Alice Gallagher

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.

Screening for herpesvirus genomes in common acute lymphoblastic leukemia

There is epidemiological evidence that infection may play a role in the etiology of childhood leukemia in particular common B cell precursor acute lymphoblastic leukemia. A panel of 20 leukemic samples (panel 1) was examined for the presence of four lymphotropic herpesviruses using conventional molecular techniques. A second independent panel of 27 leukemic samples (panel 2), along with 28 control peripheral blood samples from children with other forms of cancer, was tested for the presence of the same four viruses using sensitive real-time quantitative PCR. While herpesvirus genomes were detected, they were present at very low levels; detection rates and levels were similar in the leukemic and control panels. In addition we surveyed 18 leukemic samples (five from panel 1, six from panel 2 and a further seven samples not previously analyzed) using a degenerate PCR assay capable of detecting the genomes of known herpesviruses plus putative new members of the family. No novel herpesvirus genomes were detected suggesting that a herpesvirus is unlikely to be etiologically involved as a transforming agent in common acute lymphoblastic leukemia.

A meta-analysis of Hodgkin lymphoma reveals 19p13.3 TCF3 as a novel susceptibility locus

Recent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified associations with genetic variation at both HLA and non-HLA loci; however, much of heritable HL susceptibility remains unexplained. Here we perform a meta-analysis of three HL GWAS totaling 1,816 cases and 7,877 controls followed by replication in an independent set of 1,281 cases and 3,218 controls to find novel risk loci. We identify a novel variant at 19p13.3 associated with HL (rs1860661; odds ratio [OR] = 0.81, 95% confidence interval [95% CI] = 0.76–0.86, Pcombined = 3.5 × 10−10), located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment known to be involved in HL pathogenesis. This meta-analysis also notes associations between previously published loci at 2p16.1, 5q31, 6p31.2, 8q24.21 and 10p14 and HL subtypes. We conclude that our data suggest a link between the 19p13.3 locus, including TCF3, and HL risk

The human leukocyte antigen class I region is associated with EBV-positive Hodgkin's lymphoma : HLA-A and HLA Complex Group 9 are putative candidate genes

Various studies have indicated that the human leukocyte antigen (HLA) region is associated with Hodgkins lymphoma. We recently showed a specific association of the HLA class I region with EBV-positive Hodgkins lymphoma cases. One haplotype of two consecutive microsatellite markers (D6S265 and D6S510) was overrepresented in the patient group, whereas another haplotype was underrepresented. Here, we did fine mapping of this region of ∼400 kb as a next step to find the causative single-nucleotide polymorphism(s) (SNP). To select candidate SNPs for screening the total study population, several known SNPs were determined by sequencing two individuals homozygous for either of the above-mentioned associated haplotypes. Seven SNPs displayed different alleles in these two individuals and were therefore analyzed in the total study population, including 238 Hodgkins lymphoma patients and 365 family-based controls. All seven SNPs showed significant association with the EBV-positive patient group. Two of these SNPs were analyzed in a Scottish Hodgkins lymphoma population and revealed significant associations as well. The associated SNPs are located nearby two putative candidate genes: HLA-A and HLA complex group 9. HLA-A represents the most interesting target because of its consistent expression in EBV-positive Hodgkins lymphoma cases and its ability to present EBV-derived peptides to cytotoxic T cells. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2280–4)

Hodgkin lymphoma and Epstein‐Barr virus (EBV): No evidence to support hit‐and‐run mechanism in cases classified as non‐EBV‐associated

The Epstein‐Barr virus (EBV) is associated with a proportion of Hodgkin lymphoma (HL) cases, and this association is believed to be causal. The aetiology of cases lacking EBV in the tumour cells (EBV HRS‐ve), which make up the majority of cases in western countries, is obscure. It has been suggested that EBV may also cause these tumours by using a hit‐and‐run mechanism. Support for this idea comes from the finding that most young adult patients, who are likely to have a good immune response to EBV, have EBV HRS‐ve HL. We investigated this possibility using a combined serologic and molecular approach. Analysis of EBV seroprevalence rates in an epidemiologic study of young adult HL revealed that cases with EBV HRS‐ve HL were more likely to be EBV‐seronegative than controls. Furthermore, additional studies clearly showed that some HL patients have never been infected by EBV. Quantitative PCR was used to look for the presence of deleted EBV genomes in a series of adult cases with both EBV HRS+ve and HRS‐ve HL. Subgenomic fragments were detected in equimolar proportions. This study, therefore, found no evidence to support the idea that a hit‐and‐run mechanism involving EBV plays a role in the pathogenesis of HL.

