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 Epstein-Barr virus (EBV), herpesvirus that is the major cause of infectious mononucleosis and is associated with a number of cancers, particularly lymphomas in immunosuppressed persons, including persons with AIDS. 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 in situ hybridization A method for localizing a sequence of DNA, mRNA, or protein in a cell or tissue; the use of a DNA or RNA probe to detect a cDNA sequence in chromosome spreads or in interphase nuclei or an RNA sequence of cloned bacterial or cultured for detection of Epstein-Barr virus-encoded RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic and immunohistochemistry for detection of Epstein-Barr virus latent membrane protein A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes. , we analyzed 28 cases of Hodgkin lymphoma from Jordan and 30 cases from the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. . Eight of 28 Jordanian cases and 9 of 30 North American North American named after North America. North American blastomycosis see North American blastomycosis. North American cattle tick see boophilusannulatus. 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 ********** Prior studies have shown an association between a subset of classic Hodgkin lymphoma and Epstein-Barr virus (EBV EBV Epstein-Barr virus. EBV abbr. Epstein-Barr virus Epstein-Barr virus (EBV) A virus in the herpes family that causes mononucleosis. ) 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 pediatric /pe·di·at·ric/ (pe?de-at´rik) pertaining to the health of children. pe·di·at·ric adj. Of or relating to pediatrics. 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 Nodular sclerosis (or "NSHL") is a form of Hodgkin's lymphoma which is the most common subtype of HL in developed countries. It usually affects females more than males and has a median age of onset at ~28 years. (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 LMP left mentoposterior (position of fetus); last menstrual period. LMP abbr. last menstrual period LMP Last menstrual period, see there )-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 hematoxylin /he·ma·tox·y·lin/ (he?mah-tok´si-lin) an acid coloring matter from the heartwood of Haematoxylon campechianum; used as a histologic stain and also as an indicator. 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 epitope: see immunity. 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 chromogen /chro·mo·gen/ (kro´mah-jen) any substance giving origin to a coloring matter. chro·mo·gen n. 1. A substance that lacks definite color but may be transformed into a pigment. . The secondary antibody was polyvalent polyvalent /poly·va·lent/ (-va´lent) multivalent. pol·y·va·lent adj. 1. Acting against or interacting with more than one kind of antigen, antibody, toxin, or microorganism. 2. , reactive with both polyclonal polyclonal /poly·clo·nal/ (-klon´'l) 1. derived from different cells. 2. pertaining to several clones. polyclonal derived from different cells; pertaining to several clones. (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 xylene (zī`lēn) or dimethylbenzene (dī'mĕthəlbĕn`zēn), C6H4(CH3)2 , hydrated hy·drat·ed adj. Chemically combined with water, especially existing in the form of a hydrate. Adj. 1. hydrated - containing combined water (especially water of crystallization as in a hydrate) hydrous in 99% and 95% ethanol, predigested pre·di·gest tr.v. pre·di·gest·ed, pre·di·gest·ing, pre·di·gests 1. To subject (food) to partial digestion, usually through an enzymatic or chemical process, before ingestion. 2. with proteinase proteinase /pro·tein·ase/ (pro´ten-as?) endopeptidase. pro·tein·ase n. A protease that begins the hydrolytic breakdown of proteins usually by splitting them into polypeptide chains. K, dehydrated de·hy·drate v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates v.tr. 1. To remove water from; make anhydrous. 2. To preserve by removing water from (vegetables, for example). 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 alkaline phosphatase /al·ka·line phos·pha·tase/ (ALP) (fos´fah-tas) an enzyme that catalyzes the cleavage of orthophosphate from orthophosphoric monoesters under alkaline conditions. substrate buffer. The slides were then incubated overnight in the dark with enzyme substrate and inhibitor (levamisole levamisole /le·vam·i·sole/ (le-vam´i-sol) an immunomodulator used with fluorouracil in the treatment of colon cancer, administered as the hydrochloride salt. ). 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 membranous /mem·bra·nous/ (mem´brah-nus) pertaining to or of the nature of a membrane. mem·bra·nous adj. 1. Relating to, made of, or similar to a membrane. 2. and Golgi pattern of staining (Fig. 1B). EBV In Situ Hybridization Findings In situ hybridization for EBV showed abundant hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun) 1. crossbreeding; the act or process of producing hybrids. 2. molecular hybridization 3. of EBER in Reed-Sternberg and Hodgkin cells in all EBV LMP-1-positive cases (Fig. 1C). None of the background lymphoid lymphoid /lym·phoid/ (lim´foid) resembling or pertaining to lymph or tissue of the lymphoid system. lym·phoid adj. Of or relating to lymph or the lymphatic tissue where lymphocytes are formed. 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 Infectious mononucleosis A disease of children and young adults, characterized by fever and enlarged lymph nodes and spleen. EB (Epstein-Barr) herpesvirus is the causative agent. . 