VOLUME 27 䡠 NUMBER 23 䡠 AUGUST 10 2009

JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T

Latent Epstein-Barr Virus Infection of Tumor Cells in

From the Departments of Pathology
and Medical Biology and Hematology,
University Medical Center Groningen,
University of Groningen; the Compre-
hensive Cancer Center North Nether-
lands, Groningen; and the Department
of Public Health, Erasmus Medical
Center, Rotterdam, the Netherlands.
Submitted October 15, 2008; accepted
February 9, 2009; published online
ahead of print at www.jco.org on May
26, 2009.
Supported by Koningin Wilhelmina
Fonds (KWF) Grant No. RUG 2000-2315
from the Dutch Cancer Society and
Nederlandse organisatie voor Weten-
schappelijk Onderzoek–Medische
Wetenschappen (NWO-MW) Grant No.
920-03-136 from the Dutch Organiza-
tion of Scientific Research.
Presented at the 7th International
Symposium on Hodgkin’s Lymphoma,
November 3-7, 2007, Cologne,
Germany.
Authors’ disclosures of potential con-
flicts of interest and author contribu-
tions are found at the end of this
article.
Classical Hodgkin’s Lymphoma Predicts Adverse Outcome
in Older Adult Patients
Arjan Diepstra, Gustaaf W. van Imhoff, Michael Schaapveld, Henrike Karim-Kos, Anke van den Berg,
Edo Vellenga, and Sibrand Poppema
A B S T R A C T
Purpose
In classical Hodgkin’s lymphoma (cHL), the impact of tumor cell Epstein-Barr virus (EBV) status on
clinical outcome is controversial.
Patients and Methods
We assessed failure-free survival (FFS) and relative survival (RS) in 412 patients with cHL and
age-defined subgroups in a population-based study in the northern Netherlands. Tumor cell EBV
status was positive in 34%, and the median follow-up time was 7.1 years. Patients’ median age
at diagnosis was 35 years (range, 7 to 91 years), and 63% had Ann Arbor stage I or II, 24% had
stage III, and 12% had stage IV disease.
Results
EBV status influenced 5-year FFS and RS only in patients from the age group 50 to 74 years.
Five-year FFS was 60% in patients with EBV-positive versus 85% in EBV-negative tumors
(P ⫽ .01). Five-year RS was 69% in patients with EBV-positive versus 82% in EBV-negative tumors
(P ⫽ .03). After adjusting for histology, HLA class II expression by tumor cells, stage, presence of
extranodal localizations and treatment, and the effect of positive EBV tumor status remained
significant in FFS multivariate analysis (hazard ratio, 3.11; 95% CI, 1.28 to 7.53; P ⫽ .01).
Conclusion
This study indicates that treatment failure in older adult patients with cHL is associated with
positive tumor cell EBV status.
J Clin Oncol 27:3815-3821. © 2009 by American Society of Clinical Oncology

