Margaret L. Gulley

Molecular Diagnosis of Epstein-Barr Virus-Related Diseases

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis, and it may also be found in a wide variety of benign and malignant lesions including oral hairy leukoplakia, inflammatory pseudotumor, Hodgkins disease, non-Hodgkins lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Molecular testing is increasingly important in the diagnosis and monitoring of patients affected by these diseases. In biopsy tissues, molecular detection of EBV-encoded RNA transcripts by in situ hybridization remains the gold standard for proving that a histopathological lesion is EBV-related. EBV-encoded RNA hybridization and EBV LMP1 immunostains are used routinely to detect latent EBV in tissues affected by posttransplant lymphoproliferative disorder (PTLD) or in enlarged nodes from patients with infectious mononucleosis. Traditional serology is the best test for evaluating acute versus remote infection in healthy individuals. High serological titers serve as a tumor marker for some EBV-related malignancies, but titers are not a dependable tumor marker in immunocompromised hosts. EBV viral load testing by quantitative DNA amplification of blood samples is a promising new laboratory test that has proven useful for early diagnosis and monitoring patients with PTLD. Recent studies suggest a role for EBV viral load testing in nasopharyngeal carcinoma, Hodgkins disease, and AIDS patients with brain lymphoma. Further research is needed to define more fully the clinical utility of viral load tests in the full spectrum of EBV-associated diseases. Gene expression profiling is on the horizon as a means to improve subclassification of EBV-related diseases and to predict response to therapy.

Laboratory Assays for Epstein-Barr Virus-Related Disease

Epstein-Barr virus (EBV) infects various cell types in a wide spectrum of benign and malignant diseases. Laboratory tests for EBV have improved and are increasingly used in diagnosis, prognosis, prediction, and prevention of diseases ranging from infectious mononucleosis to selected subtypes of lymphoma, sarcoma, and carcinoma. Indeed, the presence of EBV is among the most effective tumor markers supporting clinical management of cancer patients. In biopsies, localization of EBER transcripts by in situ hybridization remains the gold standard for identifying latent infection. Other RNA- and protein-based assays detect lytic viral replication and can distinguish carcinoma-derived from lymphocyte-derived EBV in saliva or nasopharyngeal brushings. Analysis of blood using EBV viral load and serology reflects disease status and risk of progression. This review summarizes prior research in the context of basic virologic principles to provide a rational strategy for applying and interpreting EBV tests in various clinical settings. Such assays have been incorporated into standard clinical practice in selected settings such as diagnosis of primary infection and management of patients with immune dysfunction or nasopharyngeal carcinoma. As novel therapies are developed that target virus-infected cells or overcome the adverse effects of infection, laboratory testing becomes even more critical for determining when intervention is appropriate and the extent to which it has succeeded.

Using Epstein-Barr Viral Load Assays To Diagnose, Monitor, and Prevent Posttransplant Lymphoproliferative Disorder

SUMMARY Epstein-Barr virus (EBV) DNA measurement is being incorporated into routine medical practice to help diagnose, monitor, and predict posttransplant lymphoproliferative disorder (PTLD) in immunocompromised graft recipients. PTLD is an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. Validated protocols, commercial reagents, and automated instruments facilitate implementation of EBV load assays by real-time PCR. When applied to either whole blood or plasma, EBV DNA levels reflect clinical status with respect to EBV-related neoplasia. While many healthy transplant recipients have low viral loads, high EBV loads are strongly associated with current or impending PTLD. Complementary laboratory assays as well as histopathologic examination of lesional tissue help in interpreting modest elevations in viral load. Circulating EBV levels in serial samples reflect changes in tumor burden and represent an effective, noninvasive tool for monitoring the efficacy of therapy. In high-risk patients, serial testing permits early clinical intervention to prevent progression toward frank PTLD. Restoring T cell immunity against EBV is a major strategy for overcoming PTLD, and novel EBV-directed therapies are being explored to thwart virus-driven neoplasia.

