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low PD-L1 expression do show beneficial responses. This discrepancy could be attributed in part to the fact
that direct determination of PD-L1 expression in the tumor, which is typically performed by IHC assays,
has been proven to be difficult and inconsistent [24,25] . Understanding the molecular mechanisms that control
PD-L1 expression in cancer may give rise to more accurate biomarkers for positive response to PD-1/PD-L1
therapy. In addition, regulators of PD-L1 expression could serve as targets for potential combination
therapies.

[6]
In a phase II clinical trial, Kim et al. identified EBV-positivity and high mutational load as reliable and
independent biomarkers for the clinical efficacy of pembrolizumab in GC patients and recommended
considering pembrolizumab as an early therapeutic option for EBVaGC. MSI-high (MSI-H) tumors are
characterized by high rates of somatic mutations, resulting in increased presentation of neoantigens and
thus stimulation of anti-tumor immunity. As is the case in EBV-positive GCs, MSI-H tumors have high
[26]
levels of tumor-infiltrating lymphocytes (TILs) . The FDA has already approved the use of front-line
pembrolizumab monotherapy in advanced MSI-H solid tumors of any origin, including stomach .
[27]
EBVaGC appears to employ some common and some unique mechanisms for PD-L1 regulation. This
review presents the most recent findings on PD-L1 regulation in EBVaGC and discusses some of the
discrepancies in the literature, parallels with other EBV-associated cancers, and questions to be addressed
in the future [Figure 1].

EVOLUTIONARY PRESSURE FOR PD-L1 OVEREXPRESSION
EBVaGC shows high levels of TILs and thus is under strong evolutionary pressure for the development of
[4]
immune evasion strategies . One such strategy is PD-L1 overexpression, which is evident in both cancer
and stromal immune cells. It is speculated that the high immune activity in the EBVaGC microenvironment
reflects the strong immunogenicity of EBV in the body. Up to 5% of the circulating CD8 T cells in EBV-
+
[28]
infected individuals are believed to be reactive to lytic or latent EBV antigens . Indeed, several groups
have detected expression of a subset of lytic genes in EBVaGC [29-31] . According to Borozan et al. , the
[29]
lytic expression pattern detected in EBVaGC, which includes subsets of both early and late lytic proteins,
does not indicate lytic, or abortive lytic, replication. However, the presentation of lytic viral antigens by
[32]
infected cancer cells might be driving further immune activation. Camargo et al. detected higher levels
of circulating antibodies targeting both latent and lytic proteins in patients with EBV-positive compared
to EBV-negative GCs, even though virtually all patients in the study were seropositive for antibodies
against EBNA and the viral capsid. This further suggests that lytic proteins are expressed in EBVaGC and
potentially contribute to the activation of the host immune response.

SOMATIC STRUCTURAL VARIATIONS
Somatic genomic alterations include short variations, such as single nucleotide substitutions and short
insertions/deletions (indels), as well as long variations, also known as structural variations (SVs) .
[33]
SVs affect large chromosomal regions (longer than 50 base-pairs) and include amplifications, deletions,
[33]
inversions, and translocations . Somatic changes occur continuously in the life of an individual and
[33]
are usually repaired, but their accumulation over time can contribute to carcinogenesis . EBVaGC only
appears in a small percentage of EBV-infected individuals and typically long after primary EBV infection
[34]
suggesting that somatic genomic changes are likely required for cancer development .
Gene amplification
Recent high-throughput sequencing studies have revealed different genomic alterations that lead to PD-L1
overexpression in EBVaGC. TCGA performed a somatic copy-number aberrations analysis in gastric
adenocarcinoma samples and identified a frequent (15% of EBVaGC cases) somatic focal amplification at
[4]
the chromosomal region 9p24.1, which includes the genes PD-L1, PD-L2, and JAK2 . This amplification