Status of Epstein-Barr Virus Coinfection with Helicobacter pylori in Gastric Cancer

Epstein-Barr virus is a ubiquitous human herpesvirus whose primary infection causes mononucleosis, Burkett's lymphoma, nasopharyngeal carcinoma, autoimmune diseases, and gastric cancer (GC). The persistent infection causes malignancies in lymph and epithelial cells. Helicobacter pylori causes gastritis in human with chronic inflammation. This chronic inflammation is thought to be the cause of genomic instability. About 45%-word population have a probability of having both pathogens, namely, H. pylori and EBV. Approximately 180 per hundred thousand population is developing GC along with many gastric abnormalities. This makes GC the third leading cause of cancer-related death worldwide. Although lots of research are carried out individually for EBV and H. pylori, still there are very few reports available on coinfection of both pathogens. Recent studies suggested that EBV and H. pylori coinfection increases the occurrence of GC as well as the early age of GC detection comparing to individual infection. The aim of this review is to present status on coinfection of both pathogens and their association with GC.


Introduction
Gastric cancer (GC) or stomach cancer is the fifth most common cancer incident and the third leading cause of cancer-associated mortality, contributing 6.8% of total cancer cases and 8.8% of total cancer-associated death worldwide [1]. An estimated 984,000 new cases (ratio 2 : 1, male versus female) and 841,000 GC-related deaths were accounted in 2013 [2]. Approximately, 77% of GC-related cases and the death occur in developing countries, particularly in Eastern Asia, while 23% occur in developed nations [2]. GC can be divided into 4 types on the basis of appearance in different cell types: (1) adenocarcinoma: within the cells of the innermost lining of the stomach (mucous surface); (2) lymphoma: cancer of the immune system in lymph stomach tissues, very rare; (3) gastrointestinal stromal tumors: stomach epithelial lining tumors in interstitial cells of Cajal, very rare; (4) carcinoid tumors: typically arising in the hormone-producing cells of the stomach. The common histopathological features of gastric malignancies are adenocarcinoma. It accounts for nearly 90% of GC [3]. Adenocarcinomas are further divided into two parts: (1) cardia, the top part of the stomach; (2) noncardia cancers, depending on location in the stomach where they first appear. H. pylori is now a well-known and primary cause of GC [4][5][6][7][8][9][10], specifically noncardia cancer [11,12], and is declared as carcinogen I [13] to humans. H. pylori is now well known to be linked to stomach cancer in many studies along with EBV [14][15][16][17][18]. Other risk factors for GC include chronic gastritis [9], older age [19], male sex [20,21], a diet high in salt [22][23][24], smoking [25,26], alcohol consumption [27], poorly preserved foods [28], diet low in fruits and vegetables [29], tobacco product [30], pernicious anaemia [31][32][33], a history of stomach surgery for benign conditions [34], and a family history of stomach cancer [34,35].  [116]. They are more sensitive and specific than serological methods as EBV immunologic response appears after several days of infections [117][118][119]. After 15-day onset of onset of EBV infection, 100% of EBV DNA is detectable in plasma [118]. Several reports suggested that EBV DNA is present in almost all carcinoma cells in EBV-positive cases [120]. After primary EBV infection due to immune response, EBV DNA declines slowly in PBMCs, rapidly in plasma or serum, and further 3 to 4 weeks, it becomes undetectable [118,121]. Interestingly, EBV may remain latent in memory cells for an extended period in blood or take a longer time before it reaches a small, stable stage. Copy number range of 1 to 50 of EBV DNA may be detected in a healthy person infected with EBV in white blood cells (WBC) [122]. PCR and real-time PCR sensitivity and specificity vary based on detection methods as well as laboratory to laboratory practise [118,123,124].  [127,128]. Serological tests require validation at the local level, which is impractical in routine practice. Moreover, serologic findings in both the children and adults are conflicting, and the cut-off is not shown to be accurate in many studies [129,130]. Serological testing is accurate in low prevalence regions where less than 20% of the population are affected. In those patients where the gastric lining has not changed to the precancerous form of intestinal metaplasia, neither biopsy nor Urea Breath Tests can be used as there are very few bacteria present [131,132]. Moreover, serial serology from antibody concentrations can be used as follow-up after treatment of H. pylori [133].

