Annette Lake

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.

A genome-wide association study of Hodgkin's lymphoma identifies new susceptibility loci at 2p16.1 ( REL ), 8q24.21 and 10p14 ( GATA3 )

To identify susceptibility loci for classical Hodgkins lymphoma (cHL), we conducted a genome-wide association study of 589 individuals with cHL (cases) and 5,199 controls with validation in four independent samples totaling 2,057 cases and 3,416 controls. We identified three new susceptibility loci at 2p16.1 (rs1432295, REL, odds ratio (OR) = 1.22, combined P = 1.91 × 10−8), 8q24.21 (rs2019960, PVT1, OR = 1.33, combined P = 1.26 × 10−13) and 10p14 (rs501764, GATA3, OR = 1.25, combined P = 7.05 × 10−8). Furthermore, we confirmed the role of the major histocompatibility complex in disease etiology by revealing a strong human leukocyte antigen (HLA) association (rs6903608, OR = 1.70, combined P = 2.84 × 10−50). These data provide new insight into the pathogenesis of cHL.

Genome-Wide Association Study of Classical Hodgkin Lymphoma and Epstein–Barr Virus Status–Defined Subgroups

BACKGROUND Accumulating evidence suggests that risk factors for classical Hodgkin lymphoma (cHL) differ by tumor Epstein-Barr virus (EBV) status. This potential etiological heterogeneity is not recognized in current disease classification. METHODS We conducted a genome-wide association study of 1200 cHL patients and 6417 control subjects, with validation in an independent replication series, to identify common genetic variants associated with total cHL and subtypes defined by tumor EBV status. Multiple logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) assuming a log-additive genetic model for the variants. All statistical tests were two-sided. RESULTS Two novel loci associated with total cHL irrespective of EBV status were identified in the major histocompatibility complex region; one resides adjacent to MICB (rs2248462: OR = 0.61, 95% CI = 0.53 to 0.69, P = 1.3 × 10(-13)) and the other at HLA-DRA (rs2395185: OR = 0.56, 95% CI = 0.50 to 0.62, P = 8.3 × 10(-25)) with both results confirmed in an independent replication series. Consistent with previous reports, associations were found between EBV-positive cHL and genetic variants within the class I region (rs2734986, HLA-A: OR = 2.45, 95% CI = 2.00 to 3.00, P = 1.2 × 10(-15); rs6904029, HCG9: OR = 0.46, 95% CI = 0.36 to 0.59, P = 5.5 × 10(-10)) and between EBV-negative cHL and rs6903608 within the class II region (rs6903608, HLA-DRA: OR = 2.08, 95% CI = 1.84 to 2.35, P = 6.1 × 10(-31)). The association between rs6903608 and EBV-negative cHL was confined to the nodular sclerosis histological subtype. Evidence for an association between EBV-negative cHL and rs20541 (5q31, IL13: OR = 1.53, 95% CI = 1.32 to 1.76, P = 5.4 x 10(-9)), a variant previously linked to psoriasis and asthma, was observed; however, the evidence for replication was less clear. Notably, one additional psoriasis-associated variant, rs27524 (5q15, ERAP1), showed evidence of an association with cHL in the genome-wide association study (OR = 1.21, 95% CI = 1.10 to 1.33, P = 1.5 × 10(-4)) and replication series (P = .03). CONCLUSION Overall, these results provide strong evidence that EBV status is an etiologically important classification of cHL and also suggest that some components of the pathological process are common to both EBV-positive and EBV-negative patients.

HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection

Infectious mononucleosis (IM) is an immunopathological disease caused by EBV that occurs in young adults and is a risk factor for Hodgkin lymphoma (HL). An association between EBV-positive HL and genetic markers in the HLA class I locus has been identified, indicating that genetic differences in the HLA class I locus may alter disease phenotypes associated with EBV infection. To further determine whether HLA class I alleles may affect development of EBV-associated diseases, we analyzed 2 microsatellite markers and 2 SNPs located near the HLA class I locus in patients with acute IM and in asymptomatic EBV-seropositive and -seronegative individuals. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the 2 SNPs were also significantly more frequent in patients with IM than in EBV-seronegative individuals. IM patients possessing the associated microsatellite allele had fewer lymphocytes and increased neutrophils relative to IM patients lacking the allele. These patients also displayed higher EBV titers and milder IM symptoms. The results of this study indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection, suggesting that genetic variation in T cell responses can influence the nature of primary EBV infection and the level of viral persistence.

