Epstein-Barr Virus-Associated Hodgkin's Disease

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EPSTEIN-BARR VIRUS-ASSOCIATED HODGKINS DISEASE: EPIDEMIOLOGIC

CHARACTERISTICS IN INTERNATIONAL DATA

Sally L. GLASER1*, Ruby J. LIN1, Susan L. STEWART1, Richard F. AMBINDER2, Ruth F. JARRETT3, Pierre BROUSSET4, Gorm PALLESEN5,Margaret L. GULLEY6, Gulfaraz KHAN7, Jane OGRADY8, Michael HUMMEL9, Maria Victoria PRECIADO10, Hans KNECHT11,John K.C. CHAN12 and Alexander CLAVIEZ13

1Northern California Cancer Center, Union City, CA2Johns Hopkins School of Medicine, Baltimore, MD3LRF Virus Centre, University of Glasgow, UK4Hopitaux de Toulouse, France5Aarhus Kommunehospital, Denmark6University of Texas Health Science Center, San Antonio, TX7Tufts University, Boston, MA8Oxfordshire Health Authority, Oxford, UK9Freie Universitat, Berlin, Germany10Hospital de Ninos Dr. Ricardo Gutierrez, Buenos Aires, Argentina11University of Massachusetts Medical Center, Worcester, MA12Queen Elizabeth Hospital, Kowloon, Hong Kong13Universitats-Kinderklinik, Kiel, Germany

Hodgkinsdisease (HD) haslongbeensuspected to haveaninfectious precursor, and indirect evidence has implicatedEpstein-Barr virus(EBV), aubiquitousherpesvirus,asa causalagent. Recent molecular studiesusingEBER in situhybridiza-tion or latency membrane protein-1 (LMP-1) immunohisto-chemistry haveidentifiedEBV latent infection in upto 50%ofHD tumors. However, the epidemiologic features of thesecases have not been examined in detail. To explore theepidemiology of EBV-positive HD so as to understand theroleof EBV in HD etiologymoreclearly, thisproject accumu-latedpatient datafrom14studiesthat hadapplied theseEBVassays to HD tumors. With information on age at diagnosis,sex, ethnicity, histologic subtype, country of residence, clini-cal stage and EBV tumor status from 1,546 HD patients, weexamined risk for EBV-positive disease using logistic regres-sion.Fortypercent of subjects had EBV-positivetumors,andEBV prevalence varied significantlyacross groups defined bythestudy variables. Oddsratios (OR) for EBV-associatedHD

weresignificantlyelevatedforHispanicsvs. whites(OR5 4.1),mixed cellularity vs. nodular sclerosis histologic subtypes(OR5 7.3,13.4, 4.9for ages014,1549,501 years), childrenfrom economically less-developed vs. more-developed re-gions and young adult males vs. females (OR5 2.5). Thesefindings suggest that age, sex, ethnicity and the physiologiceffects of poverty may represent biologic modifiers of theEBV association and confirm that thisassociation is stronglybut variably linked to histologic subtype. The data augmentbiologic evidence that EBV is actively involved in HD patho-genesis in some casesbut describe epidemiologic complexityin thisprocess. Int . J. Cancer, 70:375382,1997.

r 1997 Wiley-Liss, Inc.

Hodgkins disease (HD) is a malignant lymphoma whoseunusually heterogeneous clinical, histologic, and epidemiologiccharacteristics have suggested either a single disease entity with a

complex host response or 2 or 3 etiologically distinct conditions.For either interpretation, an infectious precursor has long beenproposed. This hypothesis was prompted by clinical symptoms ofHD such as cyclic fevers and night sweats, by the morphologicappearance of reactive tissue surrounding the malignant (Reed-Sternberg) cells and by epidemiologic findings of a bimodalage-incidence curve, geographic variation in the incidence inyoung persons and childhood social-class risk factors consistentwith HD as an uncommon outcome of delayed infection in youngadults (Kaplan, 1980; MacMahon, 1966; Correa and OConor,1971; Gutensohn and Cole, 1981).

Considerable evidence has pointed to an etiologic role forEpstein-Barr virus (EBV), a ubiquitous herpesvirus that has beenassociated with a number of lymphoid and epithelial malignancies.

