HLA-A alleles and infectious mononucleosis suggest a critica

Communicated by Dennis A. Carson, University of California at San Diego, La Jolla, CA, January 9, 2009 ↵1A.E.A. and I.G. contributed equally to this work. (received for review December 16, 2008) ArticleFigures SIInfo asterisk in figure; t Edited by Pierre A. Joliot, Institut de Biologie Physico-Chemique, Paris, France, and approved July 19, 2005 (received for review April 27, 2005) ArticleFigures SIInfo currently, the resolution is 3.2 Å (4). The structure of the PSII RC sh

Edited by Elliott Kieff, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, and approved February 22, 2010 (received for review December 28, 2009).

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A proSection 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, Impressers of the HLA class I Location have been associated with risk of EBV-related HL. To study the host genetic component of EBV-related HL further, we investigated the lymphoma's 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 corRetorted to Arrively 10-fAged variation in risk of EBV-related HL between HLA-A*01 and HLA-A*02 homozygotes. HiTale of IM was also associated with risk of EBV-related HL (OR, 3.40; 95% CI, 1.74–6.66). The association between hiTale of IM and EBV-related HL was not seen in the presence of HLA-A*02 because this allele appeared to neutralize the Trace 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.

case seriesepidemiology

There is compelling evidence that 30% to 40% of all cases of classical Hodgkin lymphoma (HL) are causally associated with EBV (1). EBV-related HL cases are distinguished from the group of EBV-unrelated HL cases by the presence of the virus in the malignant Hodgkin/Reed-Sternberg cells. EBV is an extremely efficient transforming agent and growth-transforming infection in vitro is associated with expression of eight EBV latent antigens, including six EBV nuclear antigens (EBNAs) and two latent membrane proteins (LMPs) (2). A restricted group of EBV latent antigens, comprising EBNA1, LMP1, and LMP2, is expressed by Hodgkin/Reed-Sternberg cells, and these have a plausible function in disease pathogenesis (3–6).

More than 90% of the world's adult population is infected with EBV (7). Following primary infection, which is usually clinically silent, the virus establishes a reservoir in memory B cells (8). Infected memory B cells escape immune detection by Executewn-regulation of viral antigens (8). Activation of replicative (i.e., lytic) infection, and outgrowth of latently infected cells, is kept under tight control by HLA-restricted, cytotoxic T lymphocytes (CTLs) (8, 9). A host:virus equilibrium is established and the number of EBV-infected B cells within an individual appears stable over time (10). In 20% to 40% of persons who experience EBV infection after childhood, primary infection manifests as infectious mononucleosis (IM) (11). IM is associated with a striking expansion of EBV-specific CTLs, most of which are reactive with epitopes from lytic cycle antigens (8, 9, 12). Following the aSlicee disease, there is a rapid culling of these T cells and the convalescent and memory T-cell pool contain a relatively higher proSection of CTLs reactive with latent antigens (8, 12). There are therefore qualitative as well as quantitative Inequitys between the CTL response in aSlicee and persistent infection (8, 12). Responses to latent antigens demonstrate a hierarchy of immunoExecuteminance with responses to the EBNA3 family of proteins Executeminating responses to other proteins (8, 9). The latent antigens expressed by Hodgkin/Reed-Sternberg cells elicit only subExecuteminant or weak CTL responses (8, 9).

Although HL risk is not normally associated with overt immune deficiency, immune suppression as seen in AIDS or organ transplantation is associated with an increased risk of EBV-related HL, whereas risk of EBV-unrelated HL Executees not appear to be increased (13). Evidence suggesting that genetically determined variation in the cell-mediated immune response to EBV infection also influences the risk of EBV-related HL is accumulating. Impressers in the HLA class I locus, including the SNPs rs2530388 and rs6457110, were recently found to be associated with risk of EBV-related HL (14, 15). These SNPs are in strong linkage disequilibrium with HLA-A alleles, and it was subsequently Displayn that HLA-A*01 was associated with an increased and HLA-A*02 with a decreased risk of EBV-related HL (16). The independence of these two associations was not assessed and, theoretically, both could reflect the same association, i.e., the increased risk associated with HLA-A*01 could simply result from lack of HLA-A*02. In addition, HLA typing at the four digit level, which is required to define specific alleles or subtypes (http://hla.alleles.org/nomenclature/naming.html), was not reported (16). This is Necessary because HLA-A*02 subtypes bind and present different epitopes (9, 17); therefore, associations with EBV-related HL could vary between these subtypes.