Urea Breath Test (UBT)
. UBT measures C13 carbon dioxide in breath after ingesting C-13-labelled urea [134]. This test is approved by FDA, USA. This can be used for the individuals aged 3 years or older. The cost of UBT is more than serological or stool antigen testing and UBT can be used both as a diagnostic tool and in efficacy of treatments [131,132].

Stool Antigen Test (SAT).
Antigens released from the wall of the stomach can be detected in SAT through ELISA.
Detection of antigen only occurs if H. pylori is present and this shows active infection [135]. Similar to the UBT, the SAT can be used both as a diagnostic tool and in efficacy of treatments [125]. This is also an FDA-approved test and SAT is recommended by the ACG and the AGA [136,137].

EBV Infection to Gastric Epithelial Cells
Latency and reactivation are the hallmarks of EBV which is a ubiquitous and potentially oncogenic human herpes virus [87]. EBV was discovered in 1964 in patients with Burkett's lymphoma (BL) [138]. Initially, it was assumed that it infects only B-cells; later, it was also found in nasopharyngeal 4 Journal of Oncology epithelial cells [139], liver cells [136], stomach epithelial cells [91], brain cells [137], and so forth.

Low Tropism of EBV Infection through Oral Route to
Mouth Oropharyngeal Epithelial Cells. Due to difficulties in establishing reproducible and robust infection in vitro, it is very difficult to simulate a real understanding of EBV pathology [114]. Most of the studies suggested that EBV may be transcytosed via EBV+ IgA complex through the oral epithelium, oropharynx bidirectional, from apical membranes to the basolateral and vice versa [140]. This EBV transmigration potentially contributes to initial EBV penetration into B-cells that starts the systemic infection. EBV secretion may occur into saliva in EBV-infected individuals [114,141]. However, EBV could not be detected in oropharyngeal in the primary stage of infection in the process of transcytosis [142].

EBV Has High Tropism for B Lymphocytes. EBV interacts
with naive or memory B lymphocytes in Waldeyer's ring. Waldeyer's ring is situated in lymphoid tissue and surrounds the oropharynx [143]. EBV have a high affinity for the B-cell and complement receptor type 2 (CR2) or CD21 present on the surface of B-cell facilitate the attachment of EBV envelope glycoprotein gp350/220 to B-cell [144,145]. Following attachment, internalization of EBV occurs in the cells via the endocytosis. Further, this fusion of EBV envelopes proteins and Bcells triggers interaction of another envelopes glycoproteins, that is, gp42. Gp42 interact with HLA class II which is present on B-cells and make a core fusion complex gh/gL/gp42 and further make internalization process [146,147]. Fate of EBV-infected B-cells depends on their niche as these cells may initiate proliferation or they can reach the memory compartment. EBV establishes a latency and expresses some limited sets of genes if B-cells reach memory compartment [144,145]. B-cell infection mostly causes latency (I, II, III) [86,148]; however, freshly isolated Bcells from an EBV-infected tumor lead to transformation and reactivation in vitro [149,150]. In vivo study suggested that EBV-infected B-cells cause infectious mononucleosis with an incubation period of 30-50 days [81,121]. Several studies suggested that it is difficult to determine EBV DNA in epithelial cells during primary infection. It is debatable how epithelial cells spread the virus to precede infection of B lymphocytes. Moreover, later EBV-related severe disease shows virus amplification in epithelial cells before shedding in saliva which at least gives some evidential support as virus shed almost daily in the saliva of carriers has the glycoprotein composition of the virus made in an epithelial cell rather than a B-cells [122,151].
Infected B-cells reach the circulation, and some B-cells may also go to transformation [152,153]. Cytotoxic T lymphocytes response occurs for the B-cells and mostly this process is due to latent B-cells infection. However, terminal differentiation of B cell occurs through immune response [154]. Infected memory B-cells may reach the site of immune response and further divide into plasma and memory cells. This process initiates reactivation of EBV into lytic cycle in Bcells and this causes more infection to noninfected B-cells and hence replenishes EBV-infected B-cells latent reservoir. This establishes a cycle of persistence in the life of a healthy carrier [155][156][157][158]. It is assumed that lytic release of the virus has a high tropism to epithelial cells than B-cells [159]. In vitro studies suggested that EBV loaded B-cells or B-cells fragments have a high rate of infectivity to epithelial cells [160,161].

