Abstract
Background/Aim: The association between Epstein-Barr virus (EBV) and gastric cancer (GC) has been reported by many researchers. Immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor β (TGFβ), play an important role in the tumor process. The aim of the present study was to detect antibodies against EBV and explore the levels of TGFβ and IL-10 in Polish GC patients. Patients and Methods: Fifty patients with GC and 50 hospitalized individuals without GC (control group) were enrolled in the study. Frozen tumor tissue fragments were tested using nested PCR assay for EBV DNA detection. ELISA test was used to detect the presence of anti-VCA IgG, anti-EBNA IgG, anti-EA IgG, TGFβ and Il-10 in sera from all individuals. Results: EBVCA was detected in 88.0%, EBNA in 90.0%, and EA in 72.0% of patients. Levels of TGFβ and IL-10 were significantly higher in patients with high levels of anti-EA antibodies (25.4 ng/ml; 7.8 pg/ml) compared to patients with low levels of anti-EA antibodies (12.61 ng/ml; 4.29 pg/ml). Conclusion: The significantly higher level of EA in patients' sera indicates EBV reactivation. TGFβ level was significantly higher in GC than in the control group, especially in EA-positive patients, indicating its possible role in gastric carcinogenesis.
Gastric cancer (GC) is the fifth most commonly diagnosed cancer and the third leading cause of cancer-related mortality worldwide (1). According to Cancer Atlas Research Network, GC is divided into 4 molecular types: GC positive for Epstein-Barr virus (EBV), GC with microsatelite instability, genomically stable GC and GC with chromosomal instability (2).
Every year, more than 950,000 cases of gastric adenocarcinoma are diagnosed in the world and it is estimated that about 10% of these cases are associated with EBV (3, 4). Each year worldwide it is estimated that there are about 84,000 cases of EBV-associated gastric carcinoma (5). EBV-positive gastric cancer cases constitute the largest group of EBV-associated cancers (4). In Poland, EBV-positive GC was 12.5%, in 2012 (2). Though, there are very few publications on the serological status of GC patients, in the available world literature. In a meta-analysis by Chenan et al. (6), the authors found only 4 publications comparing the presence of anti-EBV antibodies between patients with GC and healthy individuals. There are specific serological markers of EBV infection that are useful indicators of exposure to infection and reactivation. For example, antibodies against Epstein-Barr virus capsid antigen (EBVCA), that is expressed in latent infection, anti-Epstein-Barr virus nuclear antigen (EBNA) that is primarily epidemiological, and anti-early antigen (EA), which might indicate the reactivation of latent infection (6, 7). In addition, many studies indicate that transforming growth factor-β (TGFβ) induces EBV reactivation in certain types of cancer (8, 9).
Therefore, the aim of the present study was to evaluate the levels of antibodies against EBV antigens: anti-EBVCA IgG, anti-EBNA IgG and anti-EA IgG, in order to determine the possible reactivation of the virus. Furthermore, levels of IL-10 and TGF-β in sera of GC patients compared to the control group were estimated. Finally, we tested the incidence of latent membrane protein (LMP-1), a well-known oncoprotein that takes part in neoplastic transformation of EBV-infected B lymphocytes, and the 30 bp deletion variant del-LMP-1 (10).
Materials and Methods
Patients. The present study consisted of a group of 50 patients with diagnosed GC that were hospitalized at the Surgery Ward of Private Hospital in Nałęczów and AMG Hospital Center in Ryki, Poland. All patients were Helicobacter pylori-negative (based on the medical history of the patients). The results were compared to the control group, consisted of 50 individuals hospitalized at the same Ward due to diseases other than GC. There were no statistically significant differences between the patients and the control group (age, sex, tobacco and alcohol consumption). The socio-demographic and clinicopathological characteristics of the study group are shown in Table I. The research material consisted of sera and fresh frozen tumor tissue fragments. This research was approved by the Ethics Committee and is in accordance with the GCP regulations (no. KE-0254/133/2013/Medical University of Lublin, Poland).
Molecular methods. DNA extraction from fresh frozen tumour tissue, detection of EBV DNA, amplification of EBNA-2 gene (the nested PCR) and serological tests for antibody level detection were performed as previously described (11). PCR screening for the EBV LMP-1 subtype based on exon 3, defined as wild-type (wt-LMP-1) or del-LMP-1, was analyzed as previously described (12).
