EBV-encoded mRNA Research Articles

Epstein-Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. SIGNIFICANCE: This study suggests that Epstein-Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.

Comprehensive Genomic Profiling of EBV-Positive Diffuse Large B-cell Lymphoma and the Expression and Clinicopathological Correlations of Some Related Genes.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV+ DLBCL) is a rare type of lymphoma with a high incidence in elderly patients, poor drug response, and unfavorable prognosis. Despite advances in genomic profiling and precision medicine in DLBCL, EBV+ DLBCL remain poorly characterized and understood. We include 236 DLBCL patients for EBV-encoded mRNA (EBER) in situ hybridization detection and analyzed 9 EBV+ and 6 EBV negative cases by next-generation sequencing (NGS). We then performed fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to analyze chromosome rearrangements and gene expressions in 22 EBV+ and 30 EBV negative cases. The EBER results showed a 9.3% (22/236) positive rate. The NGS results revealed recurrent alterations in MYC and RHOA, components of apoptosis and NF-κB pathways. The most frequently mutated genes in EBV+ DLBCL were MYC (3/9; 33.3%), RHOA (3/9; 33.3%), PIM1 (2/9; 22.2%), MEF2B (2/9; 22.2%), MYD88 (2/9; 22.2%), and CD79B (2/9; 22.2%) compared with KMT2D (4/6; 66.7%), CREBBP (3/6; 50.0%), PIM1 (2/6; 33.3%), TNFAIP3 (2/6; 33.3%), and BCL2 (2/6; 33.3%) in EBV-negative DLBCL. MYC and KMT2D alterations stood out the most differently mutated genes between the two groups. FISH detection displayed a lower rearrangement rate in EBV+ cohort. Furthermore, KMT2D expression was highly expressed and associated with poor survival in both cohorts. MYC was only overexpressed and related to an inferior prognosis in the EBV+ DLBCL cohort. In summary, we depicted a distinct mutation profile for EBV+ and EBV-negative DLBCL and validated the differential expression of KMT2D and MYC with potential prognostic influence, thereby providing new perspectives into the pathogenesis and precision medicine of DLBCL.

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.

In situ hybridisation for Epstein-Barr virus as a differential diagnostic tool for T- and natural killer/T-cell lymphomas in non-immunocompromised patients

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus infecting over 90% of humans and the infection persists for life. Although most people are asymptomatic, EBV infection may cause a continuous range of symptoms from transient to severe and protracted diseases depending on the immunological response of the individuals. EBV infects primarily B lymphocytes and rarely T and natural killer (NK) cells. It is implicated in around 1% of human tumours with the majority being haematological malignancies including Hodgkin lymphoma, B- and T-cell non-Hodgkin lymphomas, and immunodeficiency-associated lymphoproliferative disorders (LPDs). As it is a ubiquitous virus the confirmation of EBV-related LPDs depends on the demonstration of the viral DNA by in situ hybridisation for EBV-encoded mRNA (EBER). In current practice, CD3 and EBER positive cytotoxic extranodal lymphomas in non-immunocompromised patients are generally considered as extranodal NK/T-cell lymphoma and accordingly EBER should be performed for such tumours except for the few clinically typical entities such as mycosis fungoides. This review focuses on the application of EBER in the diagnosis of various types of T- and NK/T-cell lymphomas in non-immunocompromised patients and the diagnostic pitfalls, especially their distinction from infectious mononucleosis-related LPD of T- and NK-cell origins and the diagnostic dilemma between various T-cell lymphoma entities with or without EBV association, including nodal cytotoxic EBV positive peripheral T-cell lymphoma.

Epstein-Barr virus-associated gastric carcinoma in the remnant stomach: de novo and metachronous gastric remnant carcinoma

