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Assessment of Epstein-Barr virus association with pediatric non-Hodgkin lymphoma in immunocompetent and in immunocompromised patients in Argentina. (Original Articles).

Epstein-Barr virus (EBV) is one of 8 known human herpesviruses. It infects the vast majority of mankind, with a prevalence in the total population ranging from 80% to 90%, (1) and although it is etiologically associated with several diseases, by and large it establishes a lifelong silent infection. (2)

Latent membrane protein-1 (LMP-1) and Epstein-Barr nuclear antigen-2 (EBNA-2) are the 2 EBV latency proteins associated with cellular growth regulation. It is well known that LMP-1 exerts transforming effects on continuous rodent fibroblast cell lines (1) and that it also protects against apoptosis by enhancing expression of the bcl-2 proto-oncogene. (3)

Two EBV types circulate in most human populations, types 1 and 2, which differ only in a few latency genes, particularly nuclear antigens EBNA-2, EBNA-3a, EBNA-3b, and EBNA-3c. Epstein-Barr virus type 1 is more common in developed countries, whereas EBV type 2 is more prevalent in Africa. In addition, type 1 has proven in vitro to be a more potent transformer of B cells than type 2. (1)

In recent years, serologic and molecular assays have provided evidence that EBV is associated with several malignancies. Besides Burkitt lymphoma, EBV is now suspected as a pathogenic agent in other non-Hodgkin lymphomas (eg, acquired immune deficiency syndrome [AIDS]-associated, posttransplant, and nasal T/natural killer cell), Hodgkin lymphoma, nasopharyngeal carcinoma, lymphoepithelioma-like carcinoma, and gastric adenocarcinoma, as well as leiomyosarcoma and leiomyoma associated with immunosuppression. Because EBV-related malignancies may occur throughout life, the epidemiology of primary EBV infection, particularly its age at onset, has a potential relevance to understanding the etiology of these cancers. (4)

Lymphomas constitute approximately 10% of all childhood cancers in developed countries; the incidence of non-Hodgkin lymphomas (NHL) increases steadily throughout life, and children younger than 16 years of age account for only 3% of all patients with NHL. Pediatric NHL may be divided into 3 major histologic categories, as follows: lymphoblastic lymphoma, Burkitt lymphoma, and large cell lymphoma. (5)

Epstein-Barr virus expression has been reported to correlate with its clinical course in adult NHL, in both immunocompromised and immunocompetent patients, (6-9) as well as in pediatric patients of developed countries, especially concerning Burkitt lymphoma. (10,11) The aims of the present study were to analyze EBV expression in a pediatric population with NHL in a developing country and to correlate our findings with those reported in other countries and with disease course and outcome.


Patients and Tissue Preparation

Formalin-fixed, paraffin-embedded NHL tissue samples from 32 pediatric patients were collected retrospectively from files at a medical center in Argentina, Ricardo Gutierrez Children's Hospital, from 1993 to 2000.

Diagnosis was made from biopsies taken from the primary tumor. The histologic classification was performed according to schemes for lymphomas (12): 2 anaplastic large null cell lymphoma, 1 anaplastic large T-cell lymphoma, 10 diffuse large B-cell lymphoma (1 with primary immunodeficiency and 1 primary central nervous system lymphoma in a human immunodeficiency virus [HIV]-positive patient), 12 Burkitt lymphoma (2 HIV positive), 1 B lymphoblastic lymphoma, and 6 T lymphoblastic lymphoma (1 with primary immunodeficiency). Median age was 9 years (range 15 months to 16 years), and the male-to-female ratio was 5:3.

LMP-1 Immunohistochemical Staining

Immunostaining was used to localize LMP-1 expression in tumor cells on formalin-fixed, paraffin-embedded tissue sections using monoclonal antibodies CS1-4 (Dako Corporation, Carpinteria, Calif). Protease XIV digestion was used as antigen unmasking procedure to detect LMP-1. Immunohistochemical detection of monoclonal antibodies was performed using a streptavidin-biotin complex-peroxidase detection system (LSAB, Dako) according to the manufacturer's instructions. As a positive control we used a well-known mixed-cellularity Hodgkin lymphoma with specific staining in Reed Sternberg cells.