Epidemiologic studies have shown that a history of mononucleo-sisa clinical manifestation of EBV infectiontriples the risk ofHD, and that HD patients have higher antibody titers than controlsubjects to the EBV antigens indicating primary infection and viralreactivation (Mueller, 1987). One study found that persons withserologic evidence of EBV infection had 2.5- to 4-times the risk ofdeveloping HD as persons without infection (Mueller et al.,1989).Biologic plausibility for an association between EBV and HD issuggested by observations that EBV immortalizes human B-lympho-cytes and renders them tumorigenic in immunodeficient mice, thatexpression of an EBV gene in immortalized rodent cell lines resultsin transformation bothin vitroand in vivoand that EBV is directlytumorigenic in some primates (Wang et al., 1985; Moorthy andThorley-Dawson, 1993; Wang et al., 1988; Kaye et al., 1993;Clearyet al.,1985).

A role for EBV in HD pathogenesis has been further supported

by the identification of latent EBV infection in a proportion of HDtumors, based on Southern blot DNA hybridization (Weiss et al.,1987; Staal et al., 1989; Anagnostopoulos et al., 1989), in situhybridization and antigen detection (Weiss et al.,1989; Wu et al.,1990; Pallesen et al.,1991a;Herbst et al.,1991), and polymerasechain reaction (PCR) (Herbst et al., 1990). Ultimately, assays forabundantly expressed viral transcripts (EBERs) and immunohisto-chemical assays for the latency membrane protein-1 (LMP-1), bothof which can be applied to archived tissue specimens, haveprovided direct evidence of EBV within Reed-Sternberg cells ortheir variants in as many as 40 to 50% of HD patients (Pallesen etal., 1991a;Gulleyet al.,1994; Hummelet al.,1992; Herbstet al.,1992; Khan et al., 1992, 1993; Deacon et al., 1993; Chang et al.,1993; Ambinder et al., 1993; Brousset et al., 1993; Zhou et al.,1993; Delsol et al., 1992, 1993; Poppema et al., 1994; OGrady etal., 1994; Tomita et al., 1996; Zarate-Osorno et al.,1995; Chanet

al., 1995; Preciado et al., 1995a and b; Claviez et al., 1994;Quintanilla-Martinez et al., 1996; Pinkus et al., 1994; Murray etal., 1992; Armstrong et al., 1992; Weiss et al., 1991; Joske et al.,1992).

These latter molecular studies suggest a new classification forHD tumors as EBV-positive or EBV-negative. Distinctive epidemio-logic features for these 2 subtypes, particularly features accounting

*Correspondence to: Northern California Cancer Center,32960 Alvarado-Niles Road, Suite 600, Union City, CA 94587, USA. Fax: 510-429-2550.e-mail: [email protected]

Received: August 5, 1996.

Int. J. Cancer:70, 375382 (1997)

r 1997 Wiley-Liss, Inc.

Publication of the International UnionAgainst Cancer

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for some of the established variation of HD by age, sex, ethnicityand histologic subtype, would help validate this classification,further support a pathogenetic function for EBV in HD, andprovide clues regarding additional risk factors. However, detailedepidemiologic characterization of EBV-classified HD has beenlimited. Some studies have reported EBV-positive HD to be morecommon in males, in 1 of the 4 histologic subtypes, in children andolder adults, in Hispanic populations, and in economically less-

developed countries (Pallesen et al., 1991a; Gulley et al., 1994;Chang et al., 1993; Ambinder et al., 1993; Khan et al., 1993;Zarate-Osorno et al., 1995; Quintanilla-Martinez et al., 1996;Armstrong et al., 1993; Jarrett et al., 1991; Weinreb et al., 1996).However, these findings, frequently based on small case seriesfrom populations with differing HD risks, vary widely and oftenconflict. Certain studies suggest EBV is independently associatedwith age, whereas others fail to support such an association (Gulleyet al., 1994; Khan et al., 1993; Quintanilla-Martinez et al., 1996;Jarrett et al., 1991). Across studies, EBV-positive HD has beendetected in 34 to 96% of males and 17 to 83% of females (Gulley etal.,1994; Changet al.,1993; Zhouet al.,1993; Vestlevet al.,1992;Pinkus et al., 1994). In white populations, EBV prevalence hasvaried within and across histologic subtypes (nodular sclerosis,1350%; mixed cellularity, 5096%; lymphocyte predominance,0100%; lymphocyte depletion, 0100%) (Pallesenet al., 1991a;

Herbstet al.,1991, 1992; Hummel et al., 1992; Khanet al., 1993;Delsol et al., 1993; Poppema et al., 1994; Vestlev et al., 1992;Pinkus et al., 1994; Murray et al., 1992; Armstrong et al., 1992;Weiss et al., 1991). Among Hispanics, it has ranged from 37 to100% (Gulley et al., 1994; Chang et al., 1993; Ambinder et al.,1993; Zarate-Osorno et al., 1995; Quintanilla-Martinez et al.,1996; Armstronget al.,1993).