Corresponding author: Arjan Diepstra,
MD, PhD, Department of Pathology and
Medical Biology, University Medical
Center Groningen, Hanzeplein 1, PO
Box 30.001, 9700 RB Groningen, the
Netherlands; e-mail: a.diepstra@
path.umcg.nl.
© 2009 by American Society of Clinical
Oncology
0732-183X/09/2723-3815/$20.00
DOI: 10.1200/JCO.2008.20.5138
INTRODUCTION
Classical Hodgkin’s lymphoma (cHL) is character-
ized by sparse B cell– derived neoplastic Hodgkin’s
Reed-Sternberg (HRS) cells scattered within an
abundant inflammatory infiltrate. After the initial
demonstration that HRS cells may be positive for
Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-
1),1 these cells were later shown to harbor monoclo-
nal EBV genomes in a variable proportion of
patients, indicating clonal expansion of a single
EBV-infected B cell.2 The virus has transforming
potential, and latent infection is accepted as a causal
event in EBV-positive cHL.3
EBV tumor positivity varies with age, sex,
and histologic subtype, and the percentage of pa-
tients with EBV-positive HRS cells differs
throughout the world, ranging from 20% in some
industrialized countries to more than 90% in less
developed countries.3-5 In HRS cells the virus ex-
presses only latent membrane protein 1 (LMP-1),
LMP-2, EBNA-1, and EBV-encoded small RNAs
(EBERs). LMP-1 acts as a constitutive active tumor
necrosis factor receptor, driving activation of the
transcription factor nuclear factor ␬ B, and LMP-2 is
an antiapoptotic factor.6,7 These proteins are con-
sidered to be essential for EBV-driven malignant
transformation. Compared with EBV-negative
cHL, HRS cells in EBV-positive cHL more often
express HLA class I and HLA class II, and proba-
bly are able to present EBV-derived antigenic pep-
tides to the immune system.8-11 Accordingly, in
EBV-positive cHL, the reactive infiltrate usually
contains more cytotoxic T cells compared with
EBV-negative cHL.12
The effect of EBV status on outcome in cHL
is controversial, and different studies have shown
no, a favorable, or an unfavorable impact on
outcome.13-35 These differences may be related to
varying demographics, patient selection, and power
and type of statistical analysis. In addition, age has
emerged as an important confounding factor. Some