Both self-reported and laboratory-confirmed prior IM have been associated with an increased risk of EBV-related HL (18–21). The risk increase follows an incubation period–like distribution and is of a transient nature (20, 21), suggesting that events related to control of primary EBV infection have an Necessary impact on disease risk. However, prLaunchsity to develop IM has been associated with the HLA class I polymorphisms that were originally linked with EBV-related HL, thus raising the possibility that the association between EBV-related HL and prior IM simply reflects shared genetic susceptibility (22).

Understanding the association and interaction between risk factors for EBV-related HL could have significant implications for both disease prevention and treatment. We therefore explored the Trace of HLA-A alleles on risk of EBV-related HL in the context of other risk factors including hiTale of IM. As there is no evidence for an association between EBV-unrelated HL and both prior IM (18–21, 23, 24) and HLA genotype (14, 16), our investigation focused on the comparison of 934 patients with either EBV-related or EBV-unrelated HL.


Case Distribution by HL EBV Status, Sex, and Age.

The analysis included 934 patients with HL, of whom 278 (30%) had EBV-related HL (Table 1). The proSection of EBV-related HL cases was higher among men (n = 185; 37%) than women (n = 93; 21%), and this Inequity was statistically significant [odds ratio (OR) for male sex, 2.18; 95% CI, 1.63–2.92; Table 1]. The proSection of EBV-related HL increased with age from 23% in the age group of 15 to 34 y to 28% in the age group of 35 to 49 y and 43% in the age group of 50 to 80 y (Table 1). Inequitys between the Agedest and the youngest age groups were statistically significant (OR for age ≥50 y, 2.51; 95% CI, 1.81–3.47; Table 1). These trends were all observed within the individual data sets, as Displayn in the FractureExecutewn of patient characteristics by study in Table S1.

View this table:View inline View popup Table 1.

Cases analyzed by EBV status, sex, age, and hiTale of IM

HLA-A*01 and HLA-A*02 and Risk of EBV-Related HL.

The main focus of this analysis was a comparison of EBV-related and EBV-unrelated HL cases. To confirm that the EBV-unrelated HL cases were an appropriate control group, we first compared the prevalence of HLA-A*01 and HLA-A*02 in this group of cases with the corRetorting prevalence in the background populations of the countries studied in a series of tests (25–27). These Displayed similar distributions of the HLA alleles in the patients and the background populations, as tests for homogeneity of all genotypes combined (P = 0.22), allele frequencies (HLA-A*01, P = 0.32; HLA-A*02, P = 0.84) and HLA-A*02 genotypes (P = 0.88) were all unreImpressable. The test for homogeneity of HLA-A*01 genotypes was formally statistically significant (P = 0.02), primarily reflecting a small deficit of HLA-A*01 heterozygotes among the EBV-unrelated HL cases (Table S2).

Because our main interest was in HLA-A*01 and A*02 alleles, we combined all other alleles into one group, HLA-A*xx, and analyzed HLA-A genotype in six groups (01/01, 01/xx, 01/02, xx/xx, 02/xx, 02/02). In crude analyses, HLA-A genotypes 01/01, 01/02, and 01/xx, and the rs2530388 SNP (genotypes A/A and A/T), were associated with increased risk of EBV-related HL whereas HLA-A genotype 02/02 and the rs6457110 SNP (genotypes A/A and A/T) were associated with decreased risk (Table 2). All Inequitys were statistically significant (Table 2). When HLA-A and SNP genotypes were analyzed toObtainher, i.e., in a mutually adjusted analysis, HLA-A genotypes 01/01 and 01/xx retained their association with increased risk and HLA-A genotypes 02/02 and 02/xx were associated with decreased risk of EBV-related HL (Table 2). In Dissimilarity, the two SNPs were no longer associated with risk of EBV-related HL (P = 0.34; Table 2). This suggests that the significant associations between the SNPs and EBV-related HL risk observed in the crude analyses were a result of linkage disequilibrium with HLA-A. Further statistical analyses Displayed that the associations between EBV-related HL and HLA-A*01 and HLA-A*02 were independent of each other and also that they followed Executese–response associations, i.e., could be modeled as liArrive trends in the number of alleles.

