Alison A. Armstrong

Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin's disease

DNA from malignant cells is present in the serum/plasma of cancer patients and DNA from this source is amenable to analysis by polymerase chain reaction (PCR). In the present study, we evaluated whether Epstein‐Barr virus (EBV) DNA is present in the serum of patients with EBV‐associated Hodgkins disease (HD). Using conventional PCR, EBV DNA was detected in serum from 30/33 patients with EBV‐associated HD but in only 6/26 patients with non‐EBV‐associated disease (p < 0.001). Samples from healthy individuals were negative and only 5/12 infectious mononucleosis samples were positive. Real‐time quantitative PCR was subsequently employed to determine the concentration of EBV DNA present in serum; among positive samples the level ranged from 1 to 705 copies per 125 μl of serum. Post‐treatment samples from 5/14 cases with EBV‐associated HD contained detectable EBV DNA; analysis of this small group of cases suggests that positivity in post‐treatment samples correlates with risk factors indicative of a poor prognosis. Overall, our results are consistent with the notion that DNA from Reed‐Sternberg cells is present in the serum of HD patients, and further suggest that serum EBV should be evaluated as a prognostic marker. Int. J. Cancer (Pred. Oncol.) 84:442–448, 1999.

Risk factors for Hodgkin's disease by Epstein-Barr virus (EBV) status: prior infection by EBV and other agents

A UK population-based case–control study of Hodgkin’s disease (HD) in young adults (16–24 years) included 118 cases and 237 controls matched on year of birth, gender and county of residence. The majority (103) of the cases were classified by Epstein–Barr virus (EBV) status (EBV present in Reed–Stenberg cells), with 19 being EBV-positive. Analyses using conditional logistic regression are presented of subject reports of prior infectious disease (infectious mononucleosis (IM), chicken pox, measles, mumps, pertussis and rubella). In these analyses HD cases are compared with matched controls, EBV-positive cases and EBV-negative cases are compared separately with their controls and formal tests of differences of association by EBV status are applied. A prior history of IM was positively associated with HD (odds ratio (OR) = 2.43, 95% confidence interval (CI) = 1.10–5.33) and with EBV-positive HD (OR = 9.16, 95% CI = 1.07–78.31) and the difference between EBV-positive and EBV-negative HD was statistically significant (P = 0.013). The remaining infectious illnesses (combined) were negatively associated with HD, EBV-positive HD and EBV-negative HD (in the total series, for ≥2 episodes compared with ≤1, OR = 0.45, 95% CI = 0.25–0.83). These results support previous evidence that early exposure to infection protects against HD and that IM increases subsequent risk; the comparisons of EBV-positive and EBV-negative HD are new and generate hypotheses for further study.

Lack of involvement of known oncogenic DNA viruses in Epstein-Barr virus-negative Hodgkin's disease.

Epstein-Barr virus (EBV) is associated with around one-third of cases, but young adult cases are rarely EBV associated. In this study, known oncogenic DNA viruses, including human adenoviruses, papovaviruses and the human herpesviruses-6 (HHV-6) and -8 (HHV-8) were not detected in Hodgkins disease lesions. These results suggest that an as yet unidentified infectious agent is involved in the pathogenesis of non-EBV-associated Hodgkins disease.

The expression of the EBV latent membrane protein (LMP‐1) is independent of CD23 and bcl‐2 in Reed‐Sternberg cells in Hodgkin's disease

A series of 33 cases of Hodgkins disease was investigated for the presence of the EBV encoded latent gene product LMP‐1 and of CD23 using immunohistochemical techniques. The expression of bcl‐2 was examined in a subset of cases. LMP‐1 was detected in the Reed‐Sternberg cells in 15 cases. Although LMP‐1 is known to upregulate CD23 and bcl‐2, there was no correlation between the expression of LMP‐1 and the detection of CD23 and bcl‐2 in Reed‐Sternberg cells.

Case clustering, Epstein-Barr virus Reed-Sternberg cell status and herpes virus serology in Hodgkin's disease: Results of a case-control study

The Leukaemia Research Fund Data Collection Study (DCS) is a specialist registry of leukaemias and lymphomas. The present study involves 494 cases of Hodgkins disease (HD) registered with the DCS between 1985 and 1989. This entire data set has been tested for localised spatial clustering using an established nearest neighbour method with 18% of all cases in young people classified as clustered (P < 0.05). No clustering was found in older cases. Subsamples were selected from the registered cases for a pilot study in which case clustering, herpes virus antibody titres and Epstein-Barr virus (EBV) presence within the Reed-Sternberg (RS) cells (EBV-RS status) were investigated together. Firstly, a case-control study of HD in young people or nodular sclerosing (NS) subtype (39 HD cases and 26 healthy controls) found significant elevation of antibody titres to EBV-viral capsid antigen (VCA), EBV-early antigen (EA) and human herpes virus 6 (HHV-6) in HD cases compared with controls. EBV viral genome was present in 5 cases and 4 of these were in clusters of HD in young people. Elevation of antibody titres to the EBV antigens was not associated with case clustering or EBV-RS status. Antibody titres to HHV-6 differed significantly between EBV-RS+ and EBV-RS- cases (P = 0.04). Geometric mean titres for HHV-6 for EBV-RS+ and EBV-RS- cases were 11.5 and 73.7, respectively, with the former lower than the control value of 20.5. Secondly, a cluster study included all other cases (n = 14) in clusters containing known EBV-RS+ cases. 3 further cases were EBV-RS+ positive but no cluster consisted entirely of positive cases. Overall, 5/16 clustered, 2/12 peripheral and 1/25 random cases in these studies were EBV-RS+ (P = 0.017). The interpretation of these results in terms of shared aetiological exposures of cases within clusters and the roles of EBV and HHV-6 is discussed, and hypotheses for testing in future studies proposed.