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 Diepstra et al

studies have shown that positive EBV status is related to adverse

Table 1. EBV Status and Clinicopathologic Characteristics
prognosis in patients older than 45 to 50 years.22,25,31,33 Conversely, a
number of reports indicate that in children, adolescents, and young All Patients
EBV-Positive
adult patients (35 years or younger), EBV positivity may be associated Characteristic No. % Patients (%) P
with a favorable prognosis.20,24,27,31,35 Total 412 34.2
We recently showed that absence of HLA class II protein expres- Age, years .030ⴱ
sion on the cell surface of HRS cells is an independent adverse prog- 7-14 18 4.4 33.3
nostic factor in a population-based study in the northern parts of the 15-34 192 46.6 27.6
35-49 86 20.9 33.7
Netherlands.11 In the same population, we now present data on the
50-74 98 23.7 44.9
effect of EBV tumor status on failure-free survival (FFS) and relative ⱖ 75 18 4.4 50.0
survival (RS) in all patients and in age-defined subgroups. Sex .001ⴱ
Male 240 58.3 40.8
Female 172 41.7 25.0
PATIENTS AND METHODS Ann Arbor stage .002ⴱ
I 69 16.8 49.2
All 562 patients with Hodgkin’s lymphoma diagnosed from January 1989 II 193 46.8 25.9
through December 2000 were selected from the regional cancer registry of III 100 24.3 40.0
the Comprehensive Cancer Centre North Netherlands (Groningen, the IV 50 12.1 34.0
Netherlands). The histology of the diagnostic lymph node biopsies was B symptoms .330ⴱ
reclassified according to the WHO classification by two pathologists (A.D. Present 168 40.8 38.1
and S.P.), and the presence of EBV in HRS cells was detected by EBER in Absent 234 56.8 31.2
situ hybridization (ISH).36 Data on membranous HLA class II protein Unknown 10 2.4 40.0
expression on HRS cells of these patients were published previously and are Bulky disease .156ⴱ
included in the analysis.11 Eighty-two patients were excluded because there Present 111 26.9 27.0
was not enough material for reclassification or EBER ISH. Reclassified Absent 288 69.9 37.2
patients were excluded for the following reasons: equivocal histologic Unknown 13 3.2 30.1
diagnosis (n ⫽ 6), diagnosis at autopsy (n ⫽ 8), presence of a second Location .567ⴱ
malignancy (hematologic, n ⫽ 6; other, n ⫽ 3), no curative treatment Nodal only 340 82.5 35.3
initiated (n ⫽ 6; four of six patients older than 75 years), or no follow-up Extranodal 60 14.6 28.3
data (n ⫽ 16). Nodular lymphocyte–predominant Hodgkin’s lymphoma Unknown 12 2.9 33.3
patients (n ⫽ 23) were excluded because this subtype is considered to be a HLA class II expression .017ⴱ
different entity from cHL and is not associated with EBV.37 Positive 170 41.3 39.4
In total, 412 patients were eligible for our study. Patients had been Negative 121 29.4 24.0
staged at initial diagnosis according to the Ann Arbor staging system with Unknown 121 29.4 37.2
Cotswold modification. Additional clinical data were collected on age, sex, Type of chemotherapy .703ⴱ
presence or absence of B symptoms, bulky disease (⬎ 10 cm, or any MOPP/ABV 215 52.2 31.6
mediastinal mass more than one third of chest diameter on posteroanterior ABVD 33 8.0 33.3
x-ray), extranodal disease, year of diagnosis, and type and intensity of EBVP 26 6.3 26.9
treatment. Patients were treated in accordance with Comprehensive Can- MOPP 17 4.1 47.1
cer Centre North Netherlands guidelines. Dates of treatment failure and LOPP 7 1.7 42.9
death were recorded until January 2005. The study was approved by the Other chemotherapy 30 7.3 33.3
medical ethics board of the University Medical Center Groningen (Gro- Unknown 13 3.2 46.2
ningen, the Netherlands). Not applicable 71 17.2 39.4
Type of treatment .471ⴱ
Survival Analysis Chemotherapy only 133 32.3 35.3
Clinical outcome was determined in all patients. In addition, patients Chemotherapy and RT 208 50.5 31.7
were divided into age subgroups according to the four-diseases model as RT only 71 17.2 39.4
proposed by the Scotland and Newcastle Epidemiology of Hodgkin’s Disease Subtype ⬍ .001†
Study Group (0 to 14, 15 to 34, 35 to 49, and 50 to 74 and an additional Nodular sclerosis 339 82.3 26.5
subgroup of 75 years or older).3,13 Mixed cellularity 47 11.4 76.6
FFS was measured from date of diagnosis to date of recurrence, date Lymphocyte rich 9 2.2 33.3
of progression, or date of death with active cHL. Patients were censored Lymphocyte depleted 6 1.4 66.7
when alive and free of cHL disease at last follow-up. Follow-up time for RS Not otherwise
was measured from date of diagnosis to date of last follow-up or date of specified 11 2.7 72.7
death.38 Expected survival probabilities of the general Dutch population
Abbreviations: EBV, Epstein-Barr virus; MOPP, mechlorethamine, vincristine,
per year and by age and sex were provided by the Dutch central statistics procarbazine, prednisone; ABV, doxorubicin, bleomycin, vinblastine; ABVD,
office, Statistics Netherlands (Voorburg, the Netherlands). Prognostic fac- doxorubicin, bleomycin, vinblastine, dacarbazine; EBVP, epirubicin, bleomycin,
tors associated with survival end points in univariate analysis at a level of vinblastine, prednisone; LOPP, chlorambucil, vincristine, procarbazine, pred-
significance of P ⫽ .1 were subsequently tested in multivariate models. A nisone; RT, radiotherapy.
ⴱ 2
␹ test for independence in EBV-positive compared with EBV-negative patients.
Cox proportional hazards model was used to assess the association of EBV †Fisher exact test for independence in EBV-positive compared with EBV-
status with FFS while adjusting for relevant cofactors. A Poisson regression negative patients.
model was used to model the association of EBV status with RS.39 P
values ⱕ .05 (two-sided) were considered to be significant. The statistical
analyses were done using STATA 8.0 (STATA, College Station, TX) and
SPSS 11.5 (SPSS, Chicago, IL) software.