Because of the correlations among the age-, sex-, ethnic-, andhistologic subtype-specific patterns of HD and the internationalvariation in its incidence, exploration of the epidemiologic featuresof EBV-classified HD requires multivariate analysis of a large anddiverse case series. Although individual laboratory studies haveoften lacked the requisite sample size, when combined they includeadequate numbers of patients to permit such an analysis. Therefore, thepurpose of this project was to aggregate patient data from studies thathad used reliable assays for EBV in order to explore in some detail

the epidemiologic characteristics of EBV-associated HD.

MATERIAL AND METHODS

Investigators were invited to participate in this study if they hadpublished results of EBV testing in HD tumors using EBER in situhybridization and/or LMP-1 assays before early 1995. For eachpatient included in their publications or tested subsequently,authors were asked to contribute data on age at diagnosis, sex,ethnicity, histologic subtype, clinical stage, year of diagnosis,country of residence at diagnosis, human immunodeficiency virus(HIV) status and outcome of EBER and/or LMP-1 tests. Of the 23research groups approached, 12 submitted case information [Jarrettet al. (Armstrong et al., 1992, 1993); Brousset et al. (Brousset etal.,1993; Delsol et al.,1992, 1993); Pallesenet al.(Pallesenet al.,1991aand b; Vestlev et al.,1992; Zhou et al.,1993; Levine et al.,

1994); Gulleyet al. (1994); Khan et al. (Khan et al.,1992, 1993);OGrady et al. (1994); Hummel et al. (1992); Ambinder et al.(1993); Preciadoet al.(Preciadoet al.,1995aand b; Knecht et al.(Knecht et al., 1991; Joske et al., 1992); Chan et al. (1995); andClaviez et al. (1994)]. This was augmented with one case seriespublished in its entirety (Zarate-Osorno et al., 1995) and oneunpublished series. In total, data were available on 1,566 HDpatients, including 368 who were not previously part of publishedwork. All tumors represented initial diagnoses, had been reviewedoriginally for uniform diagnosis and histologic subtyping, and hadbeen preserved in fixative before being assayed for EBV. TwentyHIV-positive subjects were excluded from analyses because of thevery high proportion of such patients with EBV-positive tumors

and the distinctive clinical behavior of HIV-associated HD (Tirelliet al.,1995).

The presence of EBV in Reed-Sternberg cells had been investi-gated for the 1,546 remaining patients; 505 were classified byEBER alone, 482 by LMP-1 alone, and 559 by both methods. Inthis last group, agreement between the 2 techniques was very high(k 5 0.929). Therefore, HD tumors were considered EBV-positiveif assay results in Reed-Sternberg cells or their variants were

positive by either method. HD tumors were considered EBV-negative if both assays were negative or, when only one assay wasused, its result was negative. Eleven patients were EBER-positivebut LMP-1-negative; 7 patients, all with the mixed cellularity (MC)subtype, were LMP-1positive but EBER-negative.

For descriptive statistics, age at diagnosis was grouped into5-year categories (04, 59 . . . 80 or over) and into 4 larger groups(014, 1539, 4054, 55 or over) consistent with the age-incidencepatterns of HD. Classification of histologic subtype used the 4standard Rye-conference categories (nodular sclerosis [NS], lym-phocyte predominance [LP], MC, and lymphocyte depletion [LD]).Although nodular LP HD may be an entity that is distinct fromother HD, in this study the 38 LP cases designated as nodular andthe 11 described as diffuse were combined with the 88 LP cases notsubclassified. Ethnicity was categorized as white, black, Asian,Hispanic, and other. Year of diagnosis was grouped as 19521979,

19801984, 19851989 and 19901994. To approximate thechildhood socioeconomic environment shown to affect HD inci-dence (Correa and OConor, 1971), regional economic develop-ment was estimated from the 1970 per capita gross nationalproduct (GNP) for countries with adult or mixed-age cases andfrom the 1988 per capita GNP for countries with childhood caseseries. Childhood socioeconomic environment was classified ashigher or lower based on a cut-point of the 30th percentile in GNPworld rank (Smith-Morris, 1990). With this approach, the UnitedStates, United Kingdom, European countries and Hong Kong wereclassified as having higher economic development; India, Chinaand countries in Central and South America and the Middle Eastwere classified as having lower economic development.

