EBV

  • Epstein-Barr Virus (EBV) or human herpesvirus 4 (HHV-4)
  • EBV is a double-stranded DNA virus that exists episomally (outside of host chromosomes) in infected cells, including gastric epithelial cells. This distinguishes EBV-associated gastric cancer (EBVaGC) as a unique molecular subtype.1

Biology

  • EBV infects gastric epithelial cells and expresses latent genes (eg, EBNA1, LMP2A) and non-coding RNAs (eg, Epstein-Barr virus-encoded RNAs [EBERs]), contributing to promoter hypermethylation, immune evasion, and oncogenesis.1,2
  • EBVaGC commonly harbors PIK3CA mutations (≈ 80%), marked hypermethylation, and a pronounced immune infiltrate.1-3
  • These tumors show high PD-L1 expression and CD8⁺ T-cell infiltration, reflecting a highly immunogenic tumor microenvironment.1,3

Etiology & Epidemiology

  • EBVaGC represents approximately 8% to 10% of gastric cancer cases globally, making GC the largest group of EBV-associated malignancies.1,4
  • EBVaGC demonstrates a male predominance, with tumors most often located in the proximal stomach, including the body, fundus, and cardia.1,2,5
  • Meta-analysis data suggest EBV infection is associated with an approximate 18-fold increased risk of developing gastric cancer.1,5
  • Compared to patients with EBV-negative gastric cancers, those with EBVaGCs exhibit improved prognosis,  lengthened median survival, increased tumor-infiltrating lymphocytes, enhanced promoter hypermethylation, and elevated expression of major histocompatibility complex class I and II molecules.1

Testing

When to Test:

  • EBV status of a gastric tumor is an emerging biomarker for personalized treatment strategies; however, routine testing is not currently recommended due to limited prospective data.5
  • EBV testing is advised when tumor morphology exhibits prominent lymphoid stroma.5

Available Testing Methods:

  • EBER in situ hybridization (ISH) remains the gold standard for detecting EBVaGC, given its high sensitivity and specificity in formalin-fixed, paraffin-embedded tissue.6
  • LMP1 immunohistochemistry testing may be used as a preliminary screen for EBVaGC; due to its limited sensitivity and specificity, however, it is not a reliable stand-alone test.7
  • Comprehensive molecular profiling—such as whole-exome and RNA sequencing—has demonstrated strong concordance with EBER ISH results and can reliably identify the EBV-positive molecular subtype. These approaches are particularly valuable in research settings or when ISH is unavailable.8

Guideline Recommendations:

  • According the NCCN guidelines, routine EBV testing is not currently recommended in gastric cancer due to limited prospective data. However, testing should be performed when tumor morphology shows prominent lymphoid stroma, which may suggest EBVaGC.4
  • Universal microsatellite instability (MSI) or mismatch-repair (MMR) testing is recommended for all newly-diagnosed gastric cancer cases.4
  • PD-L1 testing may be considered in patients with locally advanced, recurrent, or metastatic disease who are candidates for PD-1 inhibitor therapy.4
  • Next-generation sequencing (NGS) may be performed when sufficient tumor tissue is available to assess such biomarkers as HER2, PD-L1, MSI, CLDN18.2, TMB, NTRK, RET, and BRAF V600E.4

Targeted Therapy

Approved Agents:

  • There are currently no FDA-approved therapies that specifically target EBV.
  • The immune-rich tumor microenvironment of EBVaGC supports the use of immune checkpoint inhibitors, including pembrolizumab and nivolumab.1,3
  • A DNA vaccine targeting EBVaGC and other EBV-associated malignancies is currently in preclinical development.1

Investigational Agents:

  • Pembrolizumab1,3,9,10
    • Early-phase studies in small EBVaGC cohorts demonstrated promising activity, including objective response rates approaching 100% with pembrolizumab in select cases.
    • Rationale:
      • Although EBVaGC is molecularly distinct from MSI-H tumors, it appears to respond similarly to immunotherapy, likely due to its immunogenic phenotype.
      • These tumors often exhibit high PD-L1 expression, which facilitates immune evasion and correlates with response to PD-1/PD-L1 blockade.
  • BARF1-targeting cancer vaccine1,11
    • A DNA vaccine targeting the EBV-encoded BARF1 antigen, which is highly expressed in EBVaGC, is currently in preclinical development.
    • Rationale:
      • BARF1 plays a role in promoting tumor cell proliferation and inhibiting apoptosis, highlighting its potential as a therapeutic target.
      • Preclinical studies of a synthetic DNA vaccine targeting the EBV antigen BARF1 (pBARF1) demonstrated that immunization elicits strong antigen-specific humoral and CD8⁺ T-cell responses.
      • In BARF1-positive carcinoma models, pBARF1 vaccination significantly inhibited tumor progression, with rapid tumor cell clearance observed via in vivo imaging.
      • These findings suggest that BARF1-targeted immunotherapy may induce effective antitumor immunity in EBV-associated cancers; this warrants further investigation.

Investigational Studies:

  • Phase 2 Study10
    • ClinicalTrials.gov: NCT03257163
    • Study design: Preoperative pembrolizumab for MMR- deficient and EBV-positive gastric cancer followed by chemotherapy and chemoradiation with pembrolizumab

References

  1. Salnikov MY, MacNeil KM, Mymryk JS. The viral etiology of EBV-associated gastric cancers contributes to their unique pathology, clinical outcomes, treatment responses and immune landscape. Front Immunol. 2024;15:1358511. doi:10.3389/fimmu.2024.1358511
  2. Nishikawa J, Iizasa H, Yoshiyama H, et al. Clinical importance of Epstein-Barr virus-associated gastric cancer. Cancers (Basel). 2018;10(6):167. doi:10.3390/cancers10060167
  3. Iizasa H, Kartika AV, Fekadu S, et al. Development of Epstein-Barr virus-associated gastric cancer: Infection, inflammation, and oncogenesis. World J Gastroenterol. 2022;28(44):6249-6257. doi:10.3748/wjg.v28.i44.6249
  4. NCCN. Clinical Practice Guidelines in Oncology. Gastric cancer, version 2.2025. Accessed July 10, 2025. https://www.nccn.org/professionals/physician_gls/pdf/gastric.pdf
  5. Tavakoli A, Monavari SH, Mohammadi FS, Kiani SJ, Armat S, Farahmand M. Association between Epstein-Barr virus infection and gastric cancer: a systematic review and meta-analysis. BMC Cancer. 2020;20:493. doi:10.1186/s12885-020-07013-x
  6. Camargo MC, Kim WH, Chiaravalli AM, et al. Improved survival of gastric cancer with tumour Epstein-Barr virus positivity: an international pooled analysis. Gut. 2014;63(2):236-243. doi:10.1136/gutjnl-2013-304531
  7. Costache S, de Havilland R, McLynn SD, et al. Implementing an on-slide molecular classification of gastric cancer: a tissue microarray study. Cancers (Basel). 2023;16(1):55. doi:10.3390/cancers16010055
  8. Corallo S, Lasagna A, Filippi B, et al. Unlocking the potential: Epstein-Barr virus (EBV) in gastric cancer and future treatment prospects, a literature review. Pathogens. 2024;13(9):728. doi:10.3390/pathogens13090728
  9. Kim ST, Cristescu R, Bass AJ, et al. Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer. Nat Med. 2018;24:1449-1458. doi:10.1038/s41591-018-0101-z
  10. Pembrolizumab, capecitabine, and radiation therapy in treating patients with mismatch-repair deficient and Epstein-Barr virus positive gastric cancer. ClinicalTrials.gov. Updated April 16, 2025. Accessed July 10, 2025. https://clinicaltrials.gov/study/NCT03257163
  11. Zhu X, Perales-Puchalt A, Wojtak K, et al. DNA immunotherapy targeting BARF1 induces potent anti-tumor responses against Epstein-Barr-virus-associated carcinomas. Mol Ther Oncolytics. 2021;24:218-229. doi:10.1016/j.omto.2021.12.017