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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 1  |  Page : 63-67
High incidence of Epstein Barr virus infection in childhood acute lymphocytic lukemia: A preliminary study


1 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication19-Jan-2010
 

   Abstract 

Introduction: Epstein Barr virus (EBV) has a unique association with several human malignancies, especially lymphoproliferative disorders, mainly lymphomas in adults. There is paucity of data pertaining to EBV association with various cancers in India . Objective : The study aims to investigate the association of EBV in childhood leukemia. Material and Methods: Patients attending pediatric oncology services of the referral center have been included in the study. Twenty-five consecutive pediatric patients with acute lymphocytic lukemia (ALL) were subjected to EBV studies employing sensitive polymerase chain reaction followed by hybridization for presence of Bam H1-W region of EBV genome and detection of anti Z EBV replication activator (ZEBRA) antibodies using Western blot. Positive control included a case of Burkitt's lymphoma and infectious mononucleosis each. Raji cells were used as positive control with each test. Results: The PCR for EBV was positive in 8/25 patients of ALL. Western blot test using anti ZEBRA antibodies was positive in 5/25(20%) cases of ALL. Considering PCR as the gold standard, 32% of the children with ALL had evidence of active EBV replication. The positive controls were consistently positive. None of the 30 healthy laboratory controls, 22 age matched disease controls, 12 cases of AML and 15 cases of multiple myeloma were positive either by PCR or Western blots assays (P < 0. 01). There was no statistically significant correlation between duration of therapy and EBV positivity (P > 0.05). Conclusion: These studies indicate that a significant number of patients with ALL show evidence of active EBV replication.

Keywords: Childhood Acute Lymphocytic Lukemia, EBV, Polymerase Chain Reaction

How to cite this article:
Sehgal S, Mujtaba S, Gupta D, Aggarwal R, Marwaha R K. High incidence of Epstein Barr virus infection in childhood acute lymphocytic lukemia: A preliminary study. Indian J Pathol Microbiol 2010;53:63-7

How to cite this URL:
Sehgal S, Mujtaba S, Gupta D, Aggarwal R, Marwaha R K. High incidence of Epstein Barr virus infection in childhood acute lymphocytic lukemia: A preliminary study. Indian J Pathol Microbiol [serial online] 2010 [cited 2014 Oct 22];53:63-7. Available from: http://www.ijpmonline.org/text.asp?2010/53/1/63/59186



   Introduction Top


Epstein and Barr [1] for the first time reported that Burkitt's lymphoma, unique to equatorial Africa, was in fact caused by a virus. This observation shed the mystery of a lethal tumor. Subsequently, Henle and Purtilo [2],[3] documented the association of Epstein Barr Virus (EBV) with nasopharyngeal carcinoma and X linked lymphoproliferative disorders respectively. Several other types of viruses have also been implicated in human neoplasms. Human papilloma virus has been linked to cervical cancer, esophageal cancer, oral cancer; hepatitis B virus and C virus (HBV and HCV) have a causal relation with liver cancer [4],[5] and so also the human T lymphotropic virus, originally reported in patients with T cell leukemia in Japan. [6]

The coinfection with EBV has also been linked with Hodgkin's disease, large cell lymphoma, acquired immunodeficiency syndrome (AIDS) related lymphoma; [7],[8],[9],[10] and possibly chronic lymphocytic leukemia in adults. [11] There are very few reports in world literature on the causal role of EBV in childhood leukemia. Schlehofer et al. [12] from Germany conducted EBV serology in 121 children with acute lymphocytic leukemia (ALL) and documented an increase in antiviral capsid antibodies (VCA) in these children. However, polymerase chain reaction (PCR) test was not undertaken in this study. Recent literature points towards the controversial role of EBV in childhood leukemia. [13],[14] So far no literature is available in India regarding an association or causal relationship between these malignancies and EBV. In the present preliminary report, employing sensitive and specific molecular tools like PCR assay followed by hybridization, we document that children with ALL have an increased association with active EBV replication, which may bear a causal relationship.