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 Burkitt lymphoma Oncology An aggressive extranodal B-cell NHL of children and young adults associated with EBV infection and a characteristic translocation between chromosomes 8 and 14. See Lymphoma, Lymphoma belt, WHO classification. , (7) posttransplant lymphoproliferative disorders, nasal T-cell lymphoma T-cell lymphoma A malignant proliferation of T cells arising in the skin, diagnosed by detecting rearrangement of the T-cell receptor's β chain; TCLs are often 'driven' by EBV and other viral infections; 90% of all Pts with TCL have extracutaneous involvement , 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 Southern blot hybridization Southern blotting Molecular biology A method delineated by EM Southern for detecting and manipulating specific DNA sequences previously separated by gel electrophoresis. , polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is , and in situ hybridization demonstrated direct evidence for an association between EBV and Hodgkin lymphoma. (3,10-12) Furthermore, the Southern blot analysis South·ern blot analysis n. An electrophoretic procedure used to separate and identify DNA sequences. of EBV-associated Hodgkin lymphoma has demonstrated that the EBV is monoclonal in these cases, indicating that the virus was likely present before neoplastic neoplastic /neo·plas·tic/ (ne?o-plas´tik) 1. pertaining to a neoplasm. 2. pertaining to neoplasia. neoplastic pertaining to neoplasia or a neoplasm. transformation. Using in situ hybridization, the EBV DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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 (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. 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 References 1. Weinreb M, Day PJ, Niggli F, et al. The role of Epstein-Barr virus in Hodgkin's disease Hodgkin's disease, a type of cancer of the lymphatic system. First identified in 1832 in England by Thomas Hodgkin, it is a type of malignant lymphoma. Incidence peaks in young adults and the elderly. from different geographical areas. Arch Dis Child 1996;74:27-31. 2. Sheibani K, Tubbs RR. Enzyme immunohistochemistry: Technical aspects. Semin Diagn Pathol 1984;1:235-250. 3. Weiss LM, Chen YY, Liu XF, et al. Epstein-Barr virus and Hodgkin's disease: A correlative Having a reciprocal relationship in that the existence of one relationship normally implies the existence of the other. Mother and child, and duty and claim, are correlative terms. in situ hybridization and polymerase chain reaction study. Am J Pathol 1991;139:1259-1265. 4. Vasef MA, Kamel OW, Chen YY, et al. Detection of Epstein-Barr virus in multiple sites involved by Hodgkin's disease. Am J Pathol 1995;147:1408-1415. 5. Pathmanathan R, Prasad Prasāda (Sanskrit: प्रसाद), prasād/prashad (Hindi), Prasāda in (Kannada), prasādam (Tamil), or prasadam U, Chandrika G, et al. Undifferentiated, nonkeratinizing, and squamous cell carcinoma squamous cell carcinoma n. A carcinoma that arises from squamous epithelium and is the most common form of skin cancer. Also called cancroid, epidermoid carcinoma. of the nasopharynx nasopharynx /na·so·phar·ynx/ (-far´inks) the part of the pharynx above the soft palate.nasopharyn´geal na·so·phar·ynx n. : Variants of Epstein-Barr virus-infected neoplasia neoplasia /neo·pla·sia/ (-pla´zhah) the formation of a neoplasm. cervical intraepithelial neoplasia . Am J Pathol 1995;146:1355-1367. 6. Vasef MA, Ferlito A, Weiss LM. Nasopharyngeal carcinoma, with emphasis on its relationship to Epstein-Barr virus. Ann Otol Rhinol Laryngol 1997;106:348-356. 7. Epstein MA, Achong BG, Barr YM. Virus particles in cultured lymphoblasts from Burkitt lymphoma. Lancet 1964;1:702-703. 8. Weiss LM, Chang KL. Association of the Epstein-Barr virus with hematolymphoid neoplasia. Adv Anat Pathol 1996;3:1-15. 9. Kvale G, Hoiby EA, Pedersen E. Hodgkin's disease in patients with previous infectious mononucleosis. Int J Cancer 1979;23:593-597. 10. Weiss LM, Movahed LA, Warnke RA, et al. Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med 1989;320:502-506. 11. Wu TC, Mann RB, Charache P, et al. Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease. Int J Cancer 1990;46:801-804. 12. Uhara H, Sato Y, Mukai K, et al. Detection of Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin's disease using the polymerase chain reaction and in situ hybridization. Jpn J Cancer Res 1990;81:272-278. 13. Pinkus GS, Lones M, Shintaku IP, et al. Immunohistochemical detection of Epstein-Barr virus-encoded latent membrane protein in Reed-Sternberg cells and variants of Hodgkin's disease. Mod Pathol 1994;7:454-461. 14. Enblad G, Sandvej K, Sundstrom C, et al. Epstein-Barr virus distribution in Hodgkin's disease in an unselected Swedish population. Acta Oncol 1999;38:425-429. 15. Benharroch D, Brousset P, Goldstein J, et al. Association of the Epstein-Barr virus with Hodgkin's disease in Southern Israel. Int J Cancer 1997;71:138-141. 16. Monterroso V, Zhou Y, Koo S, et al. Hodgkin's disease in Costa Rica: A report of 40 cases analyzed for Epstein-Barr virus. Am J Clin Pathol 1998;109:618-624. 17. Bosch Princep R, Alvaro Naranjo T, Balanza Roure R, et al. Hodgkin's disease: Etiopathogenic role of Epstein-Barr virus in Tarragona [in Spanish]. Med Clin (Barc) 2000;114:411-413. 18. Liu SM, Chow KC, Chiu CF, et al. Expression of Epstein-Barr virus in patients with Hodgkin's disease in Taiwan. Cancer 1998;83:367-371. 19. Engel M, Essop MF, Close P, et al. Improved prognosis of Epstein-Barr virus associated childhood Hodgkin's lymphoma: Study of 47 South African cases. J Clin Pathol 2000;53:182-186. 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 . The Jordan University of Science and Technology (Arabic: جامعة العلوم والتكنولوجيا الأردنية), , 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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