To evaluate the representativeness of our study series, wecompared the age and sex distributions for selected regionalsubgroups of our cases with HD cases from appropriate population-based cancer registries. Study subjects from the United Kingdom,Denmark, France and China (Peoples Republic and Hong Kong)were compared with registry cases diagnosed in 19831987 inthese areas (Parkin et al., 1992). United States study cases, anethnically diverse group primarily from the San Francisco BayArea, San Antonio, Texas and Georgia, were compared with19881992 population-based HD cases from California, a similarlyvaried population (Perkinset al.,1995).

Statistical analyses were undertaken using SAS version 6.11(SAS Institute, 1989). Differences across component case seriesand across case subgroups defined by study variables wereevaluated with chi-square statistics for frequency distributions andWilcoxon rank-sum tests for means. Because study cases wereneither population-based nor randomly selected, comparisons ofcharacteristics between EBV-positive and EBV-negative casespotentially would be confounded by the compositions of thecomponent case series, particularly as some had been assembled onthe basis of age, ethnicity and/or country for their increasedlikelihood of being EBV-positive (Gulley et al.,1994; Ambinderetal., 1993; Zarate-Osorno, 1995; Preciado et al., 1995a and b;Claviez et al., 1994; Armstrong et al., 1993). Therefore, wefocussed our analyses on exploring the risk of EBV-positive HDamong subsets of cases defined by each of the independentvariables.

To control for confounding, we modeled this risk using uncondi-tional logistic regression (Breslow and Day, 1980). For the models,age was grouped as 014, 1549 and 501 years; ethnicity as white,Hispanic and other; and histologic subtype as NS, MC and other to

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maximize statistical power and facilitate interpretation. Backwardelimination was used to delete variables from the initial model,producing a final model that was tested for goodness-of-fit by arestricted chi-square test. The models evaluated the effects of agegroup, sex, ethnicity, histologic subtype and regional economiclevel on the risk of EBV-positive HD, controlling for country,clinical stage and component case series. Because earlier reportssuggested that prevalence of EBV-positive HD in age groups variedby histologic subtype, sex and regional economic development, theinitial model included two-way interactions between age, histol-ogy, sex and economic level.

RESULTS

Differences across case series

Table I presents selected characteristics of the patient popula-tions for each of the 14 component case series. The numbers ofsubjects per series ranged from 21 to 304. These case series differedsignificantly (p # 0.01) in subjectsmean age and in their distribu-tions by sex, ethnicity, histologic subtype, country at diagnosis,economic level, year of diagnosis and EBV status, which variedfrom 23.1 to 66.7% positive. There were no differences in thedistributions of clinical stage across the 5 case series reportingstage data (Khan et al., 1992, 1993; OGrady et al., 1994;Zarate-Osornoet al.,1995; Preciadoet al.,1995aand b;Claviezetal.,1994).

Epidemiologic features of HD of the study sample

Study subjects were diagnosed between 1952 and 1994 (medianyear, 1989). Age at diagnosis ranged from 2 to 95, with a mean of33.7 years. Figure 1 illustrates the excess of male cases in

childhood and the young adult incidence peak characteristic of HDin incidence data (Perkins et al., 1995). The combined case seriescomprised a wide range of racial and ethnic groups, including 149Hispanics from the United States, Mexico, Honduras, Costa Ricaand Colombia; 26 Brazilians and 20 East Indians, among others.Patients resided in 24 countries, although 1,150 were from theUnited Kingdom, France, Denmark, or the United States. Table IIpresents the numbers of study cases by age, sex, ethnicity, andhistologic subtype. A total of 58.1% of the subjects was male, witha male excess in all groups except blacks and young adults withNS. Ethnic groups differed significantly in their distributions byage and histologic subtype. Children comprised approximately13% of whites and Hispanics, 26.9% of Asians, and 60.6% of

others. The proportion of NS cases ranged from 75.7% in blacks to49.3% in others, whereas MC occurred among 42.3% of Hispanicsbut only 13.5% of blacks. There was also a significant difference byregional economic level; the 221 cases from less-developed regionswere more likely than the 1,322 from more-developed areas to beyounger than age 15 (46.3%vs.10.5%), have the MC subtype (46.6vs.32.5%), and be of non-white background (83.3% vs.10.1%).

Study patients had lower mean ages than population-based casesfrom corresponding areas (United Kingdom, 34.2 vs. 41.8 years;Denmark, 38.3 vs. 44.3 years; France, 36.9 vs. 40.2 years; China,32.2 vs. 40.5 years; United States, 34.3 vs. 37.1 years). These

differences primarily reflected greater proportions of registry casesolder than age 50. Age distributions were similar between studyand registry cases only for Danish males and French females. Sexdistributions were similar between study and registry patients onlyamong those from the United Kingdom, Denmark, and France.