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 EBV Predicts Adverse Outcome in Hodgkin’s Lymphoma

Table 2. EBV Status and Clinicopathologic Characteristics in Subgroup of Table 3. Univariate Analysis of FFS
Patients Age 50 to 74 Years
No. of 5-Year ␹2
All Patients Characteristic Patients FFS (%) HR 95% CI Test P
EBV-Positive
Characteristic No. % Patients (%) P Age, years ⬍ .001
7-14 18 78 1.57 0.50 to 4.88
Total 98 44.9
15-34 192 80 1.51 0.79 to 2.98
Sex .561ⴱ
35-49 86 86 1.00 Ref
Male 61 62.2 42.6
50-74 98 74 1.95 0.97 to 3.91
Female 37 37.8 48.6
ⱖ 75 18 48 6.85 2.87 to 16.29
Ann Arbor stage .493ⴱ
Sex .629
I 23 23.5 47.8
Male 241 80 1.00 Ref
II 32 32.7 34.4
Female 171 77 1.11 0.72 to 1.69
III 22 22.5 54.5
Stage .047
IV 21 21.4 47.6
I 69 78 1.06 0.57 to 1.96
B symptoms .067†
II 193 81 1.00 Ref
Present 53 54.1 52.8
III 100 80 1.11 0.64 to 1.92
Absent 44 44.9 34.1
IV 50 65 2.22 1.24 to 3.94
Unknown 1 1.0 100
B symptoms .099
Bulky disease .298†
Absent 234 82 1.00 Ref
Present 18 18.4 33.3
Present 168 75 1.43 0.93 to 2.20
Absent 79 80.6 46.8
Unknown 10 60 —
Unknown 1 1.0 100
Bulky disease .213
Location .631†
Absent 288 78 1.00 Ref
Nodal only 73 74.5 45.2
Present 111 83 0.72 0.43 to 1.21
Extranodal 24 24.5 41.7
Unknown 13 59 —
Unknown 1 1.0 100
Location .007
HLA class II expression .304ⴱ
Nodal only 340 81 1.00 Ref
Positive 38 38.8 52.6
Extranodal 60 65 2.15 1.30 to 3.56
Negative 27 27.6 33.3
Unknown 12 64 —
Unknown 33 33.7 45.5
Hodgkin subtype .030
Type of treatment .094ⴱ
Nodular sclerosis 339 79 1.00 Ref
Chemotherapy only 38 38.0 57.9
Mixed cellularity 47 85 0.68 0.31 to 1.48
Chemotherapy and RT 46 46.0 39.1
Other 26 62 2.15 1.11 to 4.18
RT only 14 14.0 28.6
HLA class II
Type of chemotherapy .701†
expression .004
MOPP/ABV 54 55.1 46.3
Positive 170 85 1.00 Ref
ABVD 10 10.2 40.0
Negative 121 68 2.31 1.39 to 3.84
EBVP 1 1.0 100
Unknown 121 79 1.51 0.87 to 2.62
MOPP 6 6.1 66.7
EBV, all patients .429
LOPP 4 4.1 50.0
Negative 271 80 1.00 Ref
Other chemotherapy 3 3.1 66.7
Positive 141 76 1.19 0.77 to 1.84
Unknown 6 6.1 33.3
EBV, age 15-34 years .262
Not applicable 14 14.3 28.6
Negative 139 78 1.00 Ref
Subtype .002†
Positive 53 85 0.64 0.29 to 1.40
Nodular sclerosis 73 74.5 37.0
EBV, age 50-74 years .010
Mixed cellularity 13 13.3 69.2
Negative 54 85 1.00 Ref
Lymphocyte rich 3 3.1 0.0
Positive 44 60 2.90 1.24 to 6.79
Lymphocyte depleted 3 3.1 100
Not otherwise Abbreviations: FFS, failure-free survival; HR, hazard ratio; Ref, reference;
specified 6 6.1 83.3 EBV, Epstein-Barr virus.

