Mee Soo Chang

Epstein-Barr Virus-Positive Gastric Carcinoma Has a Distinct Protein Expression Profile in Comparison with Epstein-Barr Virus-Negative Carcinoma

Purpose: EBV has been detected in 2–16% of gastric carcinomas. However, there is little information available about the gene expression profile of EBV-positive gastric carcinomas. Experimental Design: EBV infection was examined using EBV-encoded small RNAs (EBERs) in situ hybridization, and 63 (5.6%) of 1127 consecutive gastric carcinomas were found to be EBV-positive. The expressions of 27 tumor-associated proteins were evaluated immunohistochemically in 63 EBV-positive gastric carcinomas and 287 EBV-negative carcinomas using the tissue array method. In addition, the genotype of EBV was investigated by PCR amplification of LMP1 (latent membrane protein 1), Epstein-Barr nuclear antigen 2 (EBNA2), and EBNA3B genes. Results: EBV-positive gastric carcinomas are characterized by the presence of lymphoid stroma, proximal location, and predominance in males. In comparison with EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal cancer), and some DNA repair proteins (P < 0.05). There was negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA, C-erbB2, and smad7. Using hierarchical cluster analysis, we divided EBV-positive gastric carcinomas into two clusters. Those patients with cluster 1 (42 cases) carcinomas had a better prognosis than those with cluster 2 (12 cases; P = 0.0002) or those with EBV-negative carcinomas (280 cases; P = 0.0251). Fifty-one (92.7%) of 55 EBV-positive carcinomas demonstrated the 30-bp deletion in LMP1 gene, and 53 (96.4%) of 55 cases were type 1 for EBNA2 and EBNA3B genes. Conclusion: EBV-positive gastric carcinomas have a distinct protein expression profile as well as distinct clinicopathological features, as compared with EBV-negative carcinomas. The subclassification of EBV-positive carcinomas, by hierarchical cluster analysis, is significantly associated with patient survival.

Epstein–Barr virus in gastric carcinomas with lymphoid stroma

Aims

Clinicopathologic Characteristics of Epstein-Barr Virus-Incorporated Gastric Cancers in Korea

We evaluated the rate of Epstein-Barr virus (EBV) infection in gastric carcinomas of Korean patients and investigated the associations between EBV infection and clinicopathological characteristics, the survival rates of patients, and p53 overexpression. EBV-encoded small RNA (EBER)-in situ hybridization and immunohistochemistry for p53 protein were done in 306 consecutive gastric carcinoma cases, of which 17 (5.6%) showed EBV infection. Of these 17 EBV-positive cases, one case strongly expressed p53 protein, while 98 (34%) of 285 EBV-negative cases overexpressed p53 (p < 0.05). The EBV-positive gastric carcinomas tended to have lymphoid stroma. They were mostly of the poorly differentiated type, negative for p53 immunoexpression, more prevalent in male patients, and diffuse according to Laurens classification (p < 0.05). There was no significant difference in the survival rate for the EBV status. In conclusion, the EBV infection rate among gastric carcinomas in Korea is similar to that ascertained in other countries. An inverse correlation between EBV and p53 overexpression was disclosed. Further study is needed to find out whether or not two genetic changes could be functionally overlapping during gastric carcinogenesis.

Epstein–Barr virus and microsatellite instability in gastric carcinogenesis

Little information is available concerning the relationship between transforming viruses and microsatellite instability (MSI). We evaluated Epstein–Barr virus (EBV) using in situ hybridization for EBV‐encoded small RNAs and MSI using the polymerase chain reaction in surgically resected gastric cancer. The study subjects included 298 consecutive cases of solitary gastric carcinoma, 63 gastric carcinomas in young patients (≤30 years old), 64 cases of gastric cancer coexisting with gastric adenoma in a single lesion, 26 cases of gastric remnant cancer, and 98 carcinomas from 47 patients with synchronous multiple gastric carcinomas. There was no overlapping case among these subsets of gastric cancer. None of these 549 gastric carcinomas demonstrated both EBV positivity and MSI positivity. Furthermore, the EBV‐positive and the MSI‐positive cases showed a mutually negative association in all subsets of gastric cancer. 5.7% of consecutive solitary gastric carcinomas were EBV positive, and 9.7% were MSI positive. EBV was positive in 1.6% of gastric cancers coexisting with gastric adenoma, 12.7% of younger patients, 28.6% of gastric remnant cancer with previous gastrectomy for benign disease, and 14.5% of synchronous cancers without adenoma. MSI was found in 1.6% of younger patients, 18.8% of gastric cancers coexisting with gastric adenoma, 25% of gastric remnant cancer with previous gastrectomy for gastric cancer, and in 53.3% of synchronous gastric carcinomas having gastric adenoma remote from the cancer. In conclusion, the carcinogenic roles of EBV and MSI may be different in terms of each subset of gastric cancer. EBV and MSI may contribute to functionally equivalent pathways in gastric carcinogenesis. Copyright