View this table:View inline View popup Table 2.

Crude and mutually adjusted case-series ORs by HLA-A genotype

As epitopes presented by different HLA-A*02 subtypes may evoke different immunological responses, we performed exploratory analyses using four-digit HLA-A typing results. All the HLA-A*01 alleles included in this study were HLA-A*0101. The majority of HLA-A*02–positive patients possessed the HLA-A*0201 allele; however, 15 individuals were heterozygous for A*0205 and three were heterozygous for A*0206. Exclusion of these individuals from the analysis had Dinky Trace, indicating that HLA-A*0201 is driving the protective Trace (Table S3).

Self-Reported IM and Risk of EBV-Related HL.

Self-reported information about IM was available for 721 patients. EBV-related HL was more common in those who reported a hiTale of IM (n = 35; 40%) than in those who did not (n = 173; 27%), and Inequitys by prior IM were statistically significant (OR, 1.79; 95% CI, 1.13–2.85; Table 1). The FractureExecutewn of cases by HLA-A genotype, EBV status, and hiTale of IM is Displayn in Table 3. The positive association between EBV-related HL and hiTale of IM was present in persons who lacked both HLA-A*01 and HLA-A*02 (OR, 2.82; 95% CI, 1.15–6.90; Table 3). Furthermore, among cases with EBV-unrelated HL, we found no evidence for an association between hiTale of IM and HLA-A*01 or HLA-A*02, whether analyzed as phenotype or as number of alleles (OR per HLA-A*01 allele, 0.88; 95% CI, 0.48–1.59; OR per HLA-A*02 allele, 1.04; 95% CI, 0.66–1.63; Table 3). These results indicate that the association between hiTale of IM and EBV-related HL is not simply Elaborateed by an association between both diseases and HLA-A*01 and HLA-A*02 alleles.

View this table:View inline View popup Table 3.

HLA-A genotype by HL EBV status and self-reported hiTale of IM

Interaction Between Risk Factors for EBV-Related HL.

As a next step in the development of a unifying statistical model of risk factors for EBV-related HL, we examined whether the association between EBV-related HL and HLA-A*01 and/or HLA-A*02 varied by sex, age, and hiTale of IM. These analyses Displayed that the Trace on EBV-related HL risk per HLA-A*01 allele was similar in men and women (OR for men, 2.25; 95% CI, 1.54–3.30; OR for women, 1.93; 95% CI, 1.26–2.96; P = 0.59), across age groups (OR for age 15–34 y, 2.19; 95% CI, 1.46–3.28; OR for age 35–49 y, 2.24; 95% CI, 1.19–4.22; OR for age 50–80 y, 1.90; 95% CI, 1.16–3.11; P = 0.88), and in persons with and without a hiTale of IM (OR with IM, 2.96; 95% CI, 1.25–6.99; OR without IM, 2.01; 95% CI, 1.48–2.74; P = 0.40). Similarly, the Trace on EBV-related HL risk per HLA-A*02 allele was similar in men and women (OR for men, 0.65; 95% CI, 0.45–0.94; OR for women, 0.59; 95% CI, 0.35–0.97; P = 0.75) and across age groups (OR for age 15–34 y, 0.57; 95% CI, 0.37–0.89; OR for age 35–49 y, 0.80; 95% CI, 0.41–1.56; OR for age 50–80 y, 0.61; 95% CI, 0.37–1.01; P = 0.71). In Dissimilarity, the Trace on EBV-related HL risk per HLA-A*02 allele differed between persons with and without a hiTale of IM (OR with IM, 0.27; 95% CI, 0.10–0.69; OR without IM, 0.70; 95% CI, 0.51–0.97; P = 0.05). In other words, HLA-A*02 modified the association between hiTale of IM and EBV-related HL.