Abbreviations: EBV, Epstein-Barr virus; RT, radiotherapy; MOPP, mechloreth-

amine, vincristine, procarbazine, prednisone; ABV, doxorubicin, bleomycin,
vinblastine; ABVD, doxorubicin, bleomycin, vinblastine, dacarbazine; EBVP,
epirubicin, bleomycin, vinblastine, prednisone; LOPP, chlorambucil, vincris-
tine, procarbazine, prednisone. and 41 years (range, 7 to 88 years) for patients with EBV-positive cHL
ⴱ 2
␹ test for independence in EBV-positive compared with EBV-negative patients.
†Fisher exact test for independence in EBV-positive compared with EBV-
(P ⬍ .001). In the majority of patients, first-line treatment consisted of
negative patients. chemotherapy combined with radiotherapy or chemotherapy only
(83%). A small number of patients diagnosed between 1989 and 1992
with pathologic stage I or II disease who had been pathologically
staged with laparotomy and splenectomy were treated with radiother-
RESULTS apy only.

Patient Characteristics EBV Tumor Status

Clinicopathologic characteristics are shown in Table 1. The me- EBER ISH staining results were unequivocal and showed that
dian age at diagnosis was 35 years (range, 7 to 91 years) for all patients 34% of patients had EBV-positive cHL. Young adult patients (age 15

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 Diepstra et al

to 34 years) were more likely to have EBV-negative cHL, whereas
EBV-positive status was more frequent in patients age 50 years or
older. EBV-positive cHL was more common in male patients. Com-
pared with patients with Ann Arbor stage II disease, patients with stage
I disease were more likely to have EBV-associated cHL. There was,
however, no significant difference between stages I and II combined
compared with stages III and IV combined. The mixed cellularity
histologic subtype showed the strongest association with EBV-positive
status (Table 1). This was the only association that was also significant
in the group of patients age 50 to 74 years (Table 2).

All ages All ages

Failure-Free Survival (%)

100 100

Cumulative Relative
80 80

Survival (%)
60 60
40 EBV positive 40 EBV positive
EBV negative EBV negative
20 20
P = .429 P = .168

0 1 2 3 4 5 0 5 10 15
Time (years) Time (years)
No. at risk
EBV+ 141 116 70 25
EBV- 270 229 130 38

15-34 years 15-34 years

Failure-Free Survival (%)

100 100
Cumulative Relative

80 80
Survival (%)

60 60
40 EBV positive 40 EBV positive
EBV negative EBV negative
20 20
P = .262 P = .100

0 1 2 3 4 5 0 5 10 15
Time (years) Time (years)
No. at risk Fig 1. Failure-free survival (left) and rela-
EBV+ 53 15 36 16 tive survival (right) in patients with classi-
EBV- 138 121 66 23 cal Hodgkin’s lymphoma and age-defined
subgroups. In the relative survival graphs,
35-49 years 35-49 years the number of patients at risk is indicated
below the x-axis. EBV, Epstein-Barr virus.
Failure-Free Survival (%)

100 100
Cumulative Relative

80 80
Survival (%)

60 60
40 40
EBV positive EBV positive
20 EBV negative 20 EBV negative
P = .473 P = .489

0 1 2 3 4 5 0 5 10 15
Time (years) Time (years)
No. at risk
EBV+ 29 25 18 6
EBV- 57 50 31 9

50-74 years 50-74 years

Failure-Free Survival (%)

100 100
EBV positive
Cumulative Relative

80 80 EBV negative
Survival (%)

60 60
40 40
EBV positive
20 EBV negative 20
P = .010 P = .010

0 1 2 3 4 5 0 5 10 15
Time (years) Time (years)
No. at risk
EBV+ 44 28 13 2
EBV- 54 44 23 3

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 EBV Predicts Adverse Outcome in Hodgkin’s Lymphoma