   Materials and Methods Top


Twenty-nine consecutive patients of lymphoid malignancies reporting to the pediatric oncology unit have been included in the study. Twenty-five patients had ALL and four had Hodgkin's disease. A single case each of Burkitt's lymphoma and acute infectious mononucleosis were also included as positive controls. The detailed clinical history was recorded followed by a thorough clinical examination. Patients of ALL were categorized according to the French American British (FAB) classification. Laboratory investigations included: peripheral blast cells of more than 30%, bone marrow biopsy, cerebrospinal fluid (CSF) examination, and fine needle aspiration cytology/and or tissue biopsy when indicated.

Controls

  1. 30 healthy laboratory workers were also included in the study.
  2. 15 patients of multiple myeloma
  3. 12 patients of acute myeloid leukemia and
  4. 22 pediatric patients with conditions other than hematological malignancies
Multiple myeloma was diagnosed on the basis of plasmacytosis in the marrow, monoclonal peak on serum electrophoresis and lytic lesions in the bones. Blood samples were withdrawn after an informed consent.

DNA Extraction and PCR

DNA from blood samples was prepared by the method of Higuchi et al. [15] DNA was also extracted from Raji cells and used as a positive control of EBV. No attempt was made to separate the leukemic cells.

Oligonucleotides specific for EBV were obtained from Gibco BRL (USA). The sequence of EBV specific primers (Tc60 and Tc61) and probes (Tc62) were based on published sequence of Baer et al.[16] and Saito et al [17] corresponding to Bam H1-W region. This region is reiterated 10 times in the EBV genome and also contains the promoter of EBNA proteins.

PCR Amplification

The 25μl PCR reaction mixture contained 2.5μl of 10x Taq buffer (Boehringer Mannheim, Germany), 1μl of 25 mM MgCl2, 70 pmol of each one ml of the primers Tc60 and Tc61, 1μl of 10 mM dNTPS, 2.5ml of Taq polymerase (Boehringer Mannheim, Germany) and autoclaved water. Each reaction tube received 25ml of PCR mixture plus 25μl of DNA. Reaction was carried out in a DNA thermal cycler (Perkin Elmer) with 5 min of denaturation at 94 0 C for first cycle, followed by one minute each for denaturation at 94 0 C, annealing at 55 0 C and extensions at 72 0 C for 33 cycles. The last cycle had an extra primer extension for 10 minutes.

Identification of Amplified Products by Hybridization

The PCR products were denatured at 95 0 C and chilled on ice. Ten ml of amplified DNA was spotted on to nylon membranes and air-dried. The membrane was placed on Whatman 3MM paper soaked in denaturing solution (0.5M NaOH and 1.5M NaCl) for five minutes and subsequently on neutralizing solution (1M Tris-HCI pH 8.0 and 1.5M NaCl). After airdrying, the membrane was baked at 80 0 C for two hours in a vacuum oven (Sheldon, USA). It was equilibrated in 5 x SSC (saline sodium citrate) for five minutes at room temperature and later subjected to prehybridization (5 x SSC, 10x Denhardts, 7% SDS, 100μg/ml heat denatured Salmon sperm DNA) at 42 0 C for - four to five hours.

The EBV specific probe TC62 corresponding to intervening sequence of amplified product was end labeled with Gamma- ATP-32 (BARC, INDIA) by T4 polynucleotide kinase (New England Biolabs). The membrane was subjected to hybridization (5xSSC, 10xDenhardts, 7% SDS, 100 ug/ml heat denatured salmon sperm DNA, 0.01 mM EDTA and the radiolabel led probe) for five hours at 42 o C. The membrane was washed twice for five minutes at room temperature with 2 x SSC plus 0.5% SDS for 10 min at 55 0 C. It was then subjected to autoradiography for 48 hours at minus 70 0 C and the intensity of signals noted. Raji cell DNA was used as a positive control during each experiment. Two cases of Burkitts Lymphoma also served as positive controls. The details of methodology have also been published earlier. [18] The specificity of the hybridization assay was 100%.