Lytic Release of EBV from B-Cells Has a High Tropism
to Infect Other Epithelial Cells. Terminal differentiation of infected memory cells triggers EBV lytic replication [154]. This can occur in any parts/organs of the host where the infected memory cells travel and EBV spread through cell to cell contact [162,163]. A study suggested that undifferentiated basal epithelial cells support latent EBV infection, while differentiation of epithelial cells promotes lytic reactivation [157]. A direct coculture experiment of epithelial cells with EBV-producing Akaka cells shows that cell-cell contact is required for the EBV entry to epithelial cells. An increase of infection efficiency was observed up to 1,000 times as compared to the only viral supernatant harvested from EBVproducing cells [158,160]. In vitro experiment suggested that complement receptor 2 (CR2) was not behind the epithelium infection for EBV, as CR2 expression was not detected in most of the infected epithelial cells [164]. Hence, it is thought to be triggered by binding of epithelial cells integrins v6 or v8 to viral glycoproteins gH/gL [165]. However, these integrins receptors present on epithelial cells show a week affinity with EBV compared to CR2, and hence a cell to cell contact is necessary for the attachment of the receptors of virus released from B-cells to the epithelial cells [158,160].
Another study of the coinfection suggested that CD21 receptor on epithelial cells plays an important role in infecting epithelial cells from EBV-producing B-cells. EBV induces a strong adhesion between B-cells and epithelial cells through activation of CD21 [166]. In a coculture experiment of EBV infected B-cells prelabelled with mABs to its cell surface and epithelial cells shows interaction of EBV glycoprotein gp350 with the CD21 complex members, CD21, CD81, and CD19, between B-cell and epithelial cell synapse [166]. Members of tetraspanins, CD82 and CD63, members of integrin's family LFA-1, integrin 1, CD11b, and integrin v 6, and members of Ig superfamily ICAM-1 and CD48 also show interaction of EBV glycoproteins and CD21 [166]. Virus genome integrates into host epithelial cell genome and amplifies with it [162]. EBV establishes a cycle of persistence in a healthy human and starts to be released in saliva or infects more B-cells. Thus, EBV spread throughout the superbasal epithelium and express latent as well as lytic proteins ( Figure 2) [163,167].

Helicobacter pylori Infection in Epithelial Cells
H. pylori infection spreads from contaminated food or may also be transferred from faces to the mouth [70]. The bacteria neutralize stomach acids and cause gastric ulcer when they penetrate the gastric mucous lining [168]. Two types of H. pylori may be found in the gastric or mucosal lining: coccoid type and helical type [169,170]. Helical type of H. pylori H. pylori infection leads to oxidative stress, toxin, and necrosis in cells. These reactions further lead to chronic inflammation, epigenetic modification, and mutation. All these alterations led to genomic instability. EBV infection leads to the expression of lytic and latent genes of EBV. These viral genes regulated epigenetic modification and chronic inflammation. Further these EBV derived mechanisms lead to genomic instability. Finally, genomic instability is one of the potent sources of carcinogenesis. may be transformed into coccoid type. Coccoid type is less vulnerable with low antigenicity and less vulnerable in gastric lining with low production of cytotoxic protein products (CagA), arginase RocF, tumor necrosis factor-(TNF-), and others [171]. This makes H. pylori escape from immune response more easily [169,170,172,173]. Nearly, 20% of H. pylori in the stomach lining adhere to the epithelial cells surface while the rest are attached through the cell to cell junction. Few numbers of H. pylori bacteria are also found in deeper intercellular space [174]. Autotransporter proteins present on H. pylori surface, BabA, SabA, AlpA, AlpB, HopZ, OipA, and others, facilitate the adherence to the epithelial surface [174][175][176][177][178][179][180][181]; however, no individual protein is found essential [182,183]. Additionally, differential expression of these proteins occurs between strains as well as within a single strain. Thus, over time, H. pylori acquire a dynamic adaption by alteration in gene expression, inactivation, or recombination ( Figure 2) [184,185].