Predictors of EBV-positive HD

A total of 618 study subjects (40.0%) had EBV-positive HD.However, the proportions of cases with EBV-positive tumorsdiffered significantly (p # 0.001) across age, sex, ethnicity, histo-logic subtype, country of residence and regional economic level.Figure 2 shows that the highest percentages of EBV-positive casesin each 5-year age group occurred in children younger than 10 and

FIGURE 1 Relative frequency distribution of Hodgkins diseasestudy subjects by age and sex.

TABLE I HD PATIENT CHARACTERISTICS BY COMPONENT CASE SERIES

Case series Number Meanage (years)

%Male

%White

%European

%NS

%MC

%EBV1

Jarrettet al.,(Armstronget al.,1992, 1993)

304 31.4 60.2 88.8 88.8 53.9 32.7 3 5.2

Broussetet al.,(Broussetet al.,1993; Delsolet al.,1992, 1993)

266 39.6 63.5 96.2 100 37.4 50.2 35.0

Pallesenet al.,(Pallesenet al.,

1991a,b;Zhouet al.,1993;Vestlevet al.,1992; Levineetal.,1994)

236 37.0 59.8 88.1 88.1 60.9 29.4 4 4.9

Gulleyet al.,(1994) 171 36.3 66.1 11.8 0 61.2 34.7 46.8Khanet al.,(1992, 1993) 130 30.4 66.1 91.9 97.7 57.7 24.6 30.8OGradyet al.,(1994) 97 38.5 65.0 100 100 44.3 35.1 33.0Hummelet al.,(1992) 94 37.8 87.2 100 100 51.1 36.2 45.7Glaser and Ambinder, not previ-

ously published65 38.9 0 66.2 0 81.5 7.7 23.1

Ambinderet al.,(1993) 39 9.8 47.4 35.9 0 48.7 41.0 53.9Preciadoet al.,(1995a,b) 37 8.2 78.4 100 0 24.3 56.8 46.0Knechtet al.,(Joskeet al.,1992;

Knechtet al.,1991)36 39.0 69.4 94.4 94.4 55.6 33.3 58.3

Zarate-Osornoet al.,(1995)1 27 32.7 48.2 0 0 48.2 25.9 66.7Chanet al.,(1995) 23 39.3 78.3 0 0 69.6 21.7 65.2Claviezet al.,(1994) 21 10.4 57.1 100 100 52.4 42.9 47.6

1Data from publication only.

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in adults older than 80; the lowest percentages were in young

adults, particularly those 15 to 29 years old. Males were nearlytwice as likely to have EBV-associated tumors as females (47.7%vs. 29.2%). EBV-positive disease affected 35.9% of whites and16.2% of blacks, but 60 to 65% each of Asians, Hispanics, Indiansand persons of other ethnic origin. The MC subtype included thelargest proportion of EBV-positive disease (70.4%), and the LPsubtype included the smallest (16.0% overall, with 13.2% fornodular LP and 36.4% for diffuse LP); 23.2% of NS and 54.9% ofLD tumors were EBV-positive. The prevalence of EBV-positivedisease ranged widely among countries, from a maximum of 87.5%in Saudi Arabia (n 5 8) to a minimum of 30.8% in the UnitedKingdom (n 5 394). Persons from less-developed countries werealmost twice as likely to have EBV-positive HD as those frommore-developed regions (63.4%vs.36.0%).

With cases stratified by age, the distribution of EBV prevalencealso varied significantly (p , 0.001) across histologic subtypes,

ethnic groups, and economic levels. Figure 3 shows that in all agegroups, NS and particularly LP had relatively low percentages ofEBV-positive cases, whereas MC was associated with high levels.Despite this variation, EBV prevalence was uniformly higher inchildren than in young adults for all histologic subtypes. Acrossethnic categories, the respective EBV prevalence in the 4 agegroups (014, 1539, 4054, 551 years) was higher among Asians(92.9%, 38.1%, 50.0%, 81.8%), Hispanics (85.7%, 49.3%, 63.2%,66.7%) and others (74.4%, 40.0%, 66.7%, 100%) than amongwhites (46.2%, 29.6%, 37.6%, 48.3%) or blacks, who had rela-tively low proportions at all ages (16.7%, 13.0%, 33.3%, 20.0%).However, in almost all ethnic groups, the highest percentages withEBV-positive tumors occurred in children or in persons older than

55, and the lowest occurred consistently in young adults. For casesfrom economically less-developed regions, the percentages of EBVpositivity were higher than for cases from more-developed regionsin all 4 age groups (70.0%, 50.7%, 75.0%, 70.4% vs. 46.9%,29.2%, 38.2%, 50.0%, respectively); however, young adults in bothgroups experienced the lowest EBV prevalence.

