11 23 2006 Oncology Ebv Phd

Epstein-Barr Virus Associated Malignancies

Historic Background of Epstein-Barr Virus 1. 1958: Denis Burkitt (an English surgeon) described an unique tumor (Burkitt’s lymphoma) of head and neck in Ugandan. 2. 1964: Epstein, Achong, and Barr identified an unique virus (Epstein-Barr virus) in Burkitt’s lymphoma. 3. 1968: Werner Henle and his wife Gertrude identified EBV as the cause of infectious mononucleosis (IM). 4. 1964-1975: a debate concerning the role of EBV in the induction of Burkitt’s lymphoma (BL). 5. EBV is a herpesvirus, ubiquitous and infects the vast majority of individuals by adulthood. Two hypotheses: 1. EBV is merely a passenger of the BL and not the primary etiologic agent. 2. Immunosuppression permitted EBV to assume an oncogenic role.

172 kb

Lat I: EBERs1/2, LMP2A; Qp promoter: EBNA1. Lat II: EBERs1/2, LMPs1,2A,2B; Qp promoter: EBNA1; Lat III: EBERs1/2, LMPs1,2A,2B; Cp or Wp promoter: EBNAs1,2, 3A/3, 3B/4, 3C/6, EBNA-LP/5.

EBV life cycle: 1. EBV virons in saliva  oropharynx  B lymphocytes  proliferation  spread through B cell compartment T cells (CD8+) respond to control B cell proliferation. 2. EBV infected B cells with limited antigen presentation persist at a frequency of 1in 1 x 105 -106 cells and long-term viral reservoir (in healthy host). 3.EBV-infected resting B cells will enter the lytic cycle an lyse, releasing virons into saliva while also infecting host B cells.

Primary EBV infection in healthy hosts is accompanied by an orchestrated serological response. IgM anti-VCA rises first. IgG anti-EBNA at least 1 month after primary infection, along with IgG anti-VCA, as markers of prior infection and as indicators of reactivation.

Table 1. Laboratory Tests for EBV Name

Purpose

In situ hybridization

Identify EBER transcripts or EBV DNA in specific cell types within histologic lesions

EBV clonality assay by Southern blot analysis

Assess clonality of lesions with respect to EBV DNA structure; distinguish latent from replicative infection based on the episomal versus linear structure of the EBV genome

EBV DNA amplification

Detect viral DNA in patient tissues; disease specificity is lacking

EBV viral load

Quantitate EBV DNA in blood or body fluids to monitor disease status over time

Immunohistochemistry (LMP1, EBNA1, EBNA2, LMP2A, BZLF1)

Identify EBV protein expression in specific cell types within histologic lesions; distinguish latent from replicative infection based on expression profiles

Culture of EBV or of EBV-infected B lymphocytes

Detect and semiquantitatively measure infectious virions or latently-infected B lymphocytes; impractical for routine clinical use

Electron microscopy

Identify whole virions representing replicative viral infection; impractical for routine clinical use

Serology (VCA, EBNA, EA, heterophile antibodies)

Measure antibody response to viral proteins in serum samples; distinguish acute from remote infection; monitor disease status over time

H&E:GCA

ISH:EBERs in dysplasia

ISH:EBERs in GCA

ISH:EBERs in single lymphocyte

EBERs +

LMP 2+

Detection of EBV gene products in Hodgkin disease. LMP1 +

EBERs in NPC

EBERs in GCA

Detection of viral gene expression in EBV-associated epithelial malignancies. LMP1 in NPC

The EBV viral load assay is accomplished by coamplificaiton of EBV DNA and a control sequence that is spiked into the sample before DNA extraction.

The EBV clonality assay evaluates clonality with respect to the structure of the EBV genome. The assay is based on the presence of variable number of tandem repeat sequence an the ends of the linear viral genome. Sample DNA digested with BamH1 and Southern blot analysis by hybridization with specific probe.

Table 2. EBV-Associated Diseases Disease

Proportion of cases EBV-related

Reference

Benign, reactive infections Infectious mononucleosis Oral hairy leukoplakia Inflammatory pseudotumor

>99 >95 40

22 8 60

Non-Hodgkin’s lymphomas and immunodeficiency-related neoplasms Non-Hodgkin’s lymphoma, all subtypes Non-Hodgkin’s lymphoma, AIDS-related Brain lymphoma, AIDS-related Brain lymphoma, immunocompetent hosts Post transplant lymphoproliferative disorder (PTLD) Burkitt’s lymphoma, African Burkitt’s lymphoma, North American Burkitt’s lymphoma, AIDS-related Lymphoma, primary immunodeficiency Lymphomatoid granulomatosis (B cell) Peripheral T cell lymphoma Nasal T/NK cell lymphoma Smooth muscle tumors in AIDS or transplant patients

5 40 95 5 95 >95 20 30 most most 40 >95 >95

5 61 62 63 3 64 64 65 66 67 28 68 69

40 70 20 <5% 50 >95

4 4 4 70 4 71

>95 75 most 7

2 29 6 7

Hodgkin’s disease Hodgkin’s disease, all subtypes Hodgkin’s disease, mixed cellularity Hodgkin’s disease, nodular sclerosis Hodgkin’s disease, lymphocyte predominant Hodgkin’s disease, lymphocyte depleted Hodgkin’s disease, AIDS-related

Carcinomas Nasopharyngeal carcinoma, Asian Nasopharyngeal carcinoma, North American Lymphoepithelioma-like carcinoma, foregut derived Gastric adenocarcinoma

Growth program: autonomous B-cell proliferation increasing EBV-infected B cells. Rescue program: EBV-infected blasts to survival and differentiate into memory B cells. Hiding program: EBNA1+/-, LMP2 promotes cell survival by blocking EBV reactivation.

