Sex-dependent different clinicopathological characterization of Epstein–Barr virus-associated gastric carcinoma: a large-scale study

Although aggressive endoscopic screening in South Korea has led to a dramatic increase in early detection and treatment success rates over the past few decades, gastric cancer (GC) remains one of the most prevalent cancers not only in South Korea but also throughout East Asia, and remains as a leading cause of death [1]. The Cancer Genome Atlas (TCGA) project, published in 2014, classified gastric adenocarcinoma into four subtypes through comprehensive molecular analysis: (1) Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC), (2) gastric carcinoma with microsatellite instability, (3) gastric carcinoma with chromosomal instability, and (4) genetically stable gastric carcinoma [2, 3].

The EBV is a double-stranded DNA virus belonging to the Herpes virus family. It proliferates in the epithelial cells of the oropharynx and is primarily transmitted through the saliva. Primary infections usually occur during childhood and often remain unnoticed without significant symptoms. Subsequently, a latent infection is observed in B lymphocytes. More than 90% of the adult population worldwide has been reported to display a positive serological response to the virus [4]. It was classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC) in 1997 due to its role in these cancers. [5, 6].

The diagnosis of EBVaGC using in situ hybridization (ISH) of the GC tissue is the gold standard [7]. Since Burke et al. first reported EBVaGC in 1990 [8], several studies have been conducted on this topic. Recent reports state that EBVaGC is the most common EBV-associated malignancy, with an estimated 75,000 to 90,000 cases occurring worldwide annually. It constitutes approximately 10% of all GCs and is prevalent in Far East Asia, where the incidence of GC is high [9]. In South Korea, 5.6–13% of all GC cases are associated with EBV infection [10‐12]. EBVaGC exhibits features that differentiate it from typical GC. Several studies have demonstrated that EBVaGC is more common in males and tends to affect younger patients. However, certain meta-analyses failed to show the significance of age in EBVaGC [13‐17]. Moreover, smoking is a risk factor for EBVaGC, and compared to non-smokers, the incidence of EBVaGC is 2.4 times higher in current smokers and two-fold higher in former smokers [18]. Using the Lauren classification, certain studies have demonstrated that the intestinal type predominates, whereas other studies indicate a higher prevalence of the diffuse-type. Other studies have reported no correlation between EBV positivity and Lauren classification [14, 15, 19, 20]. In addition, according to the World Health Organization (WHO) histology, it has been frequently associated with the poorly differentiated type [11]. In terms of cancer location, EBVaGC is commonly found in the proximal part of the stomach [11, 14]. Certain studies have reported that it has little relationship with Helicobacter pylori [21]. Furthermore, EBVaGC has fewer lymph nodes and less vascular invasion, leading to a better prognosis [13, 16, 22‐25]. This could be related to the fact that EBVaGC is an immunogenic tumor leading to an active host cell immune response [26‐28].

GC has sex-specific characteristics, which are highlighted by a higher proportion of diffuse-type GC in females, whereas GC in males is primarily located in the antrum [29, 30]. Furthermore, certain studies suggested that the number of activated immune processes was higher in females in GC [31, 32]. Based on this background, we hypothesized that EBVaGC has unique characteristics compared to other types of GC, and that these characteristics could differ depending on the sex. However, most previous studies had sample sizes of fewer than a few hundred study groups, and meta-analyses have often revealed conflicting results. Therefore, we conducted this study to obtain a more detailed understanding of the characteristics and prognosis of EBVaGC and investigate the different effects of EBV positivity depending on the sex in large-scale prospective long-term follow-up study at a single institution.

In our study, the EBVaGC group showed a higher proportion of males (P < 0.001), with tumors predominantly in the proximal stomach (P < 0.001), a higher proportion of undifferentiated cancer (P < 0.001), and a lower cancer stage (P = 0.004) than the EBV-negative group. For lymphatic and vascular invasion, EBV-positive group had significantly fewer cases of lymphatic and vascular invasion (lymphatic; P < 0.001, vascular; P = 0.004). Furthermore, the EBVaGC group displayed statistically significant survival advantages over the EBV-negative cancer group in terms of both overall and GC-specific survival. However, this effect was evident only in males.