EBV and H. pylori Factors Contributing to the Development of GC
Interaction between EBV and H. pylori in host stomach lining may have some synergistic effects in the development of GC. Many genes were found methylated in EBV and H. pylori coinfected gastric adenocarcinomas. Most frequently hypermethylated genes include COX 2, DAPK, CDH1, CDKN2A, and hMLH1. These genes are commonly found altered in various cancer types including GC [270]. Further, H. pylori positive individuals show a significantly higher EBV DNA load which suggests H. pylori role in lytic phase conversion of EBV [271]. Also, EBV DNA load was more in H. pylori positive patients than those uninfected with GC [272]. Another study on coinfection suggests that EBV with H. pylori induces severe inflammatory responses in the individual and, hence, increases the risk of developing the intestinal type GC [78]. It is thought that there are two possible mechanisms, first an additional inflammatory response in coinfection and increased tissue damaging by both H. pylori and EBV [79,215]. In this scenario, significant elevation was observed in IL-1 [273], TNF- [274], and IL-8 [275]. A study in pediatrics patients demonstrated that H. pylori infection was not but the presence of EBV, an essential factor for severe inflammation [79]. The second mechanism is based on gene products interaction which is more significant between EBV and H. pylori. In vitro study found that EBV reactivation occurs by the PLC signalling pathway and H. pylori toxin CagA strongly activates PLC [237] and also activates several kinases [276]. An ectopic expression on transgenic mice supports the oncoprotein nature of CagA [277][278][279][280]. CagA of H. pylori and LMP1 and LMP2 of EBV activate NF-B and MAP kinases, which are well-known pathways of cell survival and proliferation during carcinogenesis [211,281]. H. pylori associated oncoprotein CagA triggers an aberrant activation of WNT signalling pathway [282]. WNT signalling pathway activation leads to the activation of CDX1, a downstream gene [283], which reprograms epithelial cells in mucosal lining to acquire stemness properties by inducing SALL4 and KLF5 factors [284]. Another study also suggests that EBV and H. pylori transform the stomach epithelium cells and play roles in carcinogenesis [78]. Both pathogens induce common pathways which leads to the activation of transforming factors in stomach epithelial cells by -catenin/TCF-4 signalling pathway [79,285]. In another study, an association between EBV and H. pylori copositivity was shown and significant infiltration in premalignant lesions in GC was observed [78]. A study by Szkaradkiewicz et al. suggested that BcL2 expression was higher in EBV and H. pylori associated GC; thus, excessive overexpression may be the result of coinfection [286]. Several studies also revealed that PCDH10 (protocadherin 10) is calcium dependent cell adhesion molecule which suppresses tumor in gastric epithelial hypermethylated in H. pylori associated GC and EBV-infected individual [287][288][289]. SWI/SNF remodelling complex which is commonly observed in GC is found associated with both pathogens, EBV and H. pylori [290]. A recent study suggested that host protein SHP 1 interacts with H. pylori CagA protein and dephosphorylates CagA, thus preventing oncogenic activity of CagA. However, EBV coinfection causes methylation of host SHP 1 and prevents its dephosphorylation activity of CagA and thus may increase oncogenic potential of CagA [199]. Further, a study suggested that both EBV and H. pylori coinfection were ominously more dominant in intestinal ulcer patients compared to GERD and dyspepsia patients [291]. H. pylori positive patients show increased anti-EBV IgG titre which suggests H. pylori role in augmenting EBV DNA load and higher immune responses [291]. However, some study is also available which suggested that H. pylori attenuated TGF-expression which reactivates EBV lytic phase and might play a role in preventing EBV lytic reactivation and preventing GC [292]. Therefore, the mechanism of coexistence for H. pylori and EBV must be studied to find the probable and potential pathogenic roles for both pathogens.

Future Direction
To date, mostly clinical findings explicitly described the EBV and H. pylori coinfection in GC. Moreover, how these pathogens target host factors and downstream pathways is still unexplored. Therefore, a detailed study which could potentially uncover the mechanism of EBV and H. pylori in the progression of GC could be interesting to peruse. How H. pylori antigens interacted with EBV antigens could be interesting to explore and helps in the understanding of progression of aggressive GC. Why only few cells from host are targeted by H. pylori and EBV is also critical to understand.