Epstein-Barr Virus Lytic Infection Contributes to Lymphoproliferative Disease in a SCID Mouse Model

ABSTRACT Most Epstein-Barr virus (EBV)-positive tumor cells contain one of the latent forms of viral infection. The role of lytic viral gene expression in EBV-associated malignancies is unknown. Here we show that EBV mutants that cannot undergo lytic viral replication are defective in promoting EBV-mediated lymphoproliferative disease (LPD). Early-passage lymphoblastoid cell lines (LCLs) derived from EBV mutants with a deletion of either viral immediate-early gene grew similarly to wild-type (WT) virus LCLs in vitro but were deficient in producing LPD when inoculated into SCID mice. Restoration of lytic EBV gene expression enhanced growth in SCID mice. Acyclovir, which prevents lytic viral replication but not expression of early lytic viral genes, did not inhibit the growth of WT LCLs in SCID mice. Early-passage LCLs derived from the lytic-defective viruses had substantially decreased expression of the cytokine interleukin-6 (IL-6), and restoration of lytic gene expression reversed this defect. Expression of cellular IL-10 and viral IL-10 was also diminished in lytic-defective LCLs. These results suggest that lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.

A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas

ABSTRACT Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34+ hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1+ control. Both the control and Z-KO viruses established long-term viral latency in all infected animals. The infection appeared well controlled in some animals, but others eventually developed CD20+ diffuse large B cell lymphomas (DLBCL). Animals infected with the control virus developed tumors more frequently than Z-KO virus-infected animals. Specific immune responses against EBV-infected B cells were generated in mice infected with either the control virus or the Z-KO virus. In both cases, forms of viral latency (type I and type IIB) were observed that are less immunogenic than the highly transforming form (type III) commonly found in tumors of immunocompromised hosts, suggesting that immune pressure contributed to the outcome of the infection. These results point to an important role for lytic EBV infection in the development of B cell lymphomas in the context of an active host immune response.

Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation.

Ninety-five cases of adenocarcinoma of the stomach were evaluated for the presence of Epstein-Barr virus (EBV) using a sensitive in situ hybridization assay targeting Epstein-Barr virus-encoded RNA 1 (EBER1) transcripts. EBER1 was detected in 11 of 95 (12%) of cases. When present, the virus was localized to malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in normal-appearing gastric epithelium or intestinal metaplasia. The EBV DNA was monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant transformation. The presence of EBV was strongly associated with increased numbers of tumor-infiltrating T lymphocytes; however, EBV was not associated with prolonged survival. Neither p53 nor bcl-2 were consistently detected in the EBV-associated tumors. Specifically, 6 of 11 EBV-positive carcinomas had accumulation of p53 protein by immunohistochemical analysis, which was similar to the prevalence of p53 accumulation in EBV-negative specimens and suggests that EBV infection does not substitute for p53 mutations during tumorigenesis. The bcl-2 oncoprotein was expressed in a third of the carcinoma specimens tested, but bcl-2 expression did not correlate with the presence of EBV or with expression of EBV latent membrane protein 1. In conclusion, EBV infection appears to precede malignant transformation in a significant fraction of gastric carcinomas, but neither bcl-2 expression nor p53 accumulation appear to be consistently associated with the presence of the virus.

Recommended principles and practices for validating clinical molecular pathology tests.

CONTEXT The use of DNA- and RNA-based tests continues to grow for applications as varied as inherited disease, infectious disease, cancer, identity testing, human leukocyte antigen typing, and pharmacogenetics. Progress is driven in part by the huge growth in knowledge about the molecular basis of disease coupled with advancements in technologic capabilities. In addition to requirements for clinical utility, every molecular test also may have limitations that must be carefully considered before clinical implementation. Analytic and clinical performance characteristics as well as test limitations are established and documented through the process of test validation. OBJECTIVE To describe the established principles of test validation, along with relevant regulations in the United States, in order to provide a rational approach to introducing molecular tests into the clinical laboratory. DATA SOURCES PubMed review of published literature, published guidelines, and online information from national and international professional organizations. CONCLUSIONS These resources and recommendations provide a framework for validating clinical tests.