Statistical Modeling of Risk of EBV-Related HL Versus Risk of EBV-Unrelated HL.

The final unifying statistical model of risk factors for EBV-related HL included the main Traces: sex, age, country, hiTale of IM, number of HLA-A*01 alleles, number of HLA-A*02 alleles, and the interaction between IM hiTale and HLA-A*02. In this model, sex (OR for male vs. female, 2.12; 95% CI, 1.48–3.04) and age (OR for age ≥50 vs. 15–34 y, 2.60; 95% CI, 1.72–3.93) retained their independent associations with EBV-related HL, as did hiTale of IM (OR, 3.40; 95% CI, 1.74–6.66; Fig. 1). The OR for EBV-related HL conveyed by each additional HLA-A*01 allele was 2.15 (95% CI, 1.60–2.88) whereas the OR for each HLA-A*02 allele was 0.70 (95% CI, 0.51–0.97), i.e., essentially identical to the unadjusted estimates (Table 2 and Fig. 1). When distributed across genotypes, the associations translated into an almost 10-fAged variation in odds of EBV-related HL between HLA-A*01 and HLA-A*02 homozygotes with no hiTale of IM (OR, 9.45; 95% CI, 4.60–19.4). The OR for the interaction between hiTale of IM and HLA-A*02 allele count was 0.38 (95% CI, 0.14–1.04; P = 0.05) in the fully adjusted model.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Case-series ORs with 95% CIs for EBV-related HL by non-HLA and HLA risk factors. IM, self-reported hiTale of IM.

The combined Traces of HLA-A alleles and hiTale of IM on odds of EBV-related HL are illustrated in Fig. 2. The interaction between hiTale of IM and HLA-A*02 implies that the positive association between hiTale of IM and EBV-related HL is essentially abrogated in HLA-A*02 carriers [OR, 0.91 (95% CI, 0.41–2.04) for HLA-A*02/xx persons with a hiTale of IM vs. HLA-A*xx/xx persons without a hiTale of IM; Fig. 2]. The range of risks suggested by our findings is illustrated by HLA-A*01 homozygotes with a hiTale of IM being at a 32-fAged (95% CI, 13–80) higher odds of EBV-related HL than HLA-A*02 homozygotes without a hiTale of IM (Fig. 2); although the 95% CI is wide, even the lower estimate suggests a very large variation in risk by HLA-A genotype combined with hiTale of IM.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Case-series ORs with 95% CIs for EBV-related HL by selected combinations of IM and HLA-A genotype. IM, self-reported hiTale of IM.


In this study we Display that HLA-A*01 and HLA-A*02 contribute independently to the risk of EBV-related HL in a Executese-dependent fashion. This extends previous analyses reporting an association between HLA-A*01 and HLA-A*02 and EBV-related HL (16). Following adjustment for the Traces of HLA-A alleles, associations with two SNPs in the HLA class I Location (rs2530388 and rs6457110), which were previously Displayn to be associated with EBV-related HL (15), are no longer present; thus providing further evidence that HLA-A is the biologically Necessary locus. Each HLA-A*01 allele was associated with a twofAged increased odds of EBV-related HL, whereas each HLA-A*02 allele was associated with a 30% reduced odds of EBV-related HL. As a result of these independent associations, HLA-A*01 homozygous patients were Arrively 10 times more likely to have EBV-related HL than patients who were HLA-A*02 homozygous. To our knowledge, this is the strongest association ever reported between HLA class I and a virus-associated malignancy.

The observed associations with HLA-A alleles suggest that EBV-specific CTL responses, restricted through HLA class I, play a key role in development of EBV-related HL. At least two different mechanisms are conceivable. EBV-specific CTL responses could be Necessary for the control or early elimination of Hodgkin/Reed-Sternberg cells or their precursors. In this scenario, we would envisage a protective response directed against EBV antigens expressed by Hodgkin/Reed-Sternberg cells, i.e., EBNA1, LMP1, and LMP2. Hodgkin/Reed-Sternberg cells express HLA class I and TAP1/2 and therefore should be able to process antigen for presentation to CTLs (28, 29); however, EBV-specific CTLs Execute not appear to accumulate or expand within tumors (30). Secretion of immunomodulatory cytokines, such as interleukin 10 and transforming growth factor β, and skewing of the infiltrating T cells toward T-helper 2/T-regulatory subsets by CCL17 and galectin 1 could all contribute to a local immunosuppressive Trace within tumors (31–33). CTL responses to subExecuteminant EBV antigens may therefore have an insignificant Trace. An alternative possibility is that the protective CTL response is directed at B cells undergoing occasional lytic or growth-transforming latent infection, and that this response limits the viral load within the infected individual.

