Probes for EBER1

Epstein-Barr virus (EBV), a common pathogen in humans, is suspected as the cause of multiple pregnancy-related pathologies including depression, preeclampsia, and stillbirth. Moreover, transmission of EBV through the placenta has been reported. However, the focus of EBV infection within the placenta has remained unknown to date. In this study, we proved the expression of latent EBV genes in the endometrial glandular epithelial cells of the placenta and investigated the cytological characteristics of these cells. Sixty-eight placentas were obtained from pregnant women. Tissue microarray was constructed. EBV latent genes including EBV-encoding RNA-1 (EBER1), Epstein-Barr virusnuclear antigen 1 (EBNA1), late membrane antigen (LMP1), and RPMS1 were detected with silver in situ hybridization and/or mRNA in situ hybridization. Nuclear features of EBV-positive cells in EBV-infected placenta were compared with those of EBV-negative cells via image analysis. Sixteen placentas (23.5%) showed positive expression of all 4 EBV latent genes; only the glandular epithelial cells of the decidua showed EBV gene expression. EBV infection status was not significantly correlated with maternal, fetal, or placental factors. The nuclei of EBV-positive cells were significantly larger, longer, and round-shaped than those of EBV-negative cells regardless of EBV-infection status of the placenta. For the first time, evidence of EBV gene expression has been shown in placental tissues. Furthermore, we have characterized its cytological features, allowing screening of EBV infection through microscopic examination.

Abstract

BACKGROUND:

Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma.

METHODS:

Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10).

RESULTS:

The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH.

CONCLUSION:

We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.

Epstein-Barr virus (EBV) is a herpesvirus associated with approximately 1% of tumors worldwide. Although EBV is consistently detected in nasopharyngeal carcinoma(NPC) biopsy, it is hardly detected in normal nasopharyngeal epithelium. The mechanism how virus establishes latent infection in tumor epithelial cells, including the source of virus and the route of entry, has not been fully elucidated largely due to the lack of appropriate in vivo models. We herein aim to investigate the potential route that epithelial cells are infected with EBV. To this end, we established in vivo model system by injection of cell-free EBV or EBV producer line Akata cells together with EBV negative NPC line HONE-1 cells. Akin to in vitro infections, we presented the first in vivo evidence that cell-mediated transfer infection via Akata cells was much more efficient than cell-free virus. These cells then expressed the EBV latency-associated small RNA EBERs, but not lytic antigens, such as BZLF1. However, when cells were inoculated at separate sites, EBV producer line Akata cell failed to demonstrate the ability of migrating from distant location to interact with HONE-1 cell to establish latent infection. In conclusion, cell-cell contact is critical for in vivo EBV infection of nasopharyngeal epithelial cells.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Abstract

BACKGROUND:

TP53 is frequently mutated across various tissue types of cancers. In normal cells, long interspersed nuclear element-1 (LINE-1, L1) is mostly repressed by DNA methylation in its 5' untranslated region but is activated by DNA demethylation process during tumorigenesis. p53 is indispensable for maintaining genomic stability and plays its role in controlling genomic stability by repressing retrotransposon activity. However, it is unclear whether p53 regulates expression or methylation of L1 differently depending on the mutational status of TP53. Four hundred ninety cases of advanced gastric cancer (AGC) were analyzed for their statuses in p53 expression and L1 methylation using immunohistochemistry and pyrosequencing, respectively. Whether L1 methylation and expression statuses were differently affected by types of TP53 mutants was analyzed in gastric cancer cell line.

RESULTS:

By p53 immunohistochemistry, tumors were classified into 4 groups according to the intensity and extent of stained tumor nuclei. L1 methylation level was significantly higher in p53 expression group 1 than in the other groups in which L1 methylation level was similar (P <  0.001). Although L1 methylation and p53 expression statuses were associated with patient survival, multivariate analysis revealed that L1 methylation was an independent prognostic parameter. In in vitro analysis of AGS cells with the introduction of wild type or mutant types of TP53, L1 methylation level and activity were different depending on types of TP53 mutation.

CONCLUSIONS:

Findings suggest that L1 methylation level is affected by TP53 mutation status; although, L1 methylation status was an independent prognostic parameter in patients with AGC. Further study is required to elucidate the mechanism of how wild type or mutant p53 affects L1 activity and methylation status of L1 CpG island.