Analysis of Epstein–Barr virus (EBV) nuclear antigen 1 subtypes in EBV-associated lymphomas from Brazil and the United Kingdom

EBNA-1 is the only viral protein consistently expressed in all cells latently infected by Epstein-Barr virus (EBV). There is a high frequency of sequence variation within functionally important domains of EBNA-1, with five subtypes identified. Individuals may be infected with multiple EBV strains (classified according to EBNA-1 subtype), but Burkitts lymphoma (BL) tumours carry a single subtype and exhibit some subtype preference. Subtype variation has also been related to geographical location. In the present study EBNA-1 polymorphisms were examined in a series of haematological malignancies from two distinct geographical regions, Brazil and the United Kingdom. Nucleotide sequence analysis of the carboxy-terminal region of EBNA-1 in 34 cases revealed six distinct sequences, some of which are novel. A new subtype, named V-Ala, was identified. EBNA-1 subtype in tumours differed markedly according to geographical location. In contrast to previous studies, we found evidence of EBNA-1 sequence variation within individual BL tumour samples.

B-lymphotropic viruses in a novel tropical splenic lymphoma

Peripheral blood from patients with a novel tropical splenic lymphoma, characterized by splenomegaly and circulating naïve CD5‐negative villous B lymphocytes, has been screened for evidence of an association with the B‐lymphotropic viruses, Epstein–Barr virus (EBV), hepatitis C virus (HCV) and human herpesvirus 8 (HHV8). No increased prevalence of EBV, HCV and HHV8 was demonstrated using serological and molecular techniques, compared with a geographical, age‐matched control group. However, lymphoma patients had markedly raised EBV antibody levels without a concomitant increase in the rate of detection of viral genomes in the peripheral blood. This phenomenon also occurred in patients with hyper‐reactive malarial splenomegaly, a condition that occurs in the same geographical area and that is clinically indistinguishable from tropical splenic lymphoma, adding further weight to the suggestion that there may be an aetiological association between these two disorders.

Determination of HLA-A*02 antigen status in Hodgkin's disease and analysis of an HLA-A*02-restricted epitope of the Epstein-Barr virus LMP-2 protein.

There is good evidence for an association between Epstein‐Barr virus (EBV) and Hodgkins disease (HD). In approximately one‐third of cases, the EBV genome is detectable in Reed‐Sternberg (RS) cells and there is expression of the viral nuclear antigen EBNA‐1 and the latent membrane protein LMP‐1. Expression of LMP‐2 has been demonstrated at the mRNA level, and it is presumed that the protein is expressed alongside LMP‐1. The LMP‐2 protein is known to contain an epitope presented to cytotoxic T‐cells which is restricted through the HLA class I antigen A*0201 in healthy seropositive individuals. Since most HLA‐A*02‐positive Caucasians are HLA‐A*0201‐positive, it was hypothesized that HLA‐A*02‐positive individuals would be under‐represented among Caucasians with EBV‐associated HD. HLA‐A*02 status was determined, using flow cytometry and/or the polymerase chain reaction, for 276 individuals including 176 cases of HD. There was no significant difference between the frequency of HLA‐A*02 positivity in HD cases and controls, and between EBV‐associated and non‐associated cases of HD. The A*02 alleles of 14 cases of EBV‐associated HD were further subtyped using nested PCR; all except one case were found to be A*0201‐positive. We therefore investigated whether there was any evidence for mutation of the epitope representing amino acids 426–434 of LMP‐2a which is restricted through HLA‐A*0201. In 10/11 cases the nucleotide sequence encoding this epitope was identical to the published sequence; in the remaining case there was a mutation which would not be expected to alter the conformation of the epitope. Overall, our data suggest that other mechanisms of immune escape must be operative in EBV‐associated HD. Int. J. Cancer 72:614–618, 1997.

The Epidemiology of EBV-Associated Hodgkin’s Disease

Preceding chapters provide evidence that the Epstein-Barr virus (EBV) is involved in the pathogenesis of Hodgkin’s disease (HD). It is clear that EBV genomes or gene products cannot be detected within the affected tissues of all cases. We have designated those cases in which clonal EBV genomes are detectable within tumours or in which the Reed-Sternberg (RS) cells express EBV LMP-1 protein or EBER RNAs as EBV-associated (Armstrong et al., 1992). Studies from a number of laboratories indicate that between 32 and 50% of cases in developed countries are EBV-associated using these criteria (Pallesen et al., 1991; Weiss et al., 1991; Herbst et al., 1992; Delsol et al., 1992; Armstrong et al., 1994; Khan et al., 1993).