Dados do Resumo

Título

Analysis of viral expression of Epstein-Barr virus in patients with gastric adenocarcinoma in the state of Pará

Introdução

Epstein-Barr virus (EBV) is an oncogenic virus, linked to malignancies like gastric cancer. Notably, EBV infection induces epigenetic modifications that play a crucial role in oncogenesis and tumor progression, underscoring the importance of analyzing viral gene expression in the context of gastric adenocarcinoma to elucidate its unique characteristics. Investigating viral gene expression in tumor cells is necessary to assess which part of the viral cycle predominates in tumor samples and how this influences cancer development.

Objetivo

To analyze the expression of EBV genes in gastric cancer samples from patients at Joao de Barros Barreto University Hospital in Belém, Pará.

Métodos

The study received approval from the Ethics and Research Committee of João de Barros Barreto University Hospital under the reference number 47580121.9.0000.5634. It utilized 76 tumor tissue samples from patients with gastric cancer who had undergone surgical resection, and both fresh and paraffin-embedded samples were gathered for total RNA sequencing (RNA-seq) and in situ hybridization (ISH). The RNA-seq was conducted in a pair-end manner on the NextSeq® platform (Illumina®, US). The NextSeq® 500 MID Output V2 kit - 150 cycles (Illumina®) were utilized following the manufacturer's instructions. ISH targeting RNA-1 of EBER1 (Y5200, DAKO, Carpinteria) was performed using the automated Dako system. For EBV molecular classification from RNA-Seq data, reads were aligned with Kraken2 using the Reference Viral Database by Goodacre et al., 2018. To assess the expression of EBV viral genes, samples identified as positive according to both methods were selected and processed using the nf-core/rnaseq v3.14 pipeline. The EBV reference genome GCF_002402265.1 and genomic annotation NC_007605.1, from the NCBI database, were used as indices.

Resultados

Among the 76 tumor samples analyzed, 8 were positive for EBV based on ISH results and RNA-seq analysis. For the molecular identification of EBV-associated gastric cancer, samples were considered positive if the number of viral gene reads was ≥100. The most frequently expressed genes included RPMS1, A73, LMP-2B, EBER-1, and EBER-2, all of which were detected in all positive samples. Immediate-early genes related to the lytic phase, such as BRLF1 and BZLF1, were detected in 25% and 12.5% of samples, respectively. Viral genes of the LMP family were also present: LMP-1 was detected at low levels in 50% of positive samples, LMP-2A in 12.5%, and LMP-2B in 75%. Furthermore, expression of EBNA family genes was observed: EBNA 1.2, a variant of EBNA1, was found in 25% of samples, and EBNA-2 in 50%. Although EBNA-3 genes were generally not expressed, EBNA-3A was detected in 25% of samples, with high levels of expression in one sample, and EBNA-3B/EBNA-3C was found in 50%.

Conclusões

In summary, our findings confirm the expression of several EBV viral genes in positive samples. The expression of EBER-1 and EBER-2, as well as the presence of RPMS1, and A73, suggest a continuous contribution to the maintenance of carcinogenesis. The identification of lytic phase genes, such as BRLF1 and BZLF1, in some samples, along with the manifestation of LMPs and EBNAs, highlights the complexity of EBV latent infection and the diversity of its gene expression patterns in gastric cancer.

Palavras Chave

Epstein-Barr Virus; Gastric Adenocarcinoma; RNA-seq