Microsatellite instability and Epstein-Barr virus infection in gastric remnant cancers

Clinicopathological analysis, microsatellite analysis, detection of Epstein–Barr virus (EBV), and immunohistochemistry on p53 protein were performed in 26 cases of gastric remnant cancer (GRC). They were divided into two groups; Group A (n= 14) who had undergone a primary gastrectomy for benign gastric disease, and Group B (n= 12) who had undergone the same operation for gastric cancer. EBV infection was present in 29% of Group A, 8% of Group B and 6% of the conventional gastric carcinoma (CGC) (Group A vs CGC, P= 0.01). Microsatellite instability (MSI) was found in 7% of Group A, 25% of Group B, and 9% of the CGC (Group B vs CGC; P= 0.08). p53 Overexpression was observed in 46% of the GRC and 33% of the CGC. p53 Overexpression was observed in 90% of the intestinal type of GRC, but in only 20% of the diffuse type of GRC (P= 0.002). The cancer stage was a significant factor in the univariate analysis of survival (P= 0.04). In conclusion, GRC is different from CGC in terms of MSI or EBV association. The pathogenetic differences between the two groups require further investigation. EBV infection may have been involved in the carcinogenesis of Group A. MSI may be an important factor in the carcinogenesis of metachronous multiple gastric cancer (Group B).

Epstein-Barr Virus-Encoded BARF1 Promotes Proliferation of Gastric Carcinoma Cells through Regulation of NF-κB

ABSTRACT In Epstein-Barr virus (EBV)-infected gastric carcinoma, EBV-encoded BARF1 has been hypothesized to function as an oncogene. To evaluate cellular changes induced by BARF1, we isolated the full-length BARF1 gene from gastric carcinoma cells that were naturally infected with EBV and transfected BARF1 into EBV-negative gastric carcinoma cells. BARF1 protein was primarily secreted into culture supernatant and only marginally detectable within cells. Compared with gastric carcinoma cells containing empty vector, BARF1-expressing gastric carcinoma cells exhibited increased cell proliferation (P < 0.05). There were no significant differences in apoptosis, invasion, or migration between BARF1-expressing gastric carcinoma cells and empty vector-transfected cells. BARF1-expressing gastric carcinoma cells demonstrated increased nuclear expression of nuclear factor kappa B (NF-κB) RelA protein and increased NF-κB-dependent cyclin D1. The expression of p21WAF1 was diminished by BARF1 transfection and increased by NF-κB inhibition. Proliferation of naturally EBV-infected gastric carcinoma cells was suppressed by BARF1 small interfering RNA (siRNA) (P < 0.05). Immunohistochemical analysis of 120 human gastric carcinoma tissues demonstrated increased expression of cyclin D1 and reduced expression of p21WAF1 in EBV-positive samples versus EBV-negative gastric carcinomas (P < 0.05). In conclusion, the secreted BARF1 may stimulate proliferation of EBV-infected gastric carcinoma cells via upregulation of NF-κB/cyclin D1 and reduction of the cell cycle inhibitor p21WAF1, thereby facilitating EBV-induced cancer progression.

Immunohistochemical Analysis of Cell Cycle-Related Molecules in Gastric Carcinoma: Prognostic Significance, Correlation with Clinicopathological Parameters, Proliferation and Apoptosis

Objective: We aimed to investigate the biological significance of cell cycle regulators in gastric carcinoma. Methods: Immunohistochemistry and TUNEL staining were performed on tissue array slides containing 293 gastric carcinoma specimens. The relationship between the protein expression of each of the cell cycle regulators and prognosis, clinicopathological features, proliferation, or apoptosis was evaluated. Results: The nuclear immunoreactivity for cyclin D1, cyclin E, p21, and p27 was observed in 22, 14, 31 and 27% of cases, respectively. The expression of cyclin D1, p21, or p27 positively correlated with early pTNM stages, tumor cell proliferation (represented by Ki-67 labeling) and good prognosis, whereas it inversely correlated with the lymph node metastasis (p < 0.05). On the other hand, p27 expression inversely correlated with the apoptosis index represented by TUNEL staining (p < 0.001). In addition, the expression of cyclin D1 positively correlated with that of p21 or p27 (p < 0.05). Conclusions: Our results showed that the expression of cyclin D1, p21 and p27, alone or in combination, are early events in gastric tumorigenesis and may serve as a candidate molecular marker for the early gastric carcinoma.