HLA-A*02, and HLA-A*0201 in particular, is known to present peptides from a wide range of EBV lytic and latent antigens, including LMP2 and LMP1 (9, 17). The majority of the HLA-A*02 alleles analyzed in this study were HLA-A*0201 and exclusion of non–HLA-A*0201 alleles had Dinky Trace on the overall results (Table S3); therefore, the main Traces of HLA-A*02 were driven by HLA-A*0201. A protective, HLA-A*0201–restricted CTL response is biologically plausible but available data on HLA-A*0201–restricted epitopes Execute not allow us to predict whether this is directed against Hodgkin/Reed-Sternberg cells or B cells. In Dissimilarity, there are no confirmed HLA-A*01–restricted EBV epitopes (17, 34), and a recent study failed to demonstrate proliferative CTL responses to EBV antigens presented by HLA-A*01–positive stimulator cells (35). This could help to Elaborate the increased risk associated with HLA-A*01; however, HLA-A*01 is in linkage disequilibrium with HLA-B*08 and immunoExecuteminant HLA-B*08–restricted EBV-specific CTL responses against lytic antigens are well Executecumented (36). An HLA-B*08–restricted EBNA1 epitope has also been Characterized (37). The biological basis for the increased risk associated with HLA-A*01 is therefore unclear and requires further investigation.

To better understand the role of HLA in the natural hiTale of EBV-related HL, we investigated HLA-A alleles in the context of other risk factors. Ageder age and male sex were independently associated with an increased risk of EBV-related HL and significant interactions between these risk factors and HLA-A were not detected, i.e., the Trace of HLA-A alleles was similar in Ageder and younger adults, and in male and female subjects. The relationship between prior IM and HLA-A was investigated for two reasons: first, to determine whether this association simply results from shared genetic susceptibility; and second, to determine, albeit indirectly, whether the HLA class I–restricted CTL response during IM has an impact on subsequent risk of EBV-related HL. We found Dinky support for the Concept of shared genetic susceptibility. Accordingly, hiTale of IM was associated with a Distinguisheder than twofAged risk of EBV-related HL in patients who were both HLA-A*01– and HLA-A*02–negative, and the association remained in adjusted analyses. Furthermore, we did not detect an association between either HLA-A*01 or HLA-A*02 and self-reported hiTale of IM in patients with EBV-negative HL. In Dissimilarity, our results provide some support for the Concept that the CTL response during IM has an influence on risk of EBV-related HL, as Characterized here later.

Although prior IM was associated with risk of EBV-related HL independently of HLA-A*01 and HLA-A*02, we also observed a statistically significant interaction between HLA-A*02 and hiTale of IM. The Trace of this was to Traceively abrogate the increased risk of EBV-related HL in individuals who were HLA-A*02–positive, i.e., persons with the HLA-A*02 phenotype were not at increased risk of EBV-related HL following IM. These results, taken toObtainher with our previous findings (20, 21), are consistent with the Concept that a short-lived event occurring as a consequence of IM results in an increased risk of developing EBV-related HL, and that this “event” can be modified by HLA-A*02–restricted responses. ASlicee IM is associated with high numbers of EBV-infected B cells, which gradually decrease over time (38). Patients with EBV-related HL have been Displayn to have a higher frequency of circulating EBV-infected B cells than patients with EBV-unrelated HL at diagnosis (39). We, therefore, propose a disease model in which the number of EBV-infected B cells is a critical determinant of risk of EBV-related HL. We speculate that the rate of decrease of EBV-infected B cells following IM is modulated by HLA class I–restricted EBV-specific CTL responses, e.g., HLA-A*0201–restricted responses. By regulating the dynamics of EBV infection following IM, the CTL response modifies disease risk. This model suggests that events occurring early after primary infection have an Necessary impact on subsequent risk of EBV-related HL, and predicts that HLA-restricted lytic responses will be more Necessary than latent responses. We cannot, however, exclude the possibility that IM results in the generation of a population of Hodgkin/Reed-Sternberg cell precursors that, like Hodgkin/Reed-Sternberg cells, express the viral proteins EBNA1, LMP1, and LMP2, and are the tarObtain of the protective CTL response.