HLA-A alleles and infectious mononucleosis suggest a critical role for cytotoxic T-cell response in EBV-related Hodgkin lymphoma

A proportion of classical Hodgkin lymphoma (HL) is believed to be causally related to infection with the ubiquitous lymphotropic EBV. The determining factors for development of EBV-related HL remain poorly understood, but likely involve immunological control of the viral infection. Accordingly, markers of the HLA class I region have been associated with risk of EBV-related HL. To study the host genetic component of EBV-related HL further, we investigated the lymphomas association with HLA-A*01 and HLA-A*02 simultaneously in the setting of infectious mononucleosis (IM), a risk factor for EBV-related HL, in a case-series analysis including 278 EBV-related and 656 EBV-unrelated cases of HL. By logistic regression, HLA-A*01 alleles [odds ratio (OR) per allele, 2.15; 95% CI, 1.60–2.88] were associated with increased and HLA-A*02 alleles (OR per allele, 0.70; 95% CI, 0.51–0.97) with decreased risk of EBV-related HL. These allele-specific associations corresponded to nearly 10-fold variation in risk of EBV-related HL between HLA-A*01 and HLA-A*02 homozygotes. History of IM was also associated with risk of EBV-related HL (OR, 3.40; 95% CI, 1.74–6.66). The association between history of IM and EBV-related HL was not seen in the presence of HLA-A*02 because this allele appeared to neutralize the effect of IM on EBV-related HL risk. Our findings suggest that HLA class I-restricted EBV-specific cytotoxic T-cell responses and events in the early immune response to EBV infection in IM play critical roles in the pathogenesis of EBV-related HL.

Mutations of NFKBIA, encoding IκBα, are a recurrent finding in classical Hodgkin lymphoma but are not a unifying feature of non-EBV-associated cases

A consistent feature of the Hodgkin and Reed‐Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is the constitutive activation of NF‐κB transcription factors. In Epstein‐Barr virus (EBV)‐associated cases of cHL, expression of viral antigens most probably leads to NF‐κB activation but for non‐EBV‐associated cases, the mechanism is not clear. Previous small studies have demonstrated deleterious mutations of NFKBIA, the gene encoding IκBα, in HRS cells. In the present study, we aimed to establish the frequency of NFKBIA mutation in cHL by investigating a larger series of cases and to determine whether these mutations are a characteristic feature of non‐EBV‐associated cHL. Single HRS cells from 20 cases of cHL were analysed by PCRs covering all 6 exons of the gene. Clonal deleterious mutations were detected in 3 cases and in 1 case both alleles of the gene were shown to harbour mutations. NFKBIA mutations were detected only in non‐EBV‐associated cases but the majority of these cases had wild‐type NFKBIA. It remains possible that defects in genes encoding other inhibitors of NF‐κB, such as TNFAIP3 (A20) and CYLD, are involved in the latter cases, as described for one case in this series.

Variation at 3p24.1 and 6q23.3 influences the risk of Hodgkin’s lymphoma

In addition to HLA, recent genome-wide association studies (GWASs) of Hodgkin’s Lymphoma (HL) have identified susceptibility loci for HL at 2p16.1, 8q24.21 and 10p14. In this study, we perform a GWAS meta-analysis with published GWAS (totaling 1,465 cases and 6,417 controls of European background), and follow up the most significant association signals in 2,024 cases and 1,853 controls. A combined analysis identifies new HL susceptibility loci mapping to 3p24.1 (rs3806624; P=1.14×10-12, odds ratio [OR]=1.26) and 6q23.3 (rs7745098; P=3.42×10-9, OR=1.21). rs3806624 localizes 5’ to the EOMES (eomesodermin) gene within a p53 response element affecting p53 binding. rs7745098 maps intergenic to HBS1L and MYB, a region previously associated with hematopoiesis. These findings provide further insight into the genetic and biological basis of inherited susceptibility to HL.

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

Effect of IL‐6 promoter polymorphism on incidence and outcome in Hodgkin's lymphoma

A single nucleotide polymorphism (SNP) is present at position ‐174 of the human interleukin‐6 gene. The risk of developing Hodgkins lymphoma (HL) in young adults decreases with an increasing number of C alleles at this position. We analysed the effect of this SNP on incidence and outcome in HL. DNA samples from 408 cases and 349 controls were screened and analysed following stratification by age, histological subtype and Epstein–Barr virus status. Although the risk of classical HL in young adults decreased with increasing C alleles, case–control differences were not significant. An excess of G alleles was observed for nodular lymphocyte predominant HL in young adults (n = 21), which was significant.

Phenotype and frequency of Epstein–Barr virus-infected cells in pretreatment blood samples from patients with Hodgkin lymphoma

An accumulating body of data suggests that the Epstein–Barr virus (EBV), a lymphotropic herpesvirus, is involved in the pathogenesis of a proportion of cases of Hodgkin lymphoma (HL). In this study, we showed that the frequency of circulating EBV‐infected cells was significantly higher (P < 0·001) in pretreatment blood samples from EBV‐associated cases when compared with non‐EBV‐associated cases. We further showed that in patients with EBV‐associated disease, the virus persisted in the peripheral blood in memory B cells. This phenotype is consistent with that seen in healthy seropositive controls, post‐transplant patients and patients with acute infectious mononucleosis. The data suggest that an increased frequency of EBV carrying B cells in peripheral blood is associated with EBV‐associated HL.