EBV Detection by Epstein-Barr-encoded RNA In Situ Hybridization

Epstein-Barr-encoded RNAs (EBERs) in situ hybridization was performed on formalin-fixed, paraffin-embedded tissue sections on 3-aminopropyltriethoxy-silane (Sigma Chemical Company, St Louis, Mo) treated slides using fluorescein isothiocyanate-conjugated EBERs oligonucleotides as probes (component of a commercial kit, Novocastra, New Castle upon Tyne, United Kingdom). Detection of hybridized sites was achieved using a monoclonal antibody anti-fluorescein isothiocyanate labeled with alkaline phosphatase (Novocastra), performed according to the manufacturer's instructions. As a positive control for EBERs the same sample as in LMP-1 immunohistochemistry was used.

DNA Isolation

From formalin-fixed, paraffin-embedded biopsies, high molecular weight DNA was isolated by xylol deparaffination, (13) SDS-proteinase K lysis, and phenol-chloroform extraction. (14)

Polymerase Chain Reaction Amplification

To confirm EBV presence, a polymerase chain reaction (PCR) was carried out by amplifying EBERs sequences as described by Bonnet et al. (15) Briefly, using primers 5'-CCCTAGTGGTTTCGGACACA-3' and 5'-ACTTGCAAATGCTCTAGGCG-3', DNA was amplified in a 50-[micro]L reaction mixture containing 10 mM Tris-HCl buffer at pH 8.4, 50 mM KCl, 1.5 mM Mg[Cl.sub.2] 250 mM of each of the deoxynucleotide triphosphates (Pharmacia/LKB, Piscataway, NJ), 1 mM of each primer, and 2.5 U of Taq DNA polymerase (GIBCO, BRL, Rockville, Md). Reaction tubes were placed in a thermal cycler (MJ Research, South San Francisco, Calif). Denaturation was carried out at 95 [degrees] C for 5 minutes, followed by 40 cycles of 30 seconds denaturation at 95 [degrees] C, 1 minute annealing at 60 [degrees] C, 2 minutes extension at 72 [degrees] C, and a 7-minute final extension at 72 [degrees] C. To typify EBV, another PCR strategy was used to detect strain-specific sequence. (16) Briefly, PCR reaction employed a set of primers common to both type 1 and type 2 strains: 5'-AGAAGGGGGCGTGTGTTGT-3' and 5'-GCCGACGTCAAAAACGAGCC-3'. Priming sites flanked regions of type-specific variation, such that resulting fragments were of different size: 153 bp and 246 bp for types 1 and 2, respectively. DNA was amplified in the same reaction mixture described previously. An initial denaturing reaction was carried out at 95 [degrees] C for 5 minutes, followed by 30 cycles of amplification consisting of a 45-second denaturation step at 95 [degrees] C, a 45-second annealing step at 55 [degrees] C, and a 1-minute extension step at 72 [degrees] C. In the final cycle, the extension step was carried out for 5 minutes.

In a separate reaction tube, a set of primers for the [beta]-globin gene, 5'-GAAGAGCCAAGGACAGGTAC-3' and 5'-CAACTTCATCCACGTTCACC-3', were incubated with the template DNA and served as a control to monitor the amplification ability of a single copy gene.

Agarose Gel Electrophoresis

PCR-amplified DNA was subjected to electrophoresis on a 2% agarose gel containing ethidium bromide.

Statistical Analysis

Statistical analysis was performed using the Fisher exact test or the [chi square] test when appropriate.


Eight of the 32 (25%) NHL cases showed LMP-1 positive staining in tumor cells localized at the cell membrane and within the cytoplasm (Figure 1). Positive cases included 3 Burkitt lymphoma, 1 T-cell lymphoblastic lymphoma, and 4 diffuse large B-cell lymphomas (Table 1). These results were confirmed by EBERs in situ hybridization, which showed intense nuclear labeling of tumor cells in these positive cases (Table 1; Figure 2, a and b; Figure 3).


Among EBERs and LMP-1 positive cases, there were 5 immunocompromised patients, either with HIV infection or with primary immunodeficiency (Table 2). Among the 3 HIV-positive patients, 2 had Burkitt lymphoma, and the other had a diffuse large B-cell primary central nervous system lymphoma. The 2 primary immunodeficiency patients presented a T-cell lymphoblastic lymphoma and a diffuse large B-cell lymphoma.

