Identification of Epstein-Barr virus (EBV)-associated gastric cancer at RNA-level by evaluating transcriptional status of seven EBV crucial genes

EBV has been an emerging biomarker to inform clinical management of GC, according to the potential mechanisms of EBV infection on GC immune check point blockade (ICB) efficacy. The gold standard for detecting EBV has long been EBV-encoded RNA (EBER) in situ hybridization (ISH). However, with the increasing demand for multi-molecular biomarkers detection recommended by standard treatment guidelines for GC, a single EBV detection has been challenged in clinical practice with limited samples. Herein, an NGS-panel based on RNA for detecting EBV was established and validated in the study. Seven genes including, EBER1, EBER2, and EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 were involved in the NGS panel to evaluate EBV infection status. 42 tumor tissue samples from GC, with half EBV positive and half EBV negative evaluated by ISH were collected, as well as their paired normal tissue sample, for NGS detection. The seven genes expression profile in the enrolled samples were comprehensively analyzed. The raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA sequencing in normal tissue samples were detected as zero. Only 12 normal tissue samples were detected EBER1and/or EBER2, of which the raw read counts did not exceed 10. In ISH-EBV negative tumor samples, raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA were also zero. And, expression signals were detected for EBER1 in 7 samples and EBER2 in 3 samples, and the raw RNA counts did not exceed 17 and 5, respectively. However, in ISH-EBV positive tumor samples, the average number of RNA raw read counts of EBER1 was 7424, with the minimum value was 458; the average number of RNA raw read counts of EBER2 was 3730, with the minimum value was 137. Besides, RNA expression signals of three gene, EBNA1, LMP2A/B, and BARF1, were also been detected with lower raw read counts. Due to the significantly high expression characteristics of EBER1/EBER2 in EBV infected positive samples, the detection of EBV based on RNA by NGS panel is feasible, which provides a basis for the subsequent design of NGS panel including EBV and other molecular biomarkers for GC.