivation. Mina53 could therefore play an important role in the carcinogenesis and progression of gastric carcinoma.