We could obtain DNA from formalin-fixed, paraffin-embedded tissues from 11 of the 32 patients. EBERs in situ hybridization results were confirmed by EBERs PCR in 11 samples, with 3 proving positive and 8 negative. The 3 positive samples were also typified by the PCR described by Sample et al, (16) all of which were EBV-2, 2 corresponding to immunocompromised patients (Table 2) and 1 to an immunocompetent patient.

Treatment results and outcome of children with NHL were correlated with the histologic subtype, EBERs in situ hybridization, and LMP-1 expression. Neither EBERs expression nor LMP-1 presence yielded prognostic value for pediatric NHL in immunocompetent patients (P = .6, [chi square] test). In particular, NHLs of the 3 HIV-positive children were all EBV positive and had a fatal outcome: 1 because of illness progression refractory to treatment, 1 from multiple organ failure and neurologic depression, and 1 from septic shock.


The results of this study indicate that the distribution of EBV within our sample of cases of pediatric NHL shows EBERs presence and LMP-1 expression in 25% of patients. Negative and positive EBV results were confirmed by PCR in the 11 cases in which DNA was available. Positivity was widely distributed among histologic subtypes: 4 diffuse large B-cell lymphoma (1 with primary immunodeficiency and 1 HIV positive), 3 Burkitt lymphoma (2 HIV positive), and 1 lymphoblastic T-cell lymphoma (with primary immunodeficiency). Most EBV-positive patients were immunocompromised (62.5%); these results are in agreement with previously reported data. (4,17)

Many EBV-associated malignancies are more common in males than in females. (4) Our study showed that the male-to-female ratio of EBV-positive cases was 1:1, but larger series are required to confirm this finding.

We failed to observe frequent EBV expression in T-cell NHL as previously reported. (6,7,18-21) T-cell NHL is not predominant in our pediatric population, so it would be helpful to increase the number of T-cell NHLs studied in order to reach a definitive conclusion. Furthermore, we found no correlation between EBV status and unfavorable outcome, as also has been described for T-cell NHL. Meanwhile, this lack of correlation has been reported in B-cell adult NHL. (6)

Non-Hodgkin lymphomas have typically been the most frequent malignancy of immunosuppressed subjects, whether secondary to HIV, prior to organ transplantation, or as a congenital condition. (22) Such immunosuppression conditions tend to increase the likelihood of EBV malignancies, by deregulating T-cell immune surveillance that normally controls EBV latent infection in immunocompetent patients, and by reactivating this latent infection to a lytic one. With regard to the immunocompromised patients included in this report, we observed 100% EBV association. Therefore, our results were in accordance with previously reported data, where a high frequency of EBV expression was found. (17,22,23)

Moreover, we observed a clear correlation between EBV-positive HIV-positive NHL and an adverse outcome. It seems that EBV infection is a secondary event that contributes with immunodeficiency to impair clinical status, because immunocompetent patients show a favorable outcome in spite of being EBV positive.

Three different types of latency--I, II, and III--have been identified in EBV-associated malignancies. Burkitt lymphoma usually displays a latency type I expression pattern of EBV latent antigen in which only EBERs and EBNA-1 are expressed. Hodgkin lymphoma and nasopharyngeal carcinoma represent 2 major entities of type II malignancies, the pattern of which is characterized by the expression of EBERs, EBNA 1, LMP-1, and LMP-2. Epstein-Barr virus-associated malignancies expressing the full complement of latency antigens (EBNAs and LMPs) are classified as latency III malignancies and are only seen in immunocompromised individuals. (24,25) In this report we detected LMP-1 expression in the 3 Burkitt lymphoma cases studied, 2 proving to be HIV positive. Niedobitek et al (26) have recently demonstrated a form of EBV latency other than type I in endemic Burkitt lymphoma, detecting LMP-1 expression in tumor cells from 2 Burkitt lymphoma cases. Carbone and Gloghini (27) also reported the expression of LMP-1 in 3 of 10 AIDS-related and in 2 of 6 non-AIDS-related nonendemic Burkitt lymphoma. Our results support such findings, where LMP-1 expression is found in both immunocompromised and immunocompetent patients (Table 1). It should be borne in mind that 2 of these patients were HIV positive, and so they could have a latency type III expression pattern in which all EBV latency antigens are expressed.