Logistic regression analysis identified age group, sex, ethnicity,histologic subtype and regional economic level as significant, inde-pendent predictors of the risk of EBV-associated HD. After themodel controlled for the effects of the other factors, Hispanics had4 times the risk of EBV-positive disease as whites (odds ratio[OR] 5 4.1; 95% confidence interval, 1.89.6). The risks ofEBV-positive HD associated with histologic subtype, economiclevel, and sex were modified by age (Table III). MC tumors weresubstantially more likely to be EBV-positive than NS tumors forpersons of all ages but particularly for young adults, for whom therisk was 13-fold. The odds of HD being EBV positive weresignificantly elevated in economically less-developed than in

more-developed regions for children but not for older persons.Compared with males, females had half the risk of havingEBV-positive disease at young adult ages. Table IV shows how theOR associated with age varied by economic level, histologicsubtype and sex. In less-developed regions, the higher risk ofEBV-positive disease in children than young adults occurred forboth the NS and MC subtypes but was especially pronounced forNS (OR 5 10.0 and 14.3 for males and females). In more-developed regions, children with NS were at slightly higher risk ofhaving EBV-positive disease than young adults, although at asignificant level only for girls (OR 5 2.2). For persons older than50 compared with young adults, the effect of age on EBV positivitywas less marked and did not vary with regional economic level.

FIGURE2 Percentage of EBV-positive patients in each 5-year agegroup.

FIGURE 3 Percentage of EBV-positive patients in each histologicsubtype- and age-specific group.

TABLE II DISTRIBUTION OF STUDY SUBJECTS BY HISTOLOGIC SUBTYPE, ETHNICITY, AGE GROUP AND SEX

Ethnicity Whites Blacks Asians Hispanics Others

Age group 014 1539 4054 551 0 1 4 1 5 39 4 0 54 5 51 0 1 4 1 5 39 4 0 54 5 51 0 1 4 1 5 39 4 0 54 5 51 0 1 4 15 3 9 4 0 54 5 51

Males

NS 33 160 52 26 10 1 1 4 5 4 4 4 22 4 4 14 9 3 LP 10 42 25 13 1 1 1 1 1 1 1 1 3 2 1 MC 47 120 47 70 1 1 2 6 3 1 2 10 16 6 8 15 5 1 1

LD 2 11 5 8 1 1 1 2 1 Females

NS 30 198 36 42 4 10 1 1 1 10 3 25 4 7 4 3 1LP 2 10 9 8 1 1 1 MC 13 31 8 38 1 2 2 3 3 7 4 9 6 1 LD 3 1 1 4 3 2 1

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However, older women with NS had a nearly 4-fold higher EBVrisk than young adult women, and older men with MC hadapproximately half the EBV risk of younger men.

DISCUSSION

Earlier studies described epidemiologic features of EBV-associated HD, but these reports were based on EBV assays of

differing sensitivities and often involved small study samples. Thisproject has assembled the largest case series in which EBV latentgene products in Reed-Sternberg cells were evaluated by the 2standard reliable assays. The size and diversity of this series havepermitted us to explore epidemiologic patterns of EBV-positive HDamong relevant case subgroups and to examine risks of EBV-positive disease in these subgroups free of the effects of majorconfounders. Although the case series was neither population-based nor randomly selected, the age- and sex-specific distribution(Fig. 1) and the comparisons to regional registry data show that itwas reasonably representative of population-based HD cases withrespect to these demographic variables.

Analyses of our data have confirmed several epidemiologicfeatures of EBV-associated HD and identified characteristics thatwere significant predictors of risk after control for co-variables.Histologic subtype was the strongest risk factor for HD being EBV

positive. In our case series, EBV gene products were present inapproximately 75% of MC tumors but in only 25% of NS cases,and persons with MC had very high odds of having EBV-positivedisease compared with persons with NS. This elevated risk was notexplained by the higher frequency of MC in groups likely to haveEBV-positive tumors (children, Hispanics, residents of poorercountries). However, the particularly high odds for EBV-positiveMC in young adults does reflect the low likelihood of an EBVassociation for NS in this age group. Differences between MC andNS in extent and patterns of EBV association add to clinical andother epidemiologic evidence that these histologic subtypes repre-sent distinct entities (Cozenet al.,1992).