Cell cycle regulators, APC/β-catenin, NF-κB and Epstein–Barr virus in gastric carcinomas

Aims: To evaluate the clinicopathological value of cell cycle regulators, the Wnt pathway, the NF‐βB pathway and Epstein–Barr virus (EBV) and to assess their relationships in gastric carcinoma. Methods: We investigated cell cycle regulators (p53, p21, Rb), APC, β‐catenin and NF‐κB using immunohistochemistry and EBV using in situ hybridisation for EBV encoded small RNAs in 117 cases of gastric carcinoma. Results: p53 overexpression was more frequently observed in advanced gastric carcinoma and lymph node metastasis than in early carcinoma or in the absence of metastasis (p < 0.05). p21 loss was positively correlated with APC loss, but inversely correlated with β‐catenin nuclear accumulation and NF‐κB positivity (p < 0.05). EBV positive gastric carcinomas were located in the upper third of the stomach, and more were of the diffuse or mixed types than the EBV negative group (p < 0.05). EBV infection was positively correlated with p21 loss and APC loss and inversely correlated with β‐catenin alteration (p < 0.05). In multivariate analysis, patient age, TNM stage and p53 were independent prognostic factors for gastric carcinoma. Conclusions: p53 status is a prognostic marker for gastric carcinoma. p21, APC, β‐catenin and NF‐κB may be functionally interrelated in gastric carcinogenesis. Loss of p21 and APC may be involved in the carcinogenesis of EBV positive gastric carcinomas.

Constitutive phosphorylation of the FOXO1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis-related molecules.

BackgroundAlthough FOXO transcription factors may have an anti-angiogenic role, little is known about their role in tumor angiogenesis. The present study was performed to investigate the correlation between the constitutive expression of phosphorylated FOXO1 (pFOXO1) and angiogenesis in gastric cancer.MethodsImmunohistochemistry was performed on tissue array slides containing 272 gastric carcinoma specimens, and the correlations between the cytoplasmic pFOXO1 expression in gastric cancer cells and CD34-immunopositive microvessel area (MVA) or the expressions of angiogenesis-related molecules were analyzed. In vitro analyses with Western blotting and semiquantitative reverse transcription-polymerase chain reaction were performed using the stable SNU-638 gastric cancer cell line transfected with lentivirus-delivered FOXO1 short hairpin RNA.ResultsThe cytoplasmic expression of pFOXO1 in tumor cells was observed in 85% of gastric carcinoma cases, and was found to be positively associated with higher MVA (P = 0.048). Moreover, pFOXO1 expression was positively correlated with the expressions of several angiogenesis-related proteins, including hypoxia inducible factor-1α (HIF-1α, P = 0.003), vessel endothelial growth factor (P = 0.004), phosphorylated protein kinase B (P < 0.001), and nuclear factor-κB (P = 0.040). In contrast, the expression of pFOXO1 was not correlated with that of phosphorylated signal transducer and activator of transcription 3 or β-catenin. In addition, cell culture experiments showed that FOXO1 suppression increased the mRNA and protein expressions of HIF-1α.ConclusionOur results suggest that pFOXO1 expression in cancer cells plays a role in gastric cancer angiogenesis via mechanisms involving various angiogenesis-related molecules. Animal experiments are needed to confirm the anti-angiogenic role of FOXO1 in human gastric cancer.

Prognostic Significance of CD24 Expression in Gastric Carcinoma

PURPOSE The human CD24 antigen is a small heavily glycosylated cell surface protein, which is expressed in hematological malignancies, as well as in a large variety of solid tumors. Its expression is now known to be related to the prognosis of several kinds of tumors. This study is designed to examine the prognostic significance of CD24 in Korean gastric cancer patients. MATERIALS AND METHODS In the present study, we examined CD24 expression in 300 consecutive cases of gastric carcinoma by immunohistochemical staining using the tissue-array method. We also investigated the clinicopathological profiles related to CD24 expression. RESULTS One hundred and three cases out of 300 (34.3%) showed the positive expression of CD24. The altered expression of CD24 was significantly associated with differentiated cancer (p=0.003), the intestinal subtype according to the Lauren classification (p<0.001), the advanced stage cancer (p=0.027), with lymphatic invasion (p=0.038) and with vascular invasion (p=0.006). The survival analysis revealed that the patients with CD24 positive expression showed significantly poorer survival than those without CD24 expression. Moreover, a combined evaluation revealed that PTEN+/CD24- cases showed the best survival compared to other groups (p=0.01). CONCLUSION Positive CD24 expression occurs in a subset of gastric carcinomas and it correlates significantly with lymphatic invasion, blood vessel invasion and poor survival.