Table 1 Overview of EBV products and functionsa Protein

Function

EBNA1 EBNA2

Essential for EBV immortalization of cell, replicates EBV genome, segregates viral episomes at mitosis. Transcriptional coactivator that upregulates expression of viral and cellular genes (especially c-myc), essential for EBV immortalization of cell, one of first viral proteins produced during EBV infection.

EBNA3 3A/3 3B/4 3C/6

Essential for EBV immortalization of cell, interacts with CBF1. Not essential for EBV immortalization of cell, interacts with CBF1, function remains largely unknown. Essential for EBV immortalization of cell, overcomes retinoblastoma protein (pRB) checkpoint in cell cycle, interacts with CBF1, increases production of LMP1.

EBNA-LP/5

Interacts with EBNA2 to inactivate p53 and Rb, interacts with transcription factors in notch signaling pathway, one of first viral proteins produced during EBV infection, redistributes EBNA3A in nucleus, contributes to EBV immortalization of cell.

LMP1

Mimics CD40 ligand binding signal, elevates levels of bcl-2 and a20, acts a constitutively active receptor, essential for EBV immortalization of cell.

LMP2A and B Drives EBV into latency. May play a role in oncogenesis in Hodgkin’s disease and nasopharyngeal carcinomas.

EBER1 and 2 Forms complexes with L22, associates with PKR, not essential for EBV immortalization of cell. CSTs or BARTs Complementary strand transcripts encoded at high levels in nasopharyngeal carcinomas. Potential protein products may modify Notch signaling.

EBNA2 activates Notch signals with cell proliferation. LMP1 acts as a constitutively activated CD40/TNF receptor. LMP2: 1. PI3-K/Akt activation with antiapoptosis, 2. sequesters the Lyn and Syk kinases and target them for proteasomal degradation with inhibition of B cell activation. The GAr of EBNA1 inhibits processing by the ubiquitin/proteasome system.

Manipulation of the ubiquitin proteasome system by human tumor virus. E1: ubiquitin activase. E2: ubquitin conjugase; E3: ubiquitin ligase; DUB: deubiquitinating enzymes. Different compnents of the ubiquitin/proteasome system are targeted by the oncogenic proteins.

matic representation of the molecular interactions and signa ways engaged by LMP1.

Up-regulation of TGF-αby LMP1 through NF-κB and AP-1 as could VEGF. TGF-αactivates EGFR leads proliferation and its own down-regulation. During carcinogenic progression, the negative feedback loop upon EGFR fails and loss of expression of p16INK4a

CIS, carcinoma in situ; EDNRB, endothelin receptor B; H/E, staining with haematoxylin and eosin; TSLC1, tumour suppressor in lung cancer 1.

Role of Epstein–Barr Virus in the Pathogenesis of Nasopharyngeal Carcinoma 1. In both nasopharyngeal carcinoma (NPC) and EpsteinBarr virus (EBV)-positive gastric carcinoma, the tumour cells carry monoclonal viral genomes, which indicates that EBV infection must have occurred prior to expansion of the malignant cell clone. 2. However, the difficulty of detecting EBV-infected epithelial cells in normal nasopharyngeal biopsies from individuals who are at high risk of developing NPC argues against a pre-existing normal reservoir of epithelial cell infection from which virus-positive carcinomas arise.

3. Indeed, EBV infection has been detected both by in situ hybridization to the EBV-encoded RNAs (EBERs) and by the presence of monoclonal EBV genomes in highgrade pre-invasive lesions (severe dysplasia and carcinoma in situ) in the nasopharynx, but not in lowgrade disease. 4. Similar results have been obtained from EBV-positive gastric carcinoma, in which associated normal gastric mucosa, inflamed mucosa and pre-malignant lesions are EBV-negative.

5. Multiple genetic changes have been found in NPC, with frequent deletion of regions on chromosomes 3p, 9p, 11q, 13q and 14q and promoter hypermethylation of specific genes on chromosomes 3p (RASSF1A and retinoic-acid receptor 2) and 9p (genes that encode INK4A, INK4B, ARF and death-associated protein kinase). 6. Deletions in both 3p and 9p have been identified in lowgrade dysplastic lesions and in normal nasopharyngeal epithelium from individuals who are at high risk of developing NPC in the absence of EBV infection, indicating that genetic events occur early in the pathogenesis of NPC and that these might cause predisposition to subsequent EBV infection.

7. This possibility is supported by in vitro data showing that the stable infection of epithelial cells by EBV requires an altered, undifferentiated cellular environment. 8. The scheme of pathogenesis of NPC has been proposed, in which loss of heterozygosity (LOH) occurs early in the pathogenesis of NPC, possibly as a result of exposure to environmental cofactors such as dietary components (such as salted fish). This results in low-grade pre-invasive lesions that, after additional genetic and epigenetic events, become susceptible to EBV infection.

9. Once cells have become infected, EBV latent genes provide growth and survival benefits, resulting in the development of NPC. Additional genetic and epigenetic changes occur after EBV infection.