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Association between Epstein-Barr virus and classic Hodgkin lymphoma in Jordan: a comparative study with Epstein-Barr virus-associated Hodgkin lymphoma in North America.

Abstract: An association between Epstein-Barr virus and Hodgkin lymphoma has been shown in several parts of the world. The reported incidence of Epstein-Barr virus in Hodgkin lymphoma varies significantly from one country to another and ranges from <30% in Swedish patients to 100% in patients from Kenya. Using in situ hybridization for detection of Epstein-Barr virus-encoded RNA and immunohistochemistry for detection of Epstein-Barr virus latent membrane protein, we analyzed 28 cases of Hodgkin lymphoma from Jordan and 30 cases from the United States. Eight of 28 Jordanian cases and 9 of 30 North American cases were Epstein-Barr virus positive. Our studies show that the incidence of Epstein-Barr virus among Jordanian patients with Hodgkin lymphoma is similar to the rate in patients from the United States. This rate appears to be low to intermediate compared with rates in other parts of the world.

Key Words: Epstein-Barr virus, Epstein-Barr virus latent membrane protein, Hodgkin lymphoma, in situ hybridization

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Prior studies have shown an association between a subset of classic Hodgkin lymphoma and Epstein-Barr virus (EBV) in several parts of the world. The EBV has been shown to be present in Reed-Sternberg and Hodgkin cells, indicating a role in the pathogenesis of Hodgkin lymphoma. The association between Hodgkin lymphoma and EBV has been rarely studied in patients from Jordan, and most of the studies have only included the pediatric age group. (1) To characterize the role of EBV and its incidence in Hodgkin lymphoma among mainly adult Jordanian patients, we studied a total of 28 histologically and immunophenotypically documented cases of classic Hodgkin lymphoma in Jordan, 7 with mixed cellularity (MC) and 21 with nodular sclerosis (NS) subtypes. In addition, 30 North American cases of classic Hodgkin lymphoma (8 MC, 22 NS subtypes) were included in this study for comparative analysis.

In situ hybridization using the EBV probe that detects EBV-encoded small RNA (EBER) showed positive nuclear staining of Reed-Sternberg and Hodgkin cells in 8 (29%) of 28 Jordanian cases (4 of 7 MC, 4 of 21 NS) and in 9 (30%) of 30 North American cases (6 of 8 MC, 3 of 22 NS). Paraffin immunohistochemistry using anti-latent membrane protein (LMP)-1 antibody showed that all EBER-positive cases also expressed LMP-1 protein with a membrane and Golgi pattern of staining. Our findings confirm and further expand that EBV plays a role in the pathogenesis of a subset of classic Hodgkin lymphoma and indicate that approximately one third of classic Hodgkin lymphomas in Jordan are EBV-associated. This incidence appears to be similar to the frequency of EBV in classic Hodgkin lymphoma in the United States.

Patients and Methods

Hematoxylin and eosin-stained histologic sections from 28 cases of classic Hodgkin lymphoma in Jordan (21 NS, 7 MC) and 30 cases of classic Hodgkin lymphoma in the United States (22 NS, 8 MC) were reviewed and included for this study. Representative paraffin-embedded tissue blocks were selected for EBV studies.

Immunohistochemical Studies

Immunohistochemical studies were performed using formalin-fixed paraffin-embedded tissue sections and EBV LMP-1 antibody (Dako, Carpinteria, CA), as described previously. (2) A heat-induced epitope retrieval method was used before incubation of tissue sections with LMP-1 antibody. The EBV LMP-1 antibody was used at 1:200 dilution. Reactivity was detected using avidin-biotin technique and 3', 3'-diaminobenzidine-tetrahydrochloride dihydrate as the chromogen. The secondary antibody was polyvalent, reactive with both polyclonal (rabbit) and monoclonal (mouse immunoglobulin [Ig] G and IgM and rat IgG) primary antibodies. Sections of a known LMP-1-positive case of Hodgkin lymphoma and multitissue control blocks were used as positive and negative external controls, respectively.

In Situ Hybridization

In situ hybridization was performed using formalin-fixed paraffin-embedded tissue sections and an oligonucleotide probe complementary to EBER transcripts (Novocastra/Vector, Burlingame, CA) with a method similar to those described previously. (3,4) Briefly, the slides were deparaffinized in xylene, hydrated in 99% and 95% ethanol, predigested with proteinase K, dehydrated in graded ethanol, and incubated with probe for 2 hours at 37[degrees]C. After washing, sections were covered with blocking solution and the slides were incubated for 10 minutes and washed in alkaline phosphatase substrate buffer. The slides were then incubated overnight in the dark with enzyme substrate and inhibitor (levamisole). After a brief washing in running water, the slides were counterstained with hematoxylin and coverslipped for microscopic evaluation. To validate specificity, a cocktail of random oligonucleotide was used in a duplicate section, in parallel with specific probes. This control probe also served as a negative control. A section of a known EBV-positive case was used as a positive control.