Plasmid pGEX expressing Z EBV replication activator (ZEBRA) protein was a gift from Fritz Schwartzman, Germany. The details of methodology of Western blot assay have been published earlier. [18]


   Results Top


The ages of patients ranged from two to 14 years with a mean of 6.5 years. Twenty-three had ALL-L1 and two had ALL-L2 type of leukemia. Out of the 25 ALL patients, three cases were drug naïve, four were on on induction therapy and 16 on continuation therapy. Two cases were off chemotherapy. Polymerase chain reaction for BamH1-W EBV nuclear antigen (EBNA) promoter was positive in 8/25 (32%) of ALL patients spread over different categories [Figure 1]. Of the four cases of Hodgkin's disease, three were positive by PCR assay. Two cases were drug naïve and one had received therapy for three months only. A case of Burkitt's lymphoma [Figure 2] and another with acute infectious mononucleosis were also positive by the PCR assay. Raji cell DNA was consistently positive. Thus out of 29 cases of leukemia and Hodgkin's disease, 11 (38%) were positive. [Table 1] illustrates the positive patients of ALL and duration of therapy . Using chi square analysis there was a significant increase in EBV in ALL patients (p < 0.05) as none of the 30 healthy controls was positive for EBV. There was no direct correlation between duration of therapy and EBV positivity (r = 0.3, p > 0.5). Out of 22 Children without ALL, six had TB, nutritional deficiencies were present in five, six children were admitted with seizures and five had other severe bacterial infections. Similarly, all the 15 cases of multiple myeloma and 12 cases of AML were negative. Healthy control children could not be studied because of ethical concerns.


   Discussion Top


In recent years some convincing leads have been obtained on a causal relationship between EBV and a variety of lympho reticular malignancies. Patients with acute infectious mononucleosis document an increased susceptibility to Hodgkin's disease. [7] Patients with AIDS are known to have an increased incidence of EBV infection as well as lymphomas. [18],[19],[20] In India, although nasopharyngeal carcinoma and Burkitt type of lymphoma are uncommon, EBV infection is common as indicated by the ubiquitous presence of IgG antibodies to VCA in control subjects (unpublished data). One striking observation in all these malignancies is the long latent period between the primary infection and development of malignancy. Two exceptions, however, are the immunoblastic lymphomas occurring after acute EBV infection in immunocompromised hosts like transplant recipients and those with XLP syndrome.

PCR followed by hybridization was considered the gold standard for compiling data in this study. The paper shows evidence of active EBV replication in some patients with ALL and Hodgkin's as EBV PCR specific for replicating EB virus was positive in 38% of the children. In this study six /11 children even without therapy or with a short course of therapy showed evidence of EBV infection, There was no correlation between the duration of therapy and EBV infection in ALL (Chi square, p greater than 0.05). The number is, however, small to conclusively prove that therapy was not directly or indirectly related to positivity in these children. Ideally, age matched patients with solid tumors should also have been included but it was not done in this preliminary study. This study was initiated because a wide variety of hematological malignancies have been linked to EBV. Such molecular studies pertaining to EBV in healthy children are wanting in India. Venkitaraman et al. [21] reported age specific prevalence of IgG antibodies to VCA in 80% by the age of 5 years. No data on molecular studies on EBV in cancers is available in India barring stray case reports. Roy et al. [22] also reported a significant number of adult controls and cancer patients positive for EBV by ELISA assays. However, the point of active replication was not addressed and no molecular studies were done. There are controversial reports in the world literature regarding EBV coinfection in childhood ALL.