EBV-positive HD occurred in a large proportion of Hispanicpatients, who were 4 times as likely to have EBV-positive tumorsas whites, after control for the other factors. Prevalence and risk ofEBV-associated disease were also elevated in other non-whitegroups except blacks. Although the numbers of non-white caseswere too small to permit detailed evaluation of EBV patterns, ourfindings do demonstrate that ethnicity affects risk of EBV-associated HD independent of age, sex, histologic subtype and

nationality. Ethnic differences in EBV tumor association may bedue to unmeasured confounders (e.g., individual socioeconomicstatus) or may reflect variation in genetic susceptibility, as withHLA type (which affects risk of HD and differs among ethnicgroups) (Gutensohn, 1982; Klitzet al.,1994).

We have found that young adult females were half as likely asmales to have EBV-positive HD and were especially unlikely tohave EBV-positive NS, as illustrated in Table IV by the higher risksfor females of EBV-positive NS in childhood (OR 5 14.3, 2.2) andat older ages (OR 5 3.6, 3.9) relative to young adulthood. Thisage-specific protective effect of female gender is consistent with arole for female reproductive experience in the development ofEBV-positive HD, notably the NS subtype. Although there is nodirect evidence regarding reproductive risk for EBV-associatedmalignancies in general or HD in particular, experimental data dosupport an interaction of pregnancy-mediated immunosuppressivemechanisms (e.g., via glucocorticoids) with expression of EBVgene products, suggesting that such a biologic mechanism isplausible (Sargent, 1993; Glaseret al.,1995).

EBV-associated HD was infrequent in young adults, especiallythose with the NS subtype, and was more common in children andolder adults. This variation of EBV positivity with age andhistologic subtype is consistent with the multiple-etiology hypoth-esis, which states that the cause of HD differs by age group. Thefinding also implies that EBV is unlikely to play a primary role asthe infectious precursor to young adult HD predicted by earlierepidemiologic studies (Gutensohn and Cole, 1981; Jarrett, 1992;Glaser and Jarrett, 1996). The substantially increased risks ofEBV-positive NS and MC in children vs.young adults suggest thattiming of infection greatly affects the association of EBV with HDtumors, with early age at infection strongly predicting EBV-

positive disease for both these histologic subtypes. The variation inmagnitude of this age effect with regional economic level alsopoints to the importance of socioeconomic conditions and thusconceivably the virulence of and/or susceptibility to infection inpredicting the association of EBV in HD. For NS, the higher risk ofEBV positivity in children even in developed regions may primar-ily reflect the low risk of EBV association in NS for young adults.However, the presence of EBV in some NS tumors and the apparentimportance of host factors such as age and economic circumstancein predicting this association illustrate the complexity of therelation of this virus with the histologic expression of HD.

Our combined data confirm several previously described epide-miologic patterns of EBV-positive HD. Because the case series

TABLE III ADJUSTED ODDS RATIOS (OR) AND 95% CONFIDENCE INTERVALS(CI) FOR EBV-POSITIVE HD ASSOCIATED WITH HISTOLOGIC SUBTYPE,

REGIONAL ECONOMIC DEVELOPMENT AND SEX

Variable ComparisonAge

stratum(years)

AdjustedOR

95% CI

Histologic subtype MCvs. NS1 0141549501

7.313.4

4.9

3.814.29.019.92.88.7

Regional economicdevelopment

Lessvs. more1 0141549501

6.00.90.8

2.018.00.42.30.23.0

Sex Femalevs. male1 0141549501

0.60.41.2

0.31.10.30.60.72.0

1Referent group.

TABLE IV ADJUSTED ODDS RATIOS (OR) AND 95% CONFIDENCE INTERVALS (CI) FOR EBV-POSITIVE HDASSOCIATED WITH AGE

Vari able Comparison

StrataMales Females

Regional economicdevelopment

Histologicsubtype

AdjustedOR

95% CI Adjusted

OR 95% CI

Age 014vs. 1549 years1Less developed

NSMC

10.05.5

2.639.71.323.3

14.37.9

3.459.91.736.0

More developed NS

MC1.60.9

0.8 3.00.4 1.7

2.21.2

1.0 4.70.5 2.7

501 vs.1549 years1Less developed

NSMC

1.20.4

0.4 3.90.1 1.4

3.61.3

1.111.30.4 4.3

More developed NS

MC1.30.5

0.7 2.40.3 0.8

3.91.4

2.2 7.10.8 2.8

1Referent group.