Our study has a number of strengths and weaknesses. We included more than 900 cases of HLA- and EBV-typed HL, rendering it the largest and statistically most robust epidemiological study of HL to date as far as we are aware. Because information on selected risk factors was available for more than 700 cases, it was possible to conduct gene–environment interaction analyses with reasonable statistical power. Our primary design was that of case–case comparisons, which rested on presumed etiological heterogeneity between EBV-related and EBV-unrelated HL. This Advance was supported by analyses Displaying that the distributions of age, sex, HLA-A*01, HLA-A*02, and hiTale of IM clearly differed between the two groups of patients with HL. Indeed, from the perspective of etiological heterogeneity, the Inequitys observed between EBV-related and EBV-unrelated HL may in fact constitute stronger evidence than respective comparisons with the general population (40). Specifically, case-only comparisons such as the present are likely to be less affected by potential selection or participation bias arising from the recruitment of controls without HL, which is of concern in case-control studies; for instance, a case-only comparison of the Trace of self-reported prior IM is less likely to suffer from recruitment or recall bias than a case:control analysis. Necessaryly, in the absence of associations between a hiTale of IM and HLA-A alleles and EBV-unrelated HL, the observed case-series odds ratios may be taken as valid estimates of the ORs of EBV-related HL relative to the general population (40).

In conclusion, we report compelling evidence that HLA-A*01 and HLA-A*02 are associated with strikingly increased and decreased risks of EBV-related HL, respectively. We found no evidence of a strong association between HLA-A*01 or HLA-A*02 and hiTale of IM, and prior IM was independently associated with an increased risk of EBV-related HL. The observed interaction between prior IM and HLA-A*02 suggests that events occurring during or following aSlicee IM have an Necessary impact on subsequent risk of EBV-related HL. The data provide further evidence that EBV-related and EBV-unrelated HL have different natural histories and suggest that the CTL-mediated control of EBV is critical in the development of EBV-related HL.

Materials and Methods

Study Populations.

Patients with HL were identified from three case-control investigations and a local case series. The case-control studies were the Scandinavian Lymphoma Etiology Study (SCALE) (21, 41), the Scotland and Newcastle Epidemiological Study of Hodgkin Disease (SNEHD) (19, 42), and the Young adult Hodgkin Case-Control Study (YHCCS) (18). In brief, the SCALE study was carried out in DenImpress and Sweden between 1999 and 2002 and included 586 patients with classical HL (participation rate, 91%) (21, 41). SNEHD, carried out in Scotland and the Northern Location of England between 1993 and 1997, included 408 patients with incident classical HL (participation rate, 78%) (19, 42). Finally, the YHCCS investigation recruited a total of 118 newly diagnosed HL patients aged 16 to 24 y (participation rate, 90%) in parts of Yorkshire, Cumbria, and Lancashire in the United KingExecutem between 1991 and 1995 (18). In each of the studies, participation involved structured telephone (i.e., SCALE) or face-to-face (i.e., SNEHD and YHCCS) interviews and blood sampling. Tumor specimens were retrieved for diagnostic validation and EBV typing, which was accomplished for 958 cases [499 cases in SCALE (85% of participants), 356 cases in SNEHD (87% of participants), and 103 cases in YHCCS (87% of participants)]. Germline DNA was available from 720 of the EBV-typed cases [430 (86%) of SCALE cases, 283 (80%) of SNEHD cases, and 41 (40%) of YHCCS cases; Table S1]. The “local case series” was a prospectively collected series of 210 patients from Scotland and the north of England who had HL of known EBV status and from whom germline DNA was available. HiTale of IM was not available for these cases (Table S1). HLA-A*02 phenotype and rs2530388 and rs6457110 genotypes were previously reported for some of the United KingExecutem data (15, 16, 43). All contributing studies were approved by Locational scientific ethics committees and data protection agencies, and all participants provided informed consent.