Our study failed to disclose high EBV expression as previously found in other EBV-associated malignancies in developing countries, (4) with low socioeconomic status proving a significant factor for frequent EBV association. Such low EBV expression in Hodgkin lymphoma in Argentina has been documented by our research team. (28)

Moreover, our results strengthen the argument that EBV may be involved as a cofactor in the lymphomagenesis of some but not all pediatric NHLs, because in EBV-negative patients other factors would contribute to the development of NHLs. However, the finding that EBV is clearly associated with 25% of our pediatric population with NHL hardly detracts from the relevance of their mutual association.

Be that as it may, the role played by EBV in NHL is still unclear, and to date there have been no differences in clinical outcome between NHL EBV-positive and EBV-negative patients.

This study was supported in part by a grant from Ministerio de Salud, Secretaria de Ciencia y Tecnologia "Beca de Investigacion Ramon Carrillo-Arturo Onativa." Ms Chabay was supported by a fellowship from The National Research Council (CONICET), and Dr Preciado is a member the CONICET Research Career Program.
Table 1. Correlation of Tumor Histology With EBERs and LMP-1
Expression *

 Positive for LMP-1

 Histologic Subtype n n %

Burkitt lymphoma 12 ([dagger]) 3 9.4
B lymphoblastic lymphoma 1 0 0
T lymphoblastic lymphoma 6 ([double 1 ([double 3.1
 dagger]) dagger])
Anaplastic large
 T-cell lymphoma 1 0 0
Anaplastic large null
 cell lymphoma 2 0 0
Diffuse large
 B-cell lymphoma 9 ([double 3 ([double 9.4
 dagger]) dagger])
Diffuse large B-cell
 primary central 1 ([section]) 1 ([section]) 3.4
 nervous system lymphoma
Total 32 8 25

 Positive for EBERS

 Histologic Subtype n %

Burkitt lymphoma 3 9.4
B lymphoblastic lymphoma 0 0
T lymphoblastic lymphoma 1 ([double dagger]) 3.1
Anaplastic large
 T-cell lymphoma 0 0
Anaplastic large null
 cell lymphoma 0 0
Diffuse large
 B-cell lymphoma 3 ([double dagger]) 9.4
Diffuse large B-cell
 primary central 1 ([section]) 3.4
 nervous system lymphoma
Total 8 25

* EBERs indicates Epstein-Barr-encoded RNAs; LMP-1, latent
membrane protein-1; and HIV, human immunodeficiency virus.

([dagger]) Two of these patients were HIV positive.

([double dagger]) One of these patients had a primary immunodeficiency.

([section]) This patient was HIV positive.
Table 2. Epstein-Barr Virus Expression in Immunocompromised Patients *

Patient y Sex Immunodeficiency Site

 1 3 F HIV+ Retro-orbital
 2 2 F HIV+ Lymph node
 3 3 F HIV+ Central nervous system
 4 6 M PI Middle ear
 5 14 M PI Lymph node

Patient Histology IHC ISH PCR PCR

 1 BL + + + EBV-2
 2 BL + + - ND
 3 B-DLC + + ND ND
 4 T-CLL + + ND ND
 5 B-DLC + + + EBV-2

* LMP-1 indicates latent membrane protein-1; IHC, immunohistochemistry;
EBERs, Epstein-Barr-encoded RNAs; PCR, polymerase chain reaction EBNA,
Epstein-Barr nuclear antigen; HIV, human immunodeficiency virus; BL,
Burkitt lymphoma; EBV, Epstein-Barr virus; ND, not done; B-DLC, B-cell
diffuse lymphoma; PI, primary immunodeficiency; and T-CLL, T-cell
lymphoblastic lymphoma.


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Accepted for publication September 26, 2001.

From the Virology Laboratory (Ms Chabay and Drs Grinstein and Preciado), the Pathology Department (Drs De Matteo and Maglio), and the Hematology Department (Dr Aversa), Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina.

Reprints: Maria Victoria Preciado, PhD, Laboratorio de Virologia, Hospital de Ninos Ricardo Gutierrez, Gallo 1330, C1425EFD Ciudad de Buenos Aires, Argentina (e-mail:
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Author:Chabay, Paola A.; De Matteo, Elena N.; Aversa, Luis; Maglio, Silvana; Grinstein, Saul; Preciado, Mar
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:3ARGE
Date:Mar 1, 2002
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