Improved survival of gastric cancer with tumour Epstein–Barr virus positivity: an international pooled analysis

Background and objective About 9% of gastric carcinomas have Epstein–Barr virus (EBV) in the tumour cells, but it is unclear whether viral presence influences clinical progression. We therefore examined a large multicentre case series for the association of tumour EBV status with survival after gastric cancer diagnosis, accounting for surgical stage and other prognostic factors. Methods We combined individual-level data on 4599 gastric cancer patients diagnosed between 1976 and 2010 from 13 studies in Asia (n=8), Europe (n=3), and Latin America (n=2). EBV positivity of tumours was assessed by in situ hybridisation. Mortality HRs for EBV positivity were estimated by Cox regression models stratified by study, adjusted for distributions of sex (71% male), age (mean 58 years), stage (52% tumour-node-metastasis stages III or IV), tumour histology (49% poorly differentiated, 57% Lauren intestinal-type), anatomic subsite (70% non-cardia) and year of diagnosis. Variations by study and continent were assessed using study-specific HRs for EBV positivity. Results During median 3.0 years follow-up, 49% of patients died. Stage was strongly predictive of mortality, with unadjusted HRs (vs stage I) of 3.1 for stage II, 8.1 for stage III and 13.2 for stage IV. Tumour EBV positivity was 8.2% overall and inversely associated with stage (adjusted OR: 0.79 per unit change). Adjusted for stage and other confounders, EBV positivity was associated with lower mortality (HR, 0.72; 95% CI 0.61 to 0.86), with low heterogeneity among the study populations (p=0.2). The association did not significantly vary across patient or tumour characteristics. There was no significant variation among the three continent-specific HRs (p=0.4). Conclusions Our findings suggest that tumour EBV positivity is an additional prognostic indicator in gastric cancer. Further studies are warranted to identify the mechanisms underlying this protective association.

Guidelines for interpreting EBER in situ hybridization and LMP1 immunohistochemical tests for detecting Epstein-Barr virus in Hodgkin lymphoma.

Histochemical stains demonstrate Epstein-Barr virus (EBV) in approximately 40% of all Hodgkin hymphomas, suggesting a role in tumorigenesis and the potentialfor EBV-targeted therapy. As research progresses, it is important to define criteria for interpreting histochemical stains. Four hematopathologists independently interpreted EBV-encoded RNA (EBER) and latent membrane protein 1 (LMP1) histochemical stains from 40 cases of Hodgkin lymphoma and then reviewed the stains as a group to resolve discrepancies and to develop interpretation guidelines. To call a Hodgkin case EBV-related, the EBER and/or LMP1 signal must be unequivocally present in Reed-Sternberg/Hodgkin (RS/H) cells. The cytologic features and distribution of stained cells should be matched with those on the corresponding H&E-stained slide to help interpret whether the EBER or LMP1 signal is in malignant or reactive cells. The EBER signal is localized to the nucleus, whereas LMP1 is in the cytoplasm and surface membrane. In some cases, only a fraction of RS/H cells express these factors for technical or biologic reasons. Before calling a case EBER-negative, it is essential to show that tumor cell RNA is preserved and available for hybridization. LMP1 staining, although usually strong among all tumor cells in a given case, may alternatively be focal and weak, contributing to false-negative interpretation. EBER and LMP1 assays in combination are more effective than either assay alone for identifying EBV-related Hodgkin lymphoma.

Epstein-barr virus is detected in undifferentiated nasopharyngeal carcinoma but not in lymphoepitheliomalike carcinoma of the urinary bladder

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.