FFS
Table 4. Univariate Analysis of RS
In univariate analysis, age ⱖ 50 years, stage, extranodal disease,
infrequent histologic subtype, and absence of HLA class II protein 5-Year ␹2
Characteristic No. RS (%) RER 95% CI Test P
expression were associated with adverse FFS. There was no effect of
Age, years ⬍ .001
EBV status in the total group of patients including all ages. However, in
7-14 18 89 0.65 0.14 to 2.98
patients age 50 to 74 years, positive EBV status was significantly asso- 15-34 192 91 0.78 0.38 to 1.58
ciated with a worse FFS. In this age group, 5-year FFS for EBV-positive 35-49 86 89 1.00 Ref
patients was 60% compared with 85% for EBV-negative patients 50-74 98 76 2.90 1.47 to 5.72
(P ⫽ .010; Table 3; Fig 1, left column). This effect proved to be ⱖ 75 18 44 8.75 3.41 to 22.41
independent of extranodal disease (stage IV), histology, HLA class II Sex .164
expression status, and treatment in multivariate analysis, with an HR Male 241 88 1.00 Ref
Female 171 81 1.39 0.87 to 2.23
of 3.11 (95% CI, 1.28 to 7.53; Table 5).
Stage .073
I 69 87 1.44 0.72 to 2.85
RS II 193 90 1.00 Ref
RS was defined as excess mortality over and above the mortality III 100 82 1.59 0.88 to 2.85
of the general Dutch population per calendar year, accounting for age IV 50 70 2.41 1.23 to 4.69
and sex. Univariate analysis showed that age, B symptoms, extranodal B symptoms .003
Absent 234 90 1.00 Ref
disease (stage IV), and histology were associated with unfavorable RS.
Present 168 81 2.11 1.28 to 3.46
There was no effect of EBV status in the total group of patients (all Unknown 10 64 —
ages). In young adults (age 15 to 34 years) there was a nonsignificant Bulky disease .460
trend for EBV-positive status with improved RS Table 4; Fig 1, right Absent 288 85 1.00 Ref
column). In contrast, EBV-positive status was significantly associated Present 111 88 0.81 0.45 to 1.43
with adverse RS in patients age 50 to 74 years. Five-year RS was 69% Unknown 13 66 —
for EBV-positive patients compared with 82% for EBV-negative pa- Location .027
Nodal only 340 88 1.00 Ref
tients (P ⫽ .033; Table 5; Fig 1, right column). However, this effect was
Extranodal 60 71 1.91 1.07 to 3.38
not significant in multivariate analysis (Table 5). Unknown 12 71 —
Hodgkin’s subtype .010
Nodular sclerosis 339 86 1.00 Ref
DISCUSSION
Mixed cellularity 47 91 0.89 0.39 to 2.03
In this retrospective population-based study, we show that presence of Other 26 60 2.74 1.40 to 5.34
EBV in HRS cells is an independent adverse prognostic factor in HLA class II
expression .114
patients with cHL age 50 to 74 years. In this age group, positive EBV Positive 170 89 1.00 Ref
status was associated with treatment failure. In univariate analysis Negative 121 78 1.80 1.01 to 3.20
there was also a negative effect of EBV involvement on RS; however, Unknown 121 87 1.63 0.89 to 2.96
this effect did not remain significant in multivariate analysis. In addi- EBV, all patients .106
tion, in young adult patients with EBV-positive cHL, there was a Negative 271 86 1.00 Ref
Positive 141 84 1.47 0.92 to 2.38
nonsignificant trend toward favorable outcome. Because of differen-
EBV, age 15-34 years .164
tial effects of EBV status in different age-defined patient groups, EBV Negative 139 88 1.00 Ref
status did not correlate with prognosis in the entire population. Positive 53 97 0.42 0.12 to 1.43
Associations of latent EBV infection of HRS cells with age, male EBV, age 50-74 years .033
sex, and mixed cellularity subtype have been previously established Negative 54 82 1.00 Ref
and are also present in our data.4 In addition, we found that among Positive 44 69 2.47 1.07 to 5.66
patients with early-stage disease, patients with EBV-positive cHL were Abbreviations: RS, relative survival; RER, relative excess risk of death; Ref,
more likely to have stage I disease than were patients with EBV- reference; EBV, Epstein-Barr virus.
negative cHL. This association had been demonstrated previously in
young adult patients with cHL in the United Kingdom by Jarrett et
al,31 but there is no obvious biologic explanation for this. study in the United States by Keegan et al33 (that included the patients
In fact, the population-based study of Jarrett et al31 shows many previously studied by Clarke et al22) showed an association of EBV-
similarities to ours. Homogeneity of ethnic background, sample size, positive cHL with adverse OS and DSS in patients older than 45 years.
and follow-up time are comparable, and subgroup analyses were per- These studies have been criticized for using OS and DSS as clin-
formed in the same age groups. In both studies, EBV tumor status was ical end points. OS is affected by salvage management after relapse and
determined using EBER ISH, and patients with nodular lymphocyte– the occurrence of disease-unrelated deaths. Because the frequency of
predominant Hodgkin’s lymphoma were excluded. In the patient disease-unrelated deaths is relatively high in the elderly, these deaths
group age 50 to 74 years, Jarrett et al31 found adverse overall survival may obscure disease effects in older adult patients. DSS is also affected
(OS) for patients with EBV-positive cHL. In addition, another by treatment after relapse, and in the elderly, it may be difficult to
population-based study from the same group in the United Kingdom distinguish between cHL-related and cHL-unrelated causes of death.
showed adverse disease-specific survival (DSS) in patients with EBV- It has been suggested that FFS would be a more optimal end point
positive cHL age 60 years and older.25 Finally, a large population-based because it is related to the biologic aggressiveness of the disease.34 In