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contributing to this project included a majority of subjects fromwhom the earlier findings were derived, confirmation was antici-pated. Given the significant differences in patient characteristicsamong these series (Table I), many of the discrepant findings inEBV prevalence from previous reports are likely due to epidemio-logic heterogeneity. Recent publications on EBV in HD case seriesnot included in this analysis also present results similar to ours.Among 50 Japanese patients with HD, Tomita et al. (1996)

detected EBV-positive tumors in 64%a rate similar to that foundin our study for Asians (mostly Chinese)and reported signifi-cantly elevated 5- to 7-fold risks of EBV-positive HD for the MCsubtype compared with all others, for males vs. females, and forpersons older than 40 vs. those younger. In 183 United Statespatients of unspecified ethnicity, Pinkus et al. (1994) foundEBV-positive tumors in 69% of MC, 13% of NS, and no cases ofLP and in significantly more males than females (33.7 vs. 17.4%).Quintanilla-Martinez et al. (1996) reported that the percentage of50 adult Mexicans with EBV-positive tumors was high (70%) anddid not vary significantly by age, sex or histologic subtype; thesefindings may largely reflect the higher risk of EBV-associated HDin Hispanics, given the absence of an independent risk of EBV-positive HD with regional economic development in adults in ourcase series. An international study of 277 pediatric HD patientsdescribed a protective effect of increasing age in childhood on risk

of EBV-positive disease, consistent with the pattern in Figure 2,and significant inter-country differences in risk (Weinreb et al.,1996). However, this analysis was not controlled for ethnicity orregional economic status, which may partly explain the associationof EBV positivity with nationality.

Our results have revealed significant and often large epidemio-logic differences in EBV-positive and EBV-negative HD, but theyalso have shown that the epidemiologic characteristics consideredhere do not discriminate neatly between the 2 virus-definedsubtypes of HD. To some extent, such incomplete concordancemay be a consequence of misclassification in our data, given thatpatient information, histologic diagnosis, and laboratory assays forthis project were not obtained under a single protocol. Although theobserved differences in EBV prevalence by histologic subtype areunlikely to be due to histologic misclassification, some of therelatively few EBV-positive NS cases might represent a subcat-

egory of NS, such as the LD variant, with a higher likelihood of beingEBV associated.Inconsistencies and errors in ethnic categorization are apossible source of bias; however, because U.S. Hispanics (one groupsubject to considerable misclassification) were identified both by

medical record report and surname linkage, bias in this groupshould be reduced (West et al., 1995). Finally, inter-laboratorydifferences in technical procedures or assay interpretation also havethe potential to affect our results (Pallesenet al.,1993).

More likely, the lack of complete definition of EBV-associatedHD by our study factors reflects a complex interplay of environmen-tal and host characteristics that are only partially understood andare inadequately measured by our study variables. In some ways,

this complexity is reminiscent of that characterizing Burkittslymphoma, which is also variably linked with EBV. As with HD,the association with EBV varies with age, incidence, and geogra-phy. Burkitts lymphoma is uniformly EBV-positive in equatorialAfrica, Papua New Guinea, and parts of South America, where it isa common cancer in children; however, it is only infrequentlyEBV-linked elsewhere, occurring as a relatively rare disease thataffects older persons (Magrath, 1991; Rickinson and Kieff, 1996;Gutierrez et al., 1992). However, unlike HD, the highly EBV-positive form of Burkitts lymphoma has been strongly associatedwith additional factors (e.g., holoendemic malaria). Moreover,irrespective of EBV association, Burkitts lymphomas share acommon histologic appearance and chromosomal translocations.

Findings regarding the association of EBV in HD in this largeinternational case series suggest that the host factors of age, sex,ethnicity and physiologic effects of poverty may represent biologic

modifiers of the involvement of EBV in HD pathogenesis andconfirm that the association is strongly linked to histologic subtype.However, the imprecision of each of these factors in predicting riskof EBV-positive HD reveals an epidemiologic complexity in therelation between virus and disease that exists over and above theeffects of small sample sizes and demographic differences of thecomponent case series. Ultimately, better knowledge of the biologyof this enigmatic lymphoma and identification of co-factors forEBV association will be required to generate a more exact epidemio-logic characterization and etiologic grasp of EBV-positive HD.

ACKNOWLEDGEMENTS

The authors acknowledge the contribution of case series by Dr.G. Barboa, Director de Patologia, Bogata, Colombia; Dr. V. Napoli,Department of Pathology, Grady Health System, Atlanta, GA; and

K. Swaminathan, Department of Pathology, Postgraduate Instituteof Basic Medical Sciences, Madras, India. They thank Ms. R.Leung, Ms. E. Satariano, M.P.H., and Mr. J. Hsu, M.P.H., for theircontributions to the project.

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Epstein-Barr Virus-Associated Hodgkin's Disease