Schlehoefer et al. [12] reported increased incidence of anti VCA antibodies and anti EBNA antibodies in children with ALL in Germany but it is known that serological tests may not be very specific and antibodies do not indicate active viral replication. Using this assay, none of the controls was positive for EBV while Raji cells were consistently positive. Negative test in allth controls is also intriguing but it may indicate absence of active infection at the time of sampling. Similarly all patients with multiple myeloma and children with AML and conditions other than hematological malignancies were also negative. Wolf et al. [23] documented EBV mRNA IN 4/6 cases of hairy cell leukemia, another indication of oncogenic potential of EBV. In the single largest epidemiological study conducted on 550000 mothers and 7 million years follow-up in Finland and Iceland, Lehtinen et al [13] concluded that activation of maternal Herpes virus infection increased the risk of ALL in the offspring. Only EBV immunoglobulin M positivity in EBV-immunoglobulin-G-positive mothers was associated with a highly significant increased risk of ALL in the offspring (adjusted odds ratio=2.9, 95% confidence interval: 1.5, 5.8). These observations were supported by EBV DNA studies. No other study has, however, substantiated these observations. Loufty et al. from Egypt documented that HSV 1 and HSV 2 but not EBV was linked to ALL. [14]

In a more recent study from Sweden, Altieri et al. [24] reported that ALL was positively correlated with the number of siblings; the younger sibs were strongly protected from the risk of malignancies suggesting an infectious etiology. Sakajiri reported increased EBV infection in a patient with T ALL [25] employing Southern blotting and in situ hybridization (ISH). Patients with T ALL or those with other solid tumors were not studied.

[Table 2] summarizes the relevant information available from the world literature. This study, using molecular biology techniques, indicates that EBV infection is indeed increased in one third of cases of childhood leukemia. We also found EBV positivity in three out of four cases of Hodgkin's disease but the number is too small for a meaningful analysis. Miyagi. [26] from Japan documented EBV in 11/12 cases of Hodgkin's disease using in situ hybridization (ISH). It appears that EBV may be an important coinfection in some patients of ALL in India. Our findings corroborate observations of Lehtinen et al. [13] In the face of compelling evidence in favor of infectious etiology of ALL, O ' Connor et al [27] from CDC raised fresh issues to prove or disprove the role of infections in childhood leukemias. They commented, "Existing evidence partially supports plausibility and warrants further investigation into potential infectious determinants of ALL and CLL".

EBV is an important opportunistic infection and the role of chemotherapy in these patients could not be ignored. However, since some untreated patients or those in the induction phase also revealed PCR positivity, it points against EBV being an opportunistic infection due to therapy in this group of children. It is quite possible that EBV by itself may not cause ALL but may be an important cofactor at least in some patients. Similarly, 12 patients with AML, seven of them on chemotherapy, were also negative. In the available literature, there is no convincing evidence of EBV infection causing AML in children. It would be relevant to conduct a large multicenter study on drug naïve children with ALL, which may give key additional information on the role of EBV in childhood leukemia in India. With tremendous advances in vaccine research there could be a drastic change in the management of these patients in the foreseeable future. The disease is a formidable economic burden on society. If an infectious agent is involved in ALL, there is hope that in future these would be preventable just as the incidence of hepatocellular carcinoma due to HbsAg can be drastically reduced by HBV immunization.