EBVaGC accounts for approximately 10% of all GCs, although there are differences in each study. For instance, Kim et al. have reported an incidence of 6.27% (21 of 335 individuals) of EBVaGC [12]. In Tokunaga's study, 67 cases (6.9%) of 970 individuals were EBV-positive [33]. In addition, Lee's meta-analyses revealed an incidence of 8.8%, with 857 of 9,738 individuals having EBVaGC [22]. In a study by van Beek [14], of 566 individuals, 41 (7.2%) were EBV positive, of which 38 (92.7%) were male [14]. In our study, 456 patients with GC (9.9%) among 4,587 GC patients were EBV positive and the number of males was 404 (88.6%), similar to that reported in van Beek’s study. In addition, van Beek reported that the undifferentiated type was significantly higher in EBVaGC (76.2%), and the tumor location predominantly appeared in the proximal region of the stomach in 82.9% of patients [14]. Consistent with this, our study displayed a predominant presence in the proximal stomach at 51.8% and a higher proportion of undifferentiated tumor type (49.5%). Despite these unfavorable conditions for the prognosis of GC, we found a negative correlation between EBV positivity and lymphovascular invasion in GC, as previously demonstrated by Lee et al. [22], and Park et al. [34], and others [35, 36]. In our study, EBV positivity in GC was an independent factor associated with favorable survival outcomes. EBVaGCs are characterized by dense lymphoid cell infiltration of the gastric stroma, leading to an active host cell immune response [8, 37, 38]. This robust immune activity triggers an inflammatory response causing the fusion of cancer cells, exhibiting a “lace pattern” [39]. These cancer cells lack tubule formation and are histologically classified as undifferentiated type for these reasons. Although undifferentiated types of cancer are commonly associated with poor prognosis in GC, the active host cell immune response serves as a protective factor in EBVaGC, such as the inhibition of lymph node metastasis or vascular invasion, thereby yielding characteristically favorable prognoses despite the undifferentiated histology [40]. To date, there have been no reports of sex differences in EBVaGC. This could be attributed to the higher prevalence of GC in males than in females worldwide, and no research group has performed a subgroup analysis of EBVaGC regarding sex. However, GC showed a sex-based difference. For instance, younger patients with GC are more likely to be females, have the diffuse- and undifferentiated types of GC, and present with AGC. In contrast, older patients with GC are more likely to be males, have intestinal-type GC, and present with simultaneous tumors [41]. Intestinal-type GC was significantly less frequent in premenopausal females (19.0%) and postmenopausal females aged < 10 years (30.4%) and 10 to 19 years old (44.1%) after menopause compared to males (61%) (all P value < 0.05) [42]. However, this significant difference in the proportion of intestinal-type GC was not observed between males and females ≥ 20 years after menopause (60.6 vs. 61%, P = 0.518) [42]. Changes in the proportions of intestinal and diffuse-type cancers suggest that estrogen could have a protective effect on intestinal-type GC. EBV was the first virus to be found in human tumors and has been implicated in several malignancies such as Burkitt's lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, most of which are intricately related to immune cells. Estrogen and testosterone have direct effects on immune cells. β-estradiol stimulates dendritic cells to secrete interleukin (IL)-12 and interferon (IFN)-γ, which in turn activate the secretion of proinflammatory cytokines. In addition, β-estradiol extends the survival of B lymphocytes, activates polyclonal B lymphocytes, increases intestinal permeability, and create a pro-inflammatory environment. In males, testosterone inhibits the proliferation of T lymphocytes and interferes with the Toll-like receptor (TLR) mechanism, which is different from the effect of β-estradiol effect on B lymphocytes. Our findings revealed that the EBV positivity was a favorable prognostic factor in males but not in females. Females generally show stronger immune functions not only in cancer, but also in viral and microbial infections than males. A greater number of differentially activated immune processes have been observed in GC in females [31, 32]. In addition, a study investigating tumor-associated neutrophils (TANs), a part of the tumor immune microenvironment (TIME), reported that an increase in the number of TANs correlated with a better prognosis only in females with GC [43]. Sex differences in the TIME had been observed in various types of cancer. Ye Y et al. summarized meta-analysis regarding melanoma and lung cancer, the most common types of cancers, in the largest number of immune checkpoint blockade (ICB) clinical trials [44]. There was a higher tumor mutation burden (TMB) and PD-L1 expression in male patients with melanoma than in females [44]. In contrast, female displayed higher activity of CD4 + and CD8 + cells than in males with lung squamous cell cancer [44]. These differences affected overall survival with ICB therapy in various cancers according to sex [44]. However to date, studies on sex differences about TIME has shown inconsistent results and there is lack of studies about EBV-related cancers such as Burkitt’s lymphoma, Hodgkin’s lymphoma, and nasopharyngeal cancer. Although the underlying mechanism is unclear so far, the reason why EBV positivity had a more pronounced impact on prognosis in males in our study could be that females already have an active immune response against GC, resulting in EBVaGC being 4.03 times higher in males than females. In addition, the immunogenicity of EBVaGC did not yield significant differences in prognosis compared with that of GC. These results could be useful for other EBV-associated tumors. Thus, further research is required to assess the sex differences in EBV-associated tumors including GC with respect to EBV infection.

In conclusion, patients with EBVaGC displayed better prognosis despite its association with proximal location and poorly differentiated histology only in males, suggesting that EBV infection causes GC using sex-specific immune mechanisms.