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 Diepstra et al

Table 5. Multivariate Analysis of Failure-Free and Relative Survival

Failure-Free Survivalⴱ Relative Survival†

Age (years) EBV Status No. HR 95% CI P RER 95% CI P

All ages Negative 271 1.00 Ref .690 1.00 Ref .671
Positive 141 1.10 0.68 to 1.78 1.15 0.64 to 2.03
15-34 Negative 139 1.00 Ref .745 1.00 Ref .267
Positive 53 0.88 0.39 to 1.96 0.49 0.13 to 1.75
50-74 Negative 54 1.00 Ref .012 1.00 Ref .095
Positive 44 3.11 1.28 to 7.53 1.88 0.87 to 5.20

Abbreviations: EBV, Epstein-Barr virus; HR, hazard ratio; RER, relative excess risk of death; Ref, reference.
ⴱ
Cox proportional hazards model for failure-free survival by EBV status, adjusted for age, presence of extranodal locations (stage IV), histology, and HLA class II
expression for all ages and adjusted for extranodal locations, histology, and HLA class II expression within age groups.
†Poisson regression model for relative survival by EBV status, adjusted for age, presence of extranodal locations (stage IV), B symptoms, histology, and HLA class
II expression for all ages and adjusted for extranodal locations, histology, and HLA class II expression within age groups.