Results

Histopathologic Findings

The results are summarized in Tables 1 and 2. The 28 patients from Jordan included 16 male patients and 12 female patients. The age in one case was not available. The remaining 27 patients were 5 to 67 years old, with a median age of 30 years. The 30 patients from the United States included 18 male patients and 12 female patients, with the age ranging from 12 to 58 years and with a median age of 33 years. The subtypes of Hodgkin cases included 21 NS and 7 MC in cases from Jordan and 22 NS and 8 MC in cases from the United States (Fig. 1A). The immunophenotype of Reed-Sternberg and Hodgkin cells had been previously characterized as being typical of the classic Hodgkin lymphoma (CD[30.sup.+], CD[15.sup.+], and CD[45.sup.-]) in all cases.

EBV LPM-1 Immunohistochemical Findings

The Reed-Sternberg and Hodgkin cells in 8 of 28 (29%) Jordanian cases (4 of 7 MC, 4 of 21 NS) and 9 of 30 (30%) American cases (6 of 8 MC, 3 of 22 NS) expressed EBV LMP-1 protein with a membranous and Golgi pattern of staining (Fig. 1B).

EBV In Situ Hybridization Findings

In situ hybridization for EBV showed abundant hybridization of EBER in Reed-Sternberg and Hodgkin cells in all EBV LMP-1-positive cases (Fig. 1C). None of the background lymphoid cells showed EBER expression.

Discussion

EBV is a member of the herpes family (Herpesviridae) that has been found to be the etiologic agent of infectious mononucleosis. The EBV has also been consistently detected in almost 100% of nasopharyngeal carcinomas using EBER. (5,6) In addition, the EBV has been associated with a wide variety of lymphoid neoplasms including endemic Burkitt lymphoma, (7) posttransplant lymphoproliferative disorders, nasal T-cell lymphoma, and a significant subset of Hodgkin lymphoma. (8)

[FIGURE 1 OMITTED]

An increased risk of Hodgkin lymphoma in patients after infectious mononucleosis had been known for many years. (9) More recently, molecular techniques such as Southern blot hybridization, polymerase chain reaction, and in situ hybridization demonstrated direct evidence for an association between EBV and Hodgkin lymphoma. (3,10-12) Furthermore, the Southern blot analysis of EBV-associated Hodgkin lymphoma has demonstrated that the EBV is monoclonal in these cases, indicating that the virus was likely present before neoplastic transformation. Using in situ hybridization, the EBV DNA and RNA has been localized exclusively within the Reed-Sternberg and Hodgkin cells. In addition, an immunohisto-chemical method has shown that EBV LMP expression in EBV-associated Hodgkin lymphoma is present exclusively in Reed-Sternberg and Hodgkin cells. (13) These observations suggest that EBV plays a role in the pathogenesis of at least a subset of cases of Hodgkin lymphoma.

The association between EBV and Hodgkin lymphoma has been previously studied in many parts of the world. These studies have shown a significant variation in the incidence of EBV-associated Hodgkin lymphoma in different countries, (1,14-19) ranging from 27% in Swedish patients (14) to 100% in patients from Kenya. (1) The relation between EBV and Hodgkin lymphoma has been rarely studied in Jordan. Furthermore, the studies have been carried out on childhood Hodgkin lymphoma. (1) In a study by Weinreb et al, (1) EBV LMP-1 protein and EBER were detected in 8 of 16 Jordanian children with Hodgkin lymphoma. Our study included 28 Jordanian patients with classic Hodgkin lymphoma. The majority of the patients (25 of 28 [89%]) were adults with ages ranging from 19 to 67 years. The Hodgkin lymphoma in 8 of 28 (29%) Jordanian cases including 5 of 25 (20%) adult patients were EBV-associated. Nine of 30 (30%) Hodgkin lymphoma cases from the United States including 9 of 28 (32%) adults were EBV LMP-1 protein- and EBER-positive. The two American children with Hodgkin lymphoma, ages 12 and 15, in this study were EBV-negative.

Our results indicate that the overall incidence of EBV in patients with Hodgkin lymphoma in Jordan is similar to the frequency of EBV in North American patients with Hodgkin lymphoma (29% versus 30%). In addition, similar to prior observations, our study shows an overall higher incidence of EBV in the MC than in the NS subtype in both Jordanian and North American cases. Two of the three pediatric Jordanian patients with Hodgkin lymphoma in our study were EBV-positive, whereas none of the two North American children expressed EBV in our study. This finding may suggest a higher frequency of EBV at a younger age in Jordanian patients compared with the North American patients. However, the number of children with Hodgkin lymphoma included in our study was low and additional studies may be necessary to confirm this observation.