The remnant stomach corresponds to the gastric cardia, but is exposed to a completely different environment. The present study was performed to investigate the role of Epstein-Barr virus (EBV) infection in patients with gastric remnant carcinoma (GRC). Clinicopathological features, gastritis, and infection by EBV were investigated in patients with two types of GRC: GRC occurring at an interval of 10 years or longer between operations (de novo GRC group) and GRC occurring within 10 years after the initial operation for gastric carcinoma (metachronous GRC group). EBV involvement in the de novo GRC group (23%) was not significantly different from that in the cardia of non-remnant carcinomas (controls; 18%). EBV involvement showed greater correlations in male patients (18/63; 28%), and in those with gastritis cystica polyposa (GCP; 13/41; 31%), and those with an interval of 20 years or longer (15/50; 30%) than with the other parameters. Multivariate analysis showed a significant correlation between GCP and EBV infection. Histologically, hyperplasia or mild atrophy, and mild lymphocytic infiltration were observed in 56% and 67% of non-neoplastic mucosa of EBV-associated GRC, respectively. In the metachronous GRC group, EBV-encoded mRNA in situ hybridization (EBER-ISH) of 27 pairs of primary gastric carcinomas (GCs) and metachronous GRCs indicated that only six EBV (+) metachronous GRCs were derived from EBV (+) GC. Epstein-Barr virus infection, together with long-standing inflammation, which causes GCP, may facilitate the development of de novo GRC. Close follow-up of patients treated with distal gastrectomy for EBV-associated GC is necessary to detect metachronous GRC.

A 70-Year-Old Man With Multiple Intra-abdominal Masses and Liver and Spleen Metastases

The biopsy core was 1.2 cm in length. Microscopically, spindle cells were arranged in short intersecting fascicles and a vague storiform pattern with some lymphocytic infiltrate (Figure 2, a). These spindle cells had oval and vesicular nuclei, inconspicuous nucleoli, and eosinophilic cytoplasm with poorly defined cell borders (Figure 2, b). There was no significant cellular atypia, mitotic activity, or necrosis. Immunohistochemically, tumor cells expressed CD21 (Figure 2, c), CD35 (Figure 2, d), and epithelial membrane antigen but not CD1a, CD3, CD20, CD23, CD68, smooth muscle actin, desmin, ALK-1, S100 protein, and Epstein-Barr virus (EBV) latent membrane protein 1. The proliferation fraction as determined by Ki67 was 10%. These tumor cells and the small lymphocytes were both negative for EBV-encoded mRNA as indicated by in situ hybridization. These small lymphocytes were mainly CD3-positive T cells with a proliferation fraction at 10%, and the majority expressed CD8, T-cell intracytoplasmic antigen, and granzyme B. What is your diagnosis?

Epstein–Barr virus-related plasmablastic lymphomas arising from long-standing sacrococcygeal cysts in immunosuppressed patients

Epstein–Barr virus-related plasmablastic lymphomas arising from long-standing sacrococcygeal cysts in immunosuppressed patients

Subcutaneous panniculitis-like T-cell lymphoma: a clinicopathological, immunophenotypic and molecular analysis of six patients.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic T-cell lymphoma of the skin. In the World Health Organization classification of T-cell and natural killer cell lymphoma it is listed as an example of extranodal lymphoma. In practice, however, it is most likely to present to a dermatologist. To describe the clinicopathological, immunophenotypic and molecular features of six U.K. patients with SPTCL. The clinical, histological and immunophenotypic features were reviewed. T-cell receptor (TCR) gene analysis was performed on blood and tissue samples using polymerase chain reaction/single-strand conformational polymorphism analysis of the TCR-gamma gene using consensus primers. In situ hybridization was performed on lesional skin to detect mRNA for Epstein-Barr virus (EBV). All patients presented with subcutaneous nodules, plaques or ulceration, and three had systemic symptoms. All biopsies exhibited an infiltrate of medium to large pleomorphic cells involving the subcutis with characteristic rimming of fat spaces. Five showed areas of necrosis, but only one showed marked cytophagia. In three cases the neoplastic cells did not express TCR-beta. One was strongly p53 positive, and the other two were CD56 positive. Both these patients showed epidermal involvement with lichenoid changes histologically, and both developed the haemophagocytic syndrome. The other three cases were TCR-beta positive, CD8 positive and CD56 negative. All cases were positive with pan T-cell markers and also for the cytotoxic granule protein T-cell intracellular antigen-1 and granzyme B. All cases were EBV negative both by immunostaining (latent membrane protein-1) and by in situ hybridization (EBV-encoded mRNA). TCR gene analysis revealed a T-cell clone in four of five cases; two of these patients had an identical T-cell clone in the peripheral blood. The median survival was 16 months. However, two of the three TCR-beta-negative patients have died, whereas none of the TCR-beta-positive patients has died. This is the first series of SPTCL patients to be reported in the U.K. and the data support the view that there are two subsets of SPTCL: those derived from gammadelta T cells which carry a poor prognosis, and are usually CD56 positive, and a more indolent group derived from alphabeta T cells.