our study we show that the adverse effect of EBV-positive status in
older adult patients with cHL as described in terms of OS and DSS by
Jarrett et al,31 Keegan et al, 33 and Stark et al25 is also found with FFS.
In addition, we used RS as a clinical end point. RS is an estimation
of DSS. It is defined as the absolute survival rate among patients
corrected for the expected survival rate from the general population
with the same sex and age structure (background mortality). As a
consequence, RS includes late deaths related to treatment and toxicity
although it is difficult to assign precise causes of death.38 Therefore, RS
may be more informative than OS and DSS in older adult patients with
cHL. However, we did not find an independent effect of EBV status on
RS in our study.
Although our results are in line with those of Keegan et al,33
Jarrett et al,31 and Stark et al,25 other reports describing clinical out-
come in relation to EBV status are conflicting. Many studies did not
show an effect of EBV status on prognosis but usually less than 120
patients were studied.13,14,16,17,26,29,30,34 Studies from Spain, Brazil,
South Africa, and India with a high percentage of EBV-involved pa-
tients conclude that EBV-positive status is associated with a favorable
clinical outcome, suggesting that the prognostic impact of EBV
status is influenced by geographic locale.15,19-21,23 Other confound-
ing factors may be the determination of EBV status by LMP-1
immunohistochemistry instead of EBER ISH.15,19,23-25,29,30,32,34
and the inclusion of the non-cHL subtype nodular lymphocyte
predominant.13-16,18,19,22,24-27,32,33 Histologic (cHL) subtype may
also affect the prognostic impact of EBV status in children, adoles-
cents, and young adults. Most studies that found a favorable
effect of EBV-positive status on prognosis had a high percentage
of children and/or young adults with mixed cellularity
disease.15,19-21,24,35,40 Sizeable population-based studies like
ours all found a nonsignificant trend of favorable prognosis in
these age groups.31,33
In the population described in this article, we previously found
that cell surface expression of HLA class II on HRS cells is an indepen-
dent prognostic factor. Absence of membranous HLA class II protein
expression was associated with adverse FFS and RS.11 Downregulation
of HLA class II expression is not associated with age but occurs some-
what more frequently in EBV-negative cHL compared with EBV-
positive cHL (47.4% v 30.2%). Although HLA class II expression
status may influence the prognostic effect of EBV, its inclusion as a
cofactor in the multivariate analysis for FFS did not change the inde-
pendency of the EBV effect.
Adverse prognostic impact of EBV positivity in older adult pa-
tients with cHL has been attributed to immunosenescence.31 Immu-
nosenescence is defined as functional impairment of the immune
system that accompanies aging.41 It is associated with decreased cyto-
toxic T-cell effector responses and may impair control of latent EBV
infection.42 Consequently, in the elderly the number of latently in-
fected B cells may increase, leading to an increased risk of developing
EBV-positive cHL. Indeed, patients with EBV-positive cHL have a
higher number of circulating latently infected B cells than do patients
withEBV-negativecHLatthetimeofdiagnosis.43 Becauseimmunosenes-
cence is expected to associate with general ill health, EBV-positive cHL in
the elderly may occur preferentially in persons with a relatively short life
expectancy. Accordingly, Jarrett et al31 found a higher proportion
of cHL-unrelated deaths in EBV-positive patients compared with
those in EBV-negative patients. However, in our study an effect of
EBV status on RS was not confirmed in multivariate analysis.
Instead, our FFS analyses suggest that the adverse prognostic
impact of EBV positivity is caused by a more aggressive biology of the
tumor. In patients age 50 years and older, EBV-positive cHL did not
respond well to first-line treatment, and most events occurred within
the first 2 years. This aggressive behavior may be related to the expres-
sion of LMP-1 and LMP-2 proteins that confer activation, prolifera-
tion, and antiapoptotic signals.6,7 In children, adolescents, and young
adult patients, the adverse effect of EBV might be counteracted by
EBV-specific immune responses, acting in addition to first-line ther-
apy. In patients age 50 years and older, these EBV-specific responses
may be diminished because of immunosenescence. In conclusion, this
study demonstrates that latent EBV infection of HRS cells is associated
with adverse FFS in patients with cHL older than age 50 years.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
The author(s) indicated no potential conflicts of interest.
AUTHOR CONTRIBUTIONS
Conception and design: Arjan Diepstra, Gustaaf W. van Imhoff, Anke
van den Berg, Edo Vellenga, Sibrand Poppema
Administrative support: Arjan Diepstra, Henrike Karim-Kos
Provision of study materials or patients: Gustaaf W. van Imhoff,
Edo Vellenga

3820 © 2009 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

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Copyright © 2017 American Society of Clinical Oncology. All rights reserved.
 EBV Predicts Adverse Outcome in Hodgkin’s Lymphoma
Collection and assembly of data: Arjan Diepstra, Michael Schaapveld,
Henrike Karim-Kos
Data analysis and interpretation: Arjan Diepstra, Gustaaf W. van
Imhoff, Michael Schaapveld, Henrike Karim-Kos, Sibrand Poppema
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Copyright © 2017 American Society of Clinical Oncology. All rights reserved.
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