EBER

Synonyms: Epstein-Barr encoded RNA

by Jan Klos

Background

Epstein-Barr virus (EBV) is a member of the herpes virus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. It is named after Anthony Epstein and Yvonne Barr, who together with Bert Achong, discovered the virus in 1964 in cells cultured from the tumor specimens sent to them by Denis Burkitt from cases of Burkitt lymphoma.
Many EBV infections in especially in children are almost asymptomatic. EBV infected adolescents develop infectious mononucleosis in 35% to 70% of cases. The symptoms of infectious mononucleosis usually resolve in 1 – 2 months but the EBV remains dormant or latent in a few epithelial cells in the throat and B-lymphocytes for the rest of the person’s life. Periodically, the virus can reactivate and is commonly found in the saliva of infected persons. This reactivation usually occurs without symptoms of illness. The virus can have many distinct programs of gene expression which can be broadly categorized as being lytic or latent cycles. The lytic cycle or productive infection results in staged expression of several viral proteins with the ultimate objective of producing infectious virions. Formally, this phase of infection does not inevitably lead to lysis of the host cells as EBV virions are produced by budding from the infected cell. Lytic proteins include gp350 and gp110. A very limited, distinct set of viral proteins is produced during latent cycle infection. These include Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP) and latent membrane proteins (LMP)-1, LMP-2A and LMP-2B and the Epstein-Barr encoded RNAs (EBERs). In addition, EBV codes for at least twenty microRNAs expressed in latently infected cells.
During primary infection, EBV replicates in oropharyngeal epithelial cells and establishes Latency I, II, and III infections in B-lymphocytes. EBER1&2 LMP2A EBNA1 (Latency I), EBER1&2 LMP2A LMP2B EBNA1 LMP1 (Latency II) and EBER1&2 LMP2A LMP2B EBNA1 LMP1 EBNA2,3,4,5,6 (Latency III) EBV Latency III and II infections of B-lymphocytes, Latency II infection of oral epithelial cells and NK- or T-cells can result in malignancies showing uniform presence of EBV genome and gene expression.

Staining pattern is nuclear

EBER staining is based on in situ hybridization. It uses fluorescein (FITC) labeled Peptide Nucleic Acid (PNA) probes against EBER. After hybridization follows incubation with alkaline phosphatase labeled anti-FITC antibody. As chromogens are used 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro-blue tetrazolium (NBT) giving dark blue/black staining. The evaluation of staining should include 3 slides:
Negative control: Hybridization with non-specific PNA/FITC – should show no staining.
Positive control: Hybridization with PNA/FITC against RNA of glyceraldehyde 3-phosphate dehydrogenase giving cytoplasmic dark blue/black staining only.
Tested section: Positive staining is nuclear dark blue/black in the cells of interest. Remember that single positive cells in tested section in the absence of staining in the cells of interest may indicate reactivated latent infection in B-cells in EBV-carriers.

Staining in tumors

EBV is important in pathogenesis of 1% all tumors worldwide and it is usually found in the following tumors.

  • B-cell tumors: Burkitt’s lymphoma ((90% endemic and 30% sporadic), EBV+ DLBCL of elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis (90%), plasmablastic lymphoma (70%), primary effusion lymphoma (90%),
  • Classic Hodgkin lymphoma 20-70% (<20% of positive cases with nodular sclerosis pattern)
  • NK/T-cells: EBV+ T-cell proliferative disorders of childhood, extranodal NK/T-cell lymphoma of nasal type.
  • Angioimmunoblastic T-cell lymphoma contains usually proliferating EBV+ B-immunoblasts and a few EBV+ T-cells.
  • HIV associated lymphomas are often EBV+.
  • Lymphoproliferative disorders related to primary immunodeficiency are often EBV+.
  • Post-transplant lymphoproliferative (PTLD) disorders have association with EBV infection in 70%.
  • Lymphoepithelial carcinoma especially of head & neck origin – conflicting results from tumors with similar morphology are reported from lung, ventricle and pancreas.
  • Inflammatory pseudotumor-like variant of follicular dendritic cell sarcoma.
  • Hairy leukoplakia (EBV driven proliferation of mucosal squamous epithelium in HIV+ individuals).

Application

  • Subtyping of high grade malignant lymphomas
  • Differential diagnosis of classic Hodgkin lymphoma vs. NLPHL.
  • Immunoproliferative disorders including EBV+ mucocutaneous ulcer and Epstein-Barr virus associated smooth muscle tumor (EBV-SMT).
  • Confirming diagnosis of lymphoepithelial carcinoma in primary and metastatic setting
  • Supporting diagnosis of inflammatory myofibroblastic tumor (EBV+ in 20% using LMP but positive in 50-60% using EBER).
  • Infectious mononucleosis.
  • Differential diagnosis of PTLD vs. rejection of allograft.

Selected references

  1. Delecluse HJ, Feederle R, O’Sullivan B, et al.  Epstein Barr virus-associated tumours: an update for the attention of the working pathologist. J Clin Pathol. 2007 Dec;60(12):1358-64. Epub 2007 Sep 14
  2. Dojcinov SD, Fend F, Quintanilla-Martinez L. EBV-Positive Lymphoproliferations of B- T- and NK-Cell Derivation in  Non-Immunocompromised Hosts. Pathogens. 2018 Mar 7;7(1). pii: E28. doi: 10.3390/pathogens7010028.
  3. http://en.wikipedia.org/wiki/Epstein-Barr_virus 
  4. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues 2008