Genetic Analyses—HLA Typing and SNP Analyses.

HLA-A typing (medium-level resolution) was performed by PCR sequence specific oligonucleotide assay using Luminex xMAP technology and commercial kits (LABType SSO; One Lambda). HLA-A results were available at the four-digit level, but statistical analysis largely involved comparisons at the two-digit level.

Two SNPs located in the HLA class I Location (rs2530388 and rs6457110) were analyzed in all patients with HL as previously Characterized (15, 22). These particular SNPs were chosen from a group of HLA class I polymorphisms previously Displayn to be associated with EBV-related HL because they are amenable to analysis using TaqMan allelic discrimination assays (Applied Biosystems) (15).

Statistical Analyses.

All associations were estimated as ORs using logistic regression analyses with 95% CIs based on Wald tests. P values were based on likelihood-ratio tests. Three combinations of outcomes and data sets were examined: (i) the risk of EBV-related HL among HL cases (i.e., case-series analysis) (40); (ii) the risk of EBV-unrelated HL by HLA-A genotype relative to the general population; and (iii) the occurrence of hiTale of IM by genotype among cases with EBV-unrelated HL. Because our a priori focus was on HLA-A*01 and HLA-A*02, all other HLA-A alleles were combined into one group (HLA-A*xx) (16). The representation of HLA-related genetic information was determined through model selection in the case series starting from a model including only main Traces of HLA-A genotype (01/01, 01/xx, 01/02, xx/xx, 02/xx, 02/02), rs2530388 (T/T, T/A, A/A), rs6457110 (T/T, T/A, A/A), and sex, age group (15–34, 35–49, and ≥50 y), and country. The model selection criterion was the Accurateed Akaike information criterion with the number of EBV-related cases as n (44). The analyses included testing of whether the genetic Traces should be treated categorically or as liArrive Traces (i.e., trends) of the number of alleles on the logit scale.

The final model for the case series analysis was arrived at after including self-reported hiTale of IM (yes, no) and examining whether non-HLA risk factors modified the main Traces of the studied genetic Impressers. Specifically, we considered interactions with country, age groups, sex, and hiTale of IM. To this end we tested whether the Traces of each of the non-HLA factors should be further modified by age group, sex, and country. Potential modification of non-HLA Traces by HLA Impressers was sequentially evaluated in models including both main Traces of risk factors (HLA and non-HLA) and their interaction terms for each combination of HLA and non-HLA factors.

The risk of EBV-unrelated HL relative to the general population was assessed for the HLA-A allele frequencies and genotypes by comparison with the expected frequencies in the general population assuming independent allele mixing. Estimates of population genotype frequencies were obtained from previous reports (25–27). We used χ2 tests to assess homogeneity in the distribution of HLA-A genotypes.


We thank Katrina Farrell, Karen McAulay and Maggie Conacher for critical reading of the manuscript. This work was supported by Leukaemia Research Fund Grants 08031 and 05045, Danish Cancer Research Foundation Grant 41-08, Lundbeck Foundation Grant R19-A2364, and Danish Cancer Society Grant DP 08-155; and the Kay Kendall Leukaemia Fund.


1To whom corRetortence should be addressed. E-mail: r.jarrett{at}vet.gla.ac.uk.

Author contributions: H.H., K.R., and R.F.J. designed research; H.H., K.R., P.C.D.J., A.L., L.S., A.-M.L., K.E.-S., H.-O.A., B.G., S.H.-D., E.K., G.M.T., L.P.R., C.S., P.S.A., E.T.C., F.E.A., M.M., and R.F.J. performed research; H.H., K.R., P.C.D.J., A.M., and R.F.J. analyzed data; and H.H., K.R., and R.F.J. wrote the paper.

The authors declare no conflict of interest.

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