Epstein–Barr virus infection in cancer

EBV was the first human oncogenic virus identified. It was discovered in tumour cells isolated from Burkitt’s lymphoma tissue by Sir Anthony Epstein and Dr Yvonne Barr in 1964. Some years after its discovery, EBV was shown to be able to transform normal leukocytes into lymphoblastoid cell lines (LCLs). Since then, EBV has been found to be associated with many malignancies originating from lymphocytes or epithelial cells (Burkitt’s lymphoma, post-transplant and HIV-associated lymphomas, Hodgkin’s lymphoma, T-cell lymphoma, extra-nodal nasal-type natural killer/T-cell lymphoma, nasopharyngeal cancer, and a subset of gastric cancers), contributing to 1.5% of all cancer cases worldwide and approximately 200,000 new cases every year. However, this virus is found in more than 90% of healthy adults worldwide, indicating that EBV infection alone is not enough to cause cancer. The specific geographical distribution of some EBV-associated malignancies (such as Burkitt’s lymphoma in equatorial Africa and nasopharyngeal cancer in East Asia) indicates that the development of an EBV-associated neoplasm requires different environmental and genetic co-factors, of which only some are currently known. In this Special Issue, we present a collection of 26 papers (9 research papers and 17 reviews) covering a range of topics related to EBV infection in cancer patients. These fall into three general areas: (1) EBV-encoded genes; (2) EBV and immune responses; and (3) EBV-associated malignancies and EBV-targeted therapies. In this Special Issue, we aim to further elucidate the role of EBV infection in EBV-driven malignancies by reviewing the literature and reporting new findings addressing some of the unanswered questions in the field of EBV</p

MicroRNA and other non-coding RNAs in epstein–barr virus-associated cancers

EBV is a direct causative agent in around 1.5% of all cancers. The oncogenic properties of EBV are related to its ability to activate processes needed for cellular proliferation, survival, migration, and immune evasion. The EBV latency program is required for the immortalization of infected B cells and involves the expression of non-coding RNAs (ncRNAs), including viral microRNAs. These ncRNAs have different functions that contribute to virus persistence in the asymptomatic host and to the development of EBV-associated cancers. In this review, we discuss the function and potential clinical utility of EBV microRNAs and other ncRNAs in EBV-associated malignancies. This review is not intended to be comprehensive, but rather to provide examples of the importance of ncRNAs

Expression of hsa-miR-9* and E2F1 in primary DLBCL and DLBCL/BL Intermediate cases in comparison with <i>MYC</i> translocation-negative cases.

<p><b>(A) Expression of hsa-miR-9* in primary DLBCL and DLBCL/BL Intermediate cases in comparison with </b><b><i>MYC</i></b><b> translocation-negative cases.</b> DLBCL cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Pelengaris1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Leucci1" target="_blank">[10]</a>; DLBCL/BL Intermediate cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Baudino1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-ODonnell1" target="_blank">[16]</a>; BL cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Sun1" target="_blank">[17]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Lazzi1" target="_blank">[25]</a>. Relative expression of hsa-miR-9* was evaluated by qRT-PCR. All of DLBCL cases showed hsa-miR-9* over-expression, in comparison with <i>MYC</i> translocation-negative BL cases. Intermediate DLBCL/BL cases showed an heterogeneous expression of hsa-miR-9*.<b>(B) Expression of E2F1 in primary DLBCL and DLBCL/BL Intermediate cases in comparison with </b><b><i>MYC</i></b><b> translocation-BL negative cases.</b> DLBCL cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Pelengaris1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Leucci1" target="_blank">[10]</a>; DLBCL/BL Intermediate cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Baudino1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-ODonnell1" target="_blank">[16]</a>; BL cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Sun1" target="_blank">[17]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Lazzi1" target="_blank">[25]</a>. Relative expression of E2F1 was evaluated by qRT-PCR. E2F1 resulted less expressed in DLBCL cases, in comparison with <i>MYC</i> translocation-negative BL cases. Intermediate DLBCL/BL cases showed an heterogeneous expression of E2F1. Differences in gene expression were calculated using the ΔΔCt method. Results are representative of three different experiments. Error bars represent standard deviation between triplicates.</p

hsa-miR-9* and E2F1 expression in primary <i>MYC</i> translocation-positive and negative BL cases.

<p><b>(A) Expression of hsa-miR-9* in primary </b><b><i>MYC</i></b><b> translocation-positive and negative BL cases.</b> Reactive lymph nodes and germinal centre cells (C); BL cases positive for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Pelengaris1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Leucci1" target="_blank">[10]</a>; BL cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Baudino1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Swerdlow1" target="_blank">[19]</a>; Relative expression of hsa-miR-9* was evaluated by qRT-PCR. BL cases positive for <i>MYC</i> translocation showed hsa-miR-9* up-regulated, whereas it was down-regulated in <i>MYC</i> translocation-negative ones, except for one case. <b>(B) Expression of E2F1 in primary </b><b><i>MYC</i></b><b> translocation-positive and negative BL cases.</b> Reactive lymph nodes and germinal centre cells (C); BL cases positive for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Pelengaris1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Leucci1" target="_blank">[10]</a>; BL cases negative for <i>MYC</i> translocation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Baudino1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012960#pone.0012960-Swerdlow1" target="_blank">[19]</a>; Relative expression by qRT-PCR for E2F1 in primary cases showed an up-regulation of E2F1 mRNA expression only in BL cases negative for <i>MYC</i> translocation. In all of the other cases, E2F1 mRNA levels were lower or comparable to control. Differences in gene expression were calculated using the ΔΔCt method. Results are representative of three different experiments. Error bars represent standard deviation between triplicates.</p

Models of regulatory interactions involving c-Myc, E2F1 and miRNAs.

