Investigating EBV biology with Flow‐FISH: Implications for EBV‐mediated malignancies and the treatment thereof

Epstein Barr virus (EBV) is a gamma herpesvirus that possesses oncogenic properties. It most commonly infects epithelial cells and B lymphocytes. After cellular infection, EBV forms a persistent, asymptomatic, latent infection cycle in immune competent individuals. However, in weakened individuals, EBV is associated with pathologies that can be life threatening [1]. As a disease-causative agent, EBV is most commonly associated with infectious mononucleosis, but is also implicated to play a large role in a range of cancers. Estimates indicate that EBV is involved in at least 1.5% of cancers worldwide [2]. Furthermore, already long suspected, recently, a longitudinal study in a cohort of more than 10 million subjects showed that risk of multiple sclerosis increased 32-fold after infection with EBV [3]. Therefore, understanding biology of the virus itself and the cells harboring EBV is of extreme importance in order to unravel the role it plays in oncogenesis and (auto-)immune disorders. Several techniques have been employed to study EBV biology [4]. These techniques are often focused on the detection of EBV genetic material via, for example, PCR, or viral proteins, via immunoblotting or ELISA. However, these techniques rely on isolated viral material, and often do not provide any information regarding the (cellular) source. To address this question, single cell approaches are more suitable. One technique that combines detection of genetic material and protein is flow cytometry-based in situ hybridization, or Flow-FISH. This technique is mainly employed to study telomere length [5], T cell biology [6], and HIV infection [7], but has also been used in the context of other micro-organisms, such as EBV [8]. This type of assay is permissive for the use of fluorescently-labeled antibodies [9], and could thus be an important tool in the EBV toolbox. The first use of EBV Flow-FISH has been in cell-lines, where the Flow-FISH assay was used to study gene expression. Assay sensitivity was determined to be sufficient to detect as little as 0.01% EBV cells (equal to approximately 1 in 10.000) [10]. Furthermore, the assay was shown to correlate with expected input, with EBV Flow-FISH results matching percentages of EBV spiked cell input between 0.01 and 10% [10]. In contrast to the idea that cell-lines are semi-synchronous, EBV Flow-FISH was used to exemplify that EBV expression varies on a cell-to-cell basis in EBVinfected cell lines [11]. Together, this underlines the sensitivity and usability of the EBV Flow-FISH assay compared to conventional (diagnostic) techniques. EBV Flow-FISH was also used to study samples from patients with EBV-related lymphoproliferative disorders. Compared to healthy volunteers, EBV cells were observed only in patients with lymphoproliferative diseases, ranging from 1.29 to 25.9 EBV peripheral blood mononuclear cells [10]. Due to the flow cytometric nature of Flow-FISH and the compatibility with fluorescently-labeled antibodies [9], authors were able to further characterize the EBV-infected cells. Three patients diagnosed with hydroa vacciniforme, a form of EBV-related lymphoproliferative disease that affects NK cells and T cells, were included in the study. Indeed, EBV Flow-FISH analysis showed that the majority of EBV lymphocytes were either CD3 (range: 82%–100% of cells) and/or CD56 (range: 20%–24% of cells). Most EBV cells were CD4 /CD8 (range: 68%–92% of cells), and were identified to be mostly TCRγδ T cells (range: 32%–81%). This was confirmed by CD3, TCRαβ, TCRγδ, CD19, and/or CD3 CD56 magnetic cell sorting and subsequent conventional PCRbased EBV DNA detection. In contrast, for a patient with EBV-related post-transplantation lymphoproliferative disease, 61% of EBV cells expressed CD19, confirming involvement of the B cell compartment. Also here the results were verified through conventional viral load determination by PCR after magnetic cell sorting for CD3, CD19, and CD3 CD56 cell populations, respectively. Similar results were found in a follow up study, where 7 out of 8 hydroa vacciniforme-like lymphoma patients exhibited a CD3 phenotype, which were further identified as γδT cells or NKT cells [12]. Furthermore, in this study, patients with other malignancies were analyzed as well, such as aggressive NK cell leukemia, extranodal NK/T cell lymphoma, and peripheral T cell lymphoma. However, in these studies, the direct advantage of flow cytometry over conventional diagnostic methods was not fully exploited, as authors only used three types of fluorophores at the same time, limiting the detection of cell populations to two markers at a time [10, 12]. Subsequent research did exploit this feature. In two subsequent studies [13, 14], a more diverse cell phenotyping was employed in combination with EBV Flow-FISH. In one of these studies, Flow-FISH was employed as a tool for rapid identification of EBV-infected cells and to aid in subsequent characterization. CD19 B cells were shown to be the main cell type expressing EBV DNA in patients with B cell lymphoproliferative disease, severe infectious mononucleosis and several patients with immunodeficiencies, while the majority of EBVexpressing cells were CD3 cells in patients with NK or T cell lymphoproliferative disorders [13]. Lastly, results of the EBV Flow-FISH assay correlated with EBV load and clinical phenotype. Summarizing, in this study, the EBV Flow-FISH assay aided in diagnosis of 22 patients with EBV-related pathologies, providing simultaneous information regarding the amount of EBV-infected cells and the type. This allowed for Received: 8 June 2022 Revised: 22 July 2022 Accepted: 29 July 2022