Statistical analysis. Descriptive statistics were used to characterize patient baseline characteristics. The Mann-Whitney U-test and Kruskal-Wallis test were used to compare the antibody and cytokine levels. Pearson's Chi-square test was used to investigate the relationship of LMP-1, clinical and demographical parameters between patients and control group. Statistical significance was defined as p<0.05.
Results
Men aged over 60 years and rural residents were predominant. GC was most frequently localized to the pylorus region (58.0%), then to the body region (24.0%), and the cardiac region of the stomach (18.0%). Anti-EBVCA IgG were detected in 88.0% of GC patients and in 86.0% of control individuals, while anti-EBNA IgG respectively in 90.0% and 84.0%. Antibodies against early antigen (EA) were more frequent in patients (72.0%) than in the control group (8.0%) (p<0.001). Levels of the studied antibodies and cytokines TGFβ and IL-10, were higher (24.4 ng/ml; 7.2 pg/ml, respectively) in patients with EBV-associated GC (EBVaGC) than in control patients (10.1 ng/ml; 3.5 pg/ml, respectively) (p<0.001) (Table II). An association was detected between serum levels of TGFβ, Il-10 and the level of the studied antibodies (p<0.001). The patients infected with wild type LMP-1 had a higher concentration of TGFβ (24.8 ng/ml vs. 18.1 ng/ml in del-LMP-1; p<0.01) (Table III).
Discussion
The number of publications on various aspects of GC is steadily increasing (13, 14). EBVaGC is more common in younger patients, between 50 and 68 years of age, while EBV-negative GC is more frequent among patients aged 56-72. Several risk factors for developing EBVaGC have been identified, i.e. tobacco smoking (15, 16, 17). In our own study 64.0% were men over the age of 60, most of them tobacco smokers (74%).
Epidemiological analyses of EBV infection in GC (also in other cancers) are based on the results of laboratory tests detecting the presence of the virus in tumor tissue, i.e. principally in situ hybridization (ISH) or PCR and serological tests to assess the presence and/or titer of specific antibodies in patients' sera (18). In the present study, PCR was used to detect the presence of EBV DNA.
Socio-demographic and clinicopathological characteristics of patients and healthy individuals (control group).
Serum level of anti-EBVCA, anti-EBNA, TGFβ and IL-10 in patients and healthy individuals (control group).
EBV is very widespread in the world, so approximately 95% of the population is detected with EBV-related antibodies. In a meta-analysis by Chenan et al. (6), EBNA IgG and EBVCA IgG were more frequently detected than the other antibodies, in both patients and control group. In our study, the prevalence of anti-EVBCA and anti-EBNA was similar in the GC and the control group, though titers of these antibodies differed between the groups, as both were significantly higher in EBVaGC patients. Anti-EA antibodies were found in 72% of patients with GC, of which 50% in high titer, while they were detected only in 17% of the control group, at very low titers. High anti-EA titers at the same time with high anti-EBVCA and anti-EBNA levels might indicate the reactivation of EBV infection in patients with GC.
A scientific research in Japan has shown statistically higher levels of EBVCA among GC than in control (19). On the other hand, Shinkura et al. (7) and Kayamba et al. (20) showed higher anti-EA antibody titers in patients with EBVaGC. This fact would suggest a re-activation of infection in the early stages of neoplastic transformation (21, 22).
An important feature of EBV, as well as of the entire Herpesviridae family, is the ability to enter into latency (23, 24), in which only some genes are expressed (25, 26). EBVaGC is considered as a latency type I or II EBV infection, (18), in which i.e. EBNA-1, EBNA-2, LMP-1 and LMP-2 are expressed. EBNA-1 is present in all types of latency, and as the only EBNA type protein is also produced during activation into the lytic cycle. EBNA-2 is the major transcription factor regulating the expression of viral genes. It activates, among others, the LMP-1 and LMP-2 promoters and also transactivates many cellular genes that play a key role in cell proliferation and immortalization after infection (18).
Wang et al. (27) state that EBVaGC exhibits type I latency and, therefore, LMP-1 is not expressed. The authors show that the expression of LMP-1 occurs much more frequently in nasopharyngeal cancer (NPC). Some studies indicate low, while others show high LMP-1 expression in the EBVaGC (18). The type of virus with a deletion is more common in NPC (28). However, in a study in Japanese GC patients, the incidence of del-LMP-1 was similar in both the study and control group (29).