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.

Viruses and Hodgkin disease: No evidence of novel herpesviruses in non-EBV-associated lesions

The Epstein‐Barr virus (EBV) is associated with a proportion of cases of Hodgkin disease (HD) and this association is believed to be causal. Epidemiological studies suggest that an infectious agent is involved in the aetiology of young adult HD, however, cases in this age group are less likely to have EBV‐associated disease than cases diagnosed in early childhood or older adult years. Molecular studies have failed to find a consistent association between HD and other candidate viruses, and the aetiology of non‐EBV‐associated cases remains obscure. We looked for evidence of herpesvirus infection in samples of non‐EBV‐associated HD using a highly sensitive, degenerate PCR assay. Despite exhaustive sequence analysis of PCR products, no novel herpesviruses were identified. These results suggest that it is extremely unlikely that a novel herpesvirus is involved in the pathogenesis of non‐EBV‐associated HD.

Measles virus and classical Hodgkin lymphoma: no evidence for a direct association.

A proportion of Hodgkin lymphoma (HL) cases are causally associated with the Epstein‐Barr virus (EBV) but the aetiology of the remaining cases remains obscure. Over the last 3 decades several studies have found an association between HL and measles virus (MV) including a recent cohort study describing the detection of MV antigens in Hodgkin and Reed‐Sternberg cells, the tumour cells in HL. In the present study we looked at the relationship between history of MV infection and risk of developing HL in a population‐based, case/control study of HL. In addition we used immunohistochemistry and RT‐PCR to look for direct evidence of MV in HL biopsies. There was no significant difference in the proportion of cases reporting previous measles compared to controls in the entire data set or when young adults were considered separately. Using a robust immunohistochemical assay for MV infection, we failed to find evidence of MV in biopsies from 97 cases of HL and RT‐PCR studies similarly gave negative results. This study therefore provides no evidence that MV is directly involved in the development of HL. However, when age at first reported MV infection was investigated, significant differences emerged with children infected before school‐age having higher risk, especially of EBV−ve HL, when compared with children infected at older ages; the interpretation of these latter results is unclear.

Modeling HLA associations with EBV-positive and -negative Hodgkin lymphoma suggests distinct mechanisms in disease pathogenesis

HLA genotyping and genome wide association studies provide strong evidence for associations between Human Leukocyte Antigen (HLA) alleles and classical Hodgkin lymphoma (cHL). Analysis of these associations is complicated by the extensive linkage disequilibrium within the major histocompatibility region and recent data suggesting that associations with EBV‐positive and EBV‐negative cHL are largely distinct. To distinguish independent and therefore potentially causal associations from associations confounded by linkage disequilibrium, we applied a variable selection regression modeling procedure to directly typed HLA class I and II genes and selected SNPs from EBV‐stratified patient subgroups. In final models, HLA‐A*01:01 and B*37:01 were associated with an increased risk of EBV‐positive cHL whereas DRB1*15:01 and DPB1*01:01 were associated with decreased risk. Effects were independent of a prior history of infectious mononucleosis. For EBV‐negative cHL the class II SNP rs6903608 remained the strongest predictor of disease risk after adjusting for the effects of common HLA alleles. Associations with “all cHL” and differences by case EBV status reflected the subgroup analysis. In conclusion, this study extends previous findings by identifying novel HLA associations with EBV‐stratified subgroups of cHL, highlighting those alleles likely to be biologically relevant and strengthening evidence implicating genetic variation associated with the SNP rs6903608.

Viruses and Hodgkin lymphoma: No evidence of polyomavirus genomes in tumor biopsies

The epidemiology of young adult Hodgkin lymphoma (HL) suggests that delayed exposure to a common childhood pathogen may be involved in disease pathogenesis. The Epstein-Barr virus (EBV) is associated with a proportion of cases but cases of young adult HL in westernized countries are less frequently EBV-associated than cases in other age groups and geographical locales. This study investigated the possibility that polyomaviruses might be involved in the etiology of HL by analysing a series of 35 cases of classical HL using both specific and degenerate PCR assays for polyomavirus genomes. No positive results were obtained, indicating that it is highly unlikely that this virus family is directly involved in the pathogenesis of HL.