<p><b>(A) Regulatory network between c-Myc, E2F1 and miRNAs.</b> The interactions among c-Myc, E2F1 and miRNAs are shown. Bidirectional arrows indicate a transcriptional regulation. MiR-17-5p, miR-20a, miR-9, miR-9* and miR-34b are involved on c-Myc activation, on the other hand c-Myc itself regulates their expression. Further, miR-17–5p and miR-20a have been shown to inhibit E2F1 translation. E2F1 can induce miR-17-5p and miR-20a expression. <b>(B) Proposed model involving c-Myc, E2F1 and miRNAs in </b><b><i>MYC</i></b><b> translocation-negative BL.</b> MiR-34b and miR-9* down-regulation induces c-Myc expression directly or by E2F1 induction, respectively. c-Myc over-expression possibly determines up-regulation of miR-17-5p, miR-20a and miR-9.</p

Functional <i>in vitro</i> study on hsa-miR-9*.

<p><b>(A) Relative expression by qRT-PCR of hsa-mir-9* in LCL transfected with increasing amounts of a synthetic hsa-mir-9*</b>, compared to non-treated LCL and LCL transfected with a negative control (NC). The level of hsa-mir-9* increased in a dose-dependent manner. <b>(B) Relative expression by qRT-PCR of hsa-mir-9* in LCL transfected with an hsa-miR-9* inhibitor</b>, at the concentration of 50 nM, compared to non-treated LCL and LCL trasfected with a negative control (I NC). The level of hsa-mir-9* decreased significantly upon transfection of the inhibitor. <b>(C) Relative quantification of E2F1 and </b><b><i>MYC</i></b><b> transcripts after transfection of increasing amounts of a synthetic hsa-mir-9*.</b> A dose-dependent decrease of both genes is detected in the presence of synthetic hsa-mir-9*. <b>(D) Relative quantification of E2F1 and </b><b><i>MYC</i></b><b> transcripts after transfection of a hsa-mir-9* inhibitor.</b> Transfection of the hsa-mir-9* inhibitor is able to increase E2F1 and <i>MYC</i> expression, in comparison with the expression level of both proteins in non-treated LCL or cells transfected with a negative control (I NC). <b>(E) E2F1 and c-Myc protein levels in LCL transfected with higher concentration of synthetic hsa-mir-9*, hsa-mir-9* inhibitor and their negative controls, evaluated by western blotting.</b> Lane 1: non-treated LCL; lane 2: LCL transfected with NC, lane 3: LCL transfected with 50 nM synthetic hsa-mir-9*, lane 4: LCL transfected with I NC; lane 5: LCL transfected with 50 nM hsa-mir-9* inhibitor. Figure is representative of three different experiments. Numbers on top indicate densitometric analysis.</p

Clinical and pathological data of the different categories of analysed lymphoma.

<p>Clinical and pathological data of the different categories of analysed lymphoma.</p

Summary of miRNA analysis in the different categories of analysed lymphoma.

<p>The median fold changes has been used as an indicator of the expression level in each category of lymphomas. To group the median fold changes, cut-offs have been defined as low (median fold changes <0.5), medium (median fold changes between 0.5 and 1.5), and high (median fold changes >1.5) expression level.</p

Methylation analysis of hsa-mir-9-1 gene in primary tumors.

<p>Methylation density of hsa-mir-9-1 gene in endemic BL cases negative for <i>MYC</i> translocation (NT, Negative for translocation) and reactive lymph nodes (C1, C2) by bisulfite sequencing; Solid circles (•) and empty circles (○) indicate that CpG site is methylated or unmethylated, respectively. The BL cases negative for <i>MYC</i> translocation showed aberrant (cases 1–5) and partial (cases 6 and 7) methylation, in respect with the normal controls (C1 and C2). Cases NT1 and NT2 showed 21/23 CpG methylated, case NT3 22/23 CpGs methylated, cases NT3 and NT4 showed all of CpGs methylated (23/23), case NT5 showed 12/23 CpG methylated and case NT6 showed 9/23 CpG methylated. On the other hand, the normal controls (C1, C2) showed 2/23 and 1/23 CpGs methylated, respectively.</p

Epstein–Barr virus and the pathogenesis of diffuse large B-Cell lymphoma

Epstein–Barr virus (EBV), defined as a group I carcinogen by the World Health Organi?zation (WHO), is present in the tumour cells of patients with different forms of B-cell lymphoma,including Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorders,and, most recently, diffuse large B-cell lymphoma (DLBCL). Understanding how EBV contributes to the development of these different types of B-cell lymphoma has not only provided fundamental insights into the underlying mechanisms of viral oncogenesis, but has also highlighted potential new therapeutic opportunities. In this review, we describe the effects of EBV infection in normal B-cells and we address the germinal centre model of infection and how this can lead to lymphoma in some instances. We then explore the recent reclassification of EBV+ DLBCL as an established entity in the WHO fifth edition and ICC 2022 classifications, emphasising the unique nature of this entity. To that end, we also explore the unique genetic background of this entity and briefly discuss the potential role of the tumour microenvironment in lymphomagenesis and disease progression. Despite the recent progress in elucidating the mechanisms of this malignancy, much work remains to be done to improve patient stratification, treatment strategies, and outcomes.</p