Correlation between serum levels of TGFβ, IL-10, EBNA, EBVCA, anti-EA and LMP-1 type.
The results obtained from this study indicate that the LMP-1 protein was detected in all cases, which would indicate the presence of type II latency. The wild-type LMP-1 was detected in a predominant proportion (78%), while a del-LMP-1 was found in 22%. Tsao et al. (30) suggest that LMP-1 might play a different role in early and late stages of carcinogenesis. High expression of LMP-1 can lead to deregulation of the cytotoxic effect of tumor cells and increased rates of mutation and methylation EBVaGC, though authors state the need for further research on this field.
We were further demonstrated that all patients with EBV del-LMP-1 had a high serum anti-EA antibody titer, while 30% of GC patients with the wild type had high anti-EA titer and 60% low. In addition, there were observed high TGFβ levels (24.8 ng/ml) in patients with wild-type LMP-1, which stimulated the reactivation of latent infection. It can, therefore, be assumed that the wild type plays a greater role in the pathogenesis of GC. However, the limitation of our study was that the sample size was quite small to make inferences about the Polish population means.
Reactivation of the virus and entry into the lytic cycle might be induced by various causes, including decrease in host resistance, various chemicals (31), and TGFβ (32).
Immunosuppressive cytokines, such as IL-10 and TGFβ, play an important role in the tumor process (33-39). In the present study, IL-10 was significantly higher in patients with GC (7.2 pg/ml), and in patients with high titers of anti-EA (7.8 pg/ml). In patients with high levels of IL-10 a high titer of anti-EBNA was antibody also observed. Viral IL-10 (vil-10), which exhibits high homology with human IL-10, is secreted late in the lytic phase and also at the onset of B lymphocytes infection. (40). IL-10 levels, as shown by other studies, are statistically higher in the serum of GC patients than in the control group and therefore might predict GC and, particularly, could be used for the assessment of disease progression (41, 42). Our results showed higher serum levels of TGFβ in patients with EBVaGC (24.4 ng/ml) compared to the control group (10 ng/ml). In studies by Shukla et al. (32) carried out on a group of 95 patients with EBVaGC, TGFβ mRNA expression was detected in 89.5% of cases, indicating the role of TGFβ in the development of GC. In addition, they found that TGFβ expression was closely related to the lytic phase of infection in patients without H. pylori. These researchers suggest that TGFβ plays a role in the reactivation of EBV. In this study, TGFβ concentration correlated with the high anti-EA antibody titer (25.44 ng/ml vs. 7.8 ng/ml, respectively). On the other hand, there was no such relationship between EBVCA and EBNA.
Genetic and epigenetic changes also take part in the process of tumor transformation of gastric epithelial cells. Up to now, 9 high-expression viral genes in EBVaGC have been described (43). It has also been found that in EBVaGC cases, mutations can occur in approximately 205 cell genes, including TGFβ receptor TGFBR1, which is a serine-threonine kinase protein. Induced EBV infection with TGFBR1 mutation may be of importance for EBVaGC pathogenesis (43). In vitro studies of EBVaGC have shown that epigenetic changes also affect the expression of many genes. A correlation between EBVaGC and CpG methylation was detected suggesting that hypermethylation of entire groups of genes promotes gastric epithelial cell transformation (43).
Conclusion
Our results show that antibodies against EBV are frequent (EBVCA - 88.0%, EBNA - 90.0%, EA - 72.0%) and the wild-type LMP-1 is predominant (78.0%) in Polish patients with GC. The significantly higher level of EA in the patients points to EBV reactivation. TGFβ level was significantly higher in GC than in control group, especially in EA-positive patients, suggesting its role in gastric carcinogenesis. However, further studies are necessary to understand the complex mechanisms of tumor transformation that gastric epithelial cells undergo.
Footnotes
This article is freely accessible online.
Ethics Approval and Consent to Participate
This research was approved by the Ethics Committee and is in accordance with the GCP regulations (no. KE-0254/133/2013/Medical University of Lublin, Poland).
All participants provided written informed consent to participate in this study according to forms required by the Local Ethics Committee.
Conflicts of Interest
The Authors declare that they have no competing interests.
Funding
This study was supported by a Research Grant from the Medical University of Lublin, Lublin, Poland (DS 233).
- Received June 1, 2017.
- Revision received July 3, 2017.
- Accepted July 6, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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