Summary

Our study demonstrates that approximately one third of Jordanian patients with classic Hodgkin lymphoma are EBV-associated. This frequency is similar to the incidence of EBV in North American Hodgkin lymphoma. This is in contrast to the higher rate of EBV-positive Hodgkin lymphoma reported in several other parts of the world. In addition, this study suggests an overall higher frequency of EBV expression among Jordanian children with Hodgkin lymphoma compared with the U.S. children with Hodgkin lymphoma.
The object of education is to prepare the young to educate themselves
throughout their lives.
--Robert Maynard Hutchins

Table 1. Summary of the results of EBV in situ hybridization and EBV
LMP-1 immunohistochemical stains in Jordanian patients with Hodgkin's
lymphoma (a)

Patient Sex/Age
no. (yr) Site Type LMP EBER

 1 M/? Supraclavicular node MC + +
 2 M/5 Mesenteric node MC + +
 3 F/15 Neck node MC - -
 4 F/17 Neck node NS + +
 5 M/19 Neck node NS - -
 6 F/22 Neck node NS - -
 7 M/22 Supraclavicular node NS - -
 8 M/23 Inguinal node NS - -
 9 F/24 Supraclavicular node NS - -
10 M/25 Inguinal node NS - -
11 F/26 Presternal/cervical node NS - -
12 F/28 Pretracheal node NS - -
13 F/29 Neck node NS - -
14 M/30 Inguinal node NS + +
15 M/30 Neck node NS - -
16 F/32 Axillary node NS - -
17 M/36 Inguinal node NS - -
18 M/41 Supraclavicular node MC - -
19 M/41 Inguinal node MC - -
20 M/42 Supraclavicular node NS + +
21 M/49 Axillary node NS - -
22 F/50 Axillary node MC + +
23 F/55 Axillary node NS - -
24 M/56 Mediastinum node NS - -
25 M/60 Supraclavicular node NS - -
26 M/62 Spleen/liver/node MC + +
27 F/63 Iliac node NS - -
28 F/67 Neck node NS + +

(a) EBV, Epstein-Barr virus; LMP, latent membrane protein; M, male; F,
female; MC, mixed cellularity; NS, nodular sclerosis; EBER, EBV--encoded
RNA; +, positive; -, negative.

Table 2. Summary of the results of EBV in situ hybridization and EBV
LMP-1 immunohistochemical stains in North American patients with
Hodgkin's lymphoma (a)

Patient Age
No. Sex (yr) Site Type LMP EBER

 1 M 12 Supraclavicular node MC - -
 2 M 15 Inguinal node NS - -
 3 M 21 Supraclavicular node NS - -
 4 F 21 Chest wall node NS - -
 5 M 21 Supraclavicular node NS - -
 6 M 21 Thoracic node NS - -
 7 F 22 Neck node NS - -
 8 M 22 Neck node NS - -
 9 M 23 Axillary node MC + +
10 F 25 Supraclavicular node NS - -
11 F 25 Pleura node NS - -
12 F 30 Neck node NS - -
13 M 30 Groin node MC + +
14 M 33 Neck node MC + +
15 M 33 Neck node NS - -
16 F 34 Neck node MC - -
17 F 36 Neck node NS - -
18 M 37 Supraclavicular node NS + +
19 F 37 Chest wall node NS - -
20 M 38 Internal mammary node NS - -
21 F 38 Neck node NS - -
22 M 39 Retroperitoneum node NS + +
23 F 40 Neck node NS - -
24 M 40 Supraclavicular node NS - -
25 M 44 Groin node NS + +
26 M 49 Supraclavicular node NS - -
27 M 49 Chest wall node NS - -
28 F 52 Retroperitoneum node MC + +
29 M 57 Groin node MC + +
30 F 58 Supraclavicular node MC + +

(a) EBU, Epstein-Barr virus; LMP, latent membrane protein; EBER, EBU-
encoded RNA; M, male; F, female; MC, mixed cellularity; NS, nodular
sclerosis; +, positive; -, negative.


Accepted July 9, 2003.

Copyright [c] 2004 by The Southern Medical Association

0038-4348/04/9703-0273

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RELATED ARTICLE: Key Points

* The incidence of Epstein-Barr virus in Jordanian patients with classic Hodgkin lymphoma was studied.

* In situ hybridization for detection of Epstein-Barr virus-encoded RNA was used.

* Immunohistochemistry for latent membrane protein was used.

Mohammad A. Vasef, MD, Manaf A. Ubaidat, MD, Hasan S. Khalidi, MD, Nidal M. Almasri, MD, Mousa Al-Abbadi, MD, and Hassan Z. Annab, MD

From the Department of Pathology, University of lowa Hospitals and Clinics, lowa City, IA; and the Jordan University of Science and Technology, Jordan Hospital, and Islamic Hospital, Amman, Jordan.

Reprint requests to Mohammad A. Vasef, MD, Department of Pathology, University of lowa Hospitals and Clinies, 200 Hawkins Drive, Iowa City, IA 52241. Email: mohammad-vasef@uiowa.edu
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Title Annotation:Original Article
Author:Annab, Hassan Z.
Publication:Southern Medical Journal
Date:Mar 1, 2004
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