 
   References Top

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9.MacMahon EM, Glass JD, Hayward SD, Mann RB, Becker PS, Charache P, et al. Epstein Barr virus in AIDS related primary central nervous system lymphoma. Lancet 1991;338:969-73.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Pedersen C, Gerstoft J, Lundgren JD, Skinhøj P, Bøttzauw J, Geisler C, et al. HIV associated lymphomas; histopathology and association with EBV genome: relation to clinical, immunological and prognostic features. Eur J Cancer 1991;27:1416-23.  Back to cited text no. 10      
11.Laytragoon-Lewin N, Chen F, Avila-Carino J, Zou JZ, Mellstedt H, Ernberg L et al. Epstein Barr virus (EBV) carrying cells of chronic lymphatic leukemia (CLL) subpopulation express EBNA 1and LMPs but not ENBA2 in vivo. Int J of Cancer 1995;63:486-90.  Back to cited text no. 11      
12.Schlehofer B, Blettner M, Geletneky K, Haaf HG, kaatsch P, Michaelis J et al. Sero-epidemiological analysis of the risk of virus infections for childhood leukemia. Int J of Cancer 1996;65:584-90.   Back to cited text no. 12      
13.Lehtinen M, Koskela P, Ogmundsdottir HM, Bloigu A, Dillner J, Gudnadottir M, et al. Maternal Herpes virus infection and risk of Acute Lymphoblastic Leukemia in the offspring. Am J Epidemiol 2003;158:207-13 .   Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Loutfy SA, Alam El-Din HM, Ibrahim MF, Hafez MMHerpes simplex type 1 and 2, Epstein Barr virus and cytomegalovirus in children with acute lymphoblastic leukemia in Egypt. Saudi Med J 2006;27:1139-45.  Back to cited text no. 14      
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18.Mujtaba S, Varma S, Sehgal S. Coinfection with Epstein Barr virus in North Indian patients with HIV/AIDS. Indian J Pathol Microbiol 2005;48:349-53.  Back to cited text no. 18      
19.Hamilton - Dutoit SJ, Pallesen G, Franzmann M B, Karkov J, Black F, Skinhøj P. AIDS related lymphoma: histopathology, immunophenotype, and association with EBV as demonstrated by in situ nucleic acid hybridization. Am J Pathol 1991;138:149-63 .   Back to cited text no. 19      
20.Weiss LM, Movahed LA. In situ demonstration of Epstein-Barr viral genomes in viral-associated B cell lymphoproliferations. Am J Pathol 1989;134:651-9 .   Back to cited text no. 20  [PUBMED]  [FULLTEXT]  
21.Venkitaraman AR, Lenoir GM, John TJ. The seroepidemiology of infection due to Epstein Barr virus in southern India. J Med Virol 1985;15:11-6.  Back to cited text no. 21  [PUBMED]    
22.Roy A, Dey S, Chatterjee R. Prevalence of serum IgG and IgM antibodies against Epstein Barr virus capsid antigen in Indian patients with respiratory tract carcinomas. Neoplasma 1994;41:29-33.  Back to cited text no. 22  [PUBMED]    
23.Wolf BC, Martin AW, Neiman RS, Janckila AJ, Yam LT, Caracansi A, et al. The detection of Epstein-Barr virus in hairy cell leukemia cells by in situ hybridization. Am J Pathol 1999;136:717-23.  Back to cited text no. 23      
24.Altieri A, Castro F, Bermejo JL, Hemminki K. Number of Siblings and the risk of Lymphoma, Leukemia and Myeloma by histopathology. Cancer Epidemiol Biomarkers Prev 2006;15:1281-6.   Back to cited text no. 24  [PUBMED]  [FULLTEXT]  
25.Sakajiri S, Mori k, Isobe y, kawamata N, Oshimi K. Epstein Barr virus associated T cell acute lymphoblastic leukemia. Br J Hematol 2002;117:127-9.   Back to cited text no. 25      
26.Miyagi J, Toda T, Uezato H, Ohshima K, Miyakuni T, Takasu N, et al. Detection of Epstein barr virus type 1 in malignant nodal lymphoma, studied in Okinawa, a sub tropical area in Japan. Int J Hematol 2002;75:78-84.   Back to cited text no. 26  [PUBMED]  [FULLTEXT]  
27.O'Connor SM, Boneva RS. Infectious etiologies of childhood leukemia: plausibility and challenges to proof. Environ Health Perspect 2007;115:146-50.  Back to cited text no. 27  [PUBMED]  [FULLTEXT]  

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S Sehgal
Department of Immunopathology, Room No 28, Research Block A, 4rth floor, PGIMER, Chandigarh - 160 012
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DOI: 10.4103/0377-4929.59186

PMID: 20090225

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