Our meta-analysis showed that the pooled prevalence of EBV among gastric cancer patients from 27 countries is 8.78% (95% CI: 7.75–9.93%; I2 = 83.0%). We chose strict inclusion and exclusion criteria to obtain pertinent studies and to increase the chance of finding a valid conclusion. The pooled prevalence and OR obtained in this meta-analysis were calculated from studies which detected EBV infection with ISH method. All studies that investigated the presence of EBV by other methods, including different types of PCR assays, and even immunohistochemistry (IHC), did not consider in our analysis. The reason for this stems from the fact that the sensitivity and specificity of each detection method are different, and it is not reliable to draw a conclusion using the pooled data.
The gold standard technique for the detection of EBV in tissues is ISH with EBV EBERs (EBER-ISH) due to its high sensitivity and specificity to determine the precise intranuclear localization of the EBV-infected cells. The diagnosis of EBV-associated gastric cancer is confirmed by the presence of EBER within the tumor cells and its absence in the normal tissue adjacent to the tumor [3]. Many studies have reported the higher prevalence of EBV among gastric cancer patients by PCR assay than EBER-ISH technique [83]. However, PCR is unable to discriminate between cancer cells and lymphocytes infiltrating in tumor stromal, and thus it is impossible to know from where the EBV genome is amplified. It should be noted that the great majority of people (nearly 90%) are EBV carriers, and their lymphocytes probably contain EBV genomes [84]. Regarding the aforementioned statements, our meta-analysis exclusively focused on the positivity of the EBV-associated gastric cancers by ISH only.
One of the major strong points in this meta-analysis is that the pooled estimate of ORs was calculated from studies with matched pairs and non-matched pairs designs, separately, with different statistical methods. The detailed descriptions about the analysis of data for matched pairs and non-matched pairs studies are available in several previous studies [85]. It has been recommended that a matched-pairs analysis should be used to assess effect sizes for studies with matched pairs design. Accordingly, the pooled OR determined for studies with non-matched pairs and matched pairs designs were 3.31 (95% CI: 0.95–11.54; I2 = 55.0%) and 18.56 (95% CI: 15.68–21.97; I2 = 55.4%), respectively.
To date, several studies have attempted to discover the role of EBV infection in gastric cancer progression. EBV enters B lymphocytes in oropharyngeal lymphoid tissues. The virus then enters the gastric epithelial cells, either by the cell-to-cell contact between B lymphocytes and gastric epithelial cells or by direct entry into the gastric epithelia [86]. It has been reported that EBV entry into the gastric epithelial cells is facilitated by the previous mucosal damage [53]. After the virus enters the cell, EBV establishes type I latency in which a limited set of latent gene is expressed [64]. A recent systematic review study showed that the most of the EBV latent proteins expressed in gastric cancer cases were EBNA1 (98.1%) and LMP2A (53.8%), whereas LMP1 and LMP2B were detected in only 10% of EBV-associated gastric cancer cases. Some of lytic proteins such as BARF1 were also reported to be present in almost half of EBV-associated gastric cancer cases [87]. It is shown that the EBV-encoded BARF1 acts as oncogene and promotes cell proliferation in gastric cancer through upregulation of NF-κB signaling and reduction of the cell cycle inhibitor p21 [88]. It is well known that DNA methylation plays a crucial role in gastric cancer development and progression [89]. Methylation of both viral and cellular genome is one of the important mechanisms involved in the development and maintenance of EBV-associated gastric cancer. It is well documented that EBV latent membrane protein 2A (LMP2A) plays a variety of key roles in the epigenetic abnormalities such as aberrant DNA methylation in host stomach cells, and in the development and maintenance of EBV-associated gastric cancer [90].
Another interesting finding of our meta-analysis is that the prevalence of EBV was 1.9-fold higher in male patients than in female patients with gastric cancer (P < 0.01). However, the OR estimate for EBV-associated gastric cancer was significantly higher among females than in males (P = 0.06). According to these results, we concluded that women are more likely than men (1.5-fold) to develop EBV-associated gastric cancer. This novel finding can be explained by different genetic background, lifestyle, or hormonal conditions between the two genders.
Subgroup analyses based on the tumor anatomical location indicate an anatomic preference for EBV during gastric carcinogenesis. Indeed, EBV-associated gastric cancers were significantly more prevalent in the cardia and in the body of the stomach than in the antrum (P < 0.01) (Table 2). However, the situation was different when OR was calculated. So that the OR estimate for EBV-associated gastric cancer was remarkably higher in the antrum than in the cardia and in the body (Table 3), although the difference was not statistically significant. This feature can be justified with the fact that the various parts of the stomach have different physiological conditions.
One prominent finding of the present meta-analysis is that EBV was detected more frequently in biopsy samples than in FFPE specimens from gastric cancer patients (2.4-fold, P < 0.01). It is well documented that there are several challenges when working with FFPE samples such as the low amount of extracted nucleic acids, and fragmentation of genomes and transcripts during the processes of fixation and embedding in paraffin. Therefore, in order to prevent false-negative results, using biopsy samples is recommended.
According to Lauren's histological classification, gastric carcinoma is classified into two distinct types, namely intestinal and diffuse types. There are many differences between intestinal and diffuse types based on their epidemiology, etiology, and pathology [67]. However, the current meta-analysis showed that the prevalence of EBV was similar in intestinal and diffuse types (8.15% and 9.40%, respectively), and no significant association of EBV infection with the histological type was found (P = 0.27).
Similarly, our results did not indicate any significant difference in the prevalence of EBV-associated gastric cancer among different geographic regions, even between developed and developing countries. The same prevalence in developed and developing countries demonstrates that economic conditions are not related to EBV-associated gastric cancer risk.