Replicative viral DNA in Epstein-Barr virus associated Burkitt\'s lymphoma biopsies

Leukemia Research Vol. 17, No. 3, pp. 285-289, 1993. Printed in Great Britain.

0145-2126/93 $6.00 + .00 Pergamon Press Ltd

R E P L I C A T I V E V I R A L D N A IN E P S T E I N - B A R R VIRUS A S S O C I A T E D BURKITT'S L Y M P H O M A BIOPSIES M. I. GUTIERREZ,K. BHATIA and I. MAGRATH Lymphoma Biology Section, Pediatric Branch, National Cancer Institute, NIH, Bethesda, MD 20892, U.S.A. (Received 9 March 1992. Revision accepted 22 August 1992) Abstract--Epstein-Barr virus (EBV) is linked to a spectrum of human diseases including epithelial and lymphoid malignancies in which it exists predominantly in a latent state. EBV is capable of establishing replicative infection at oropharyngeal and genital sites. Replicative EBV infection also occurs in oral hairy leukoplakia, in EBV associated iymphoproliferative disorders, and to a minor degree in nasopharyngeal carcinomas. Recent evidence also suggests that EBV replication, also, may be associated with AIDS related lymphomas and Hodgkin's disease. However it is widely believed that virus in circulating B-lymphocytes and in B-cell malignancies is stringently latent. We now show that by Southern blot analysis we can detect replicative forms of virion DNA in 14.5% (8 of 55) of EBV-positive Burkitt's lymphoma biopsies. This may be the explanation for the elevation of the titres of lytic cycle EBV antigens that is associated with presentation and relapse of EBV associated Burkitt's lymphoma. Key words: EBV, Burkitt's lymphoma, B-cells, viral replication, monoclonality.

sustaining source for transmission of the infection to B-lymphocytes. Infection of B-lymphocytes also persists for life, but is believed to be stringently latent in these cells, although EBV replication has been documented in some AIDS-related lymphomas and Hodgkin's lymphomas [11,12]. In contrast to Burkitt's lymphoma, NPC occurs later in life, but in both diseases serological changes (i.e. increases in antibodies against EBV early antigen), usually interpreted to signify virus reactivation, precede or accompany the appearance of the tumor. In NPC this has also been confirmed by the demonstration of replicative forms in the tumor cells. [8]. In this report we show that EBV replication also occurs in some Burkitt's lymphomas, including one AIDS-associated lymphoma. We suggest that this reactivation of EBV may account for the serological changes that precede or accompany the clinical presentation of Burkitt's lymphoma.

INTRODUCTION EPSTEIN-BARR virus (EBV) is a human herpes virus capable of infecting epithelial cells and circulating lymphocytes. The viral genome has been shown to be present in 100% of anaplastic nasopharyngeal carcinomas (NPC) [1] and 95% of endemic Burkitt's lymphomas (BL) [2]. EBV DNA is also present in 20-30% of sporadic BL [3], in a significant fraction (40%-100%) of lymphomas resulting from either congenital or acquired immunodeficiency [4, 5] and in Reed-Sternberg cells in a proportion of cases of Hodgkin's disease [6]. While it seems unlikely that EBV is merely a passenger virus in these malignancies its role in pathogenesis has yet to be determined. Support for a causative role for EBV is provided by the findings that in B-cell lymphomas and NPC, EBV infection precedes expansion of the malignant clone [7, 8]. Moreover, EBV can immortalize B-cells and prevent apoptosis by upregulating the transcription of the protooncogene bcl-2 [9]. Primary EBV infection is thought to occur in pharyngeal epithelial cells. This reservoir for EBV normally persists indefinitely and provides a self-

MATERIALS AND METHODS Representative samples of tumors were collected from biopsies obtained for diagnostic purposes and stored at -80°C until processed. Endemic BL samples were obtained from the National Cancer Institute Burkitt Tumor project in Ghana. Sporadic BL biopsies and AIDS-associated BL originated from North and South America. All Burkitt's lymphoma samples used in this study have been previously characterized for rearrangements of the c-myc locus and for the presence of EBV sequences [3].

Abbreviations: EBV, Epstein-Barr virus; NPC, nasopharyngeal carcinoma; TR, tandem repeats. Correspondence to: Kishor Bhatia, Bldg 10, Room 13N240 Pediatric Branch, NCI, NIH, Bethesda, MD 20892, U.S.A. 285

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Bgl II

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BamHI

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BamHI

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BanlHI

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BamHl LINEAR TERMINI

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FIG. 1. Restriction map of the termini of EBV. The figure depicts the fragments from the NJ-het region of the EBV genome used as probes. The figure also illustrates graphically the possible restriction fragments identified by this probe following BamHI digestion of EBV DNA.

DNA was extracted from these samples using a standard protocol [10]. In brief, frozen tissue specimens were pulverized and cells were lysed. Proteins were digested using proteinase K. The lysates were then subjected to extraction with phenol, phenol:chloroform (1:1) and chloroform. Nucleic acids were recovered from the aqueous phase following ethanol precipitation. To determine the status of the EBV genome within the tumor, the samples were analyzed by digesting 10 ~tg of the tumor DNA with BamHI. DNA was then electrophoresed in 1% agarose and transferred to a nylon membrane. Hybridization with an EBV probe was performed under stringent conditions in 3 x standard saline citrate at 65°C for 14 h. Post hybridization washes were carried out in the presence of 0.5% sodium dodecyl sulfate at 60°C and the membranes were subject to autoradiography. A 5.2 Kb BamHI-EcoRI fragment, obtained from the terminal fragment NJ-het and a 3.5 Kb EcoRI-BamHI from the left end of the EBV genome were used as EBV probds (Fig. 1). Forty nanograms of this DNA was used as a template in a random priming oligo-labelling reaction to obtain labelled fragments (1 × 109 dpm/~tg DNA). RESULTS A N D DISCUSSION We have analyzed 55 EBV-positive tumor biopsies for the structural heterogeneity of the termini of the EBV genome. These included 2 AIDS-associated lymphomas, 24 endemic Burkitt's lymphomas and 29 sporadic Burkitt's lymphomas. The EBV genome is a 172 Kb double-stranded linear molecule with homologous direct tandem repeats (TR), each of approximately 500b.p. in length, at each end of the molecule [13, 14]. Following infection, the linear termini are fused at the T R region to produce circular genomes. The process of circularization of the linear DNA results in the incorporation of a variable number of TR units. Only one viral particle survives in each cell and the number of TRs is characteristic of each virion, being the same for all episomal copies in each cell but varying from cell to cell. The terminal heterogeneity can be identified by the size of the fragments obtained following digestion of the EBV DNA with BamHI which cuts outside the TR (Fig.

1). The terminal BamHI fragments obtained from a circular episome of EBV D N A contain, besides the TR unit(s), 7.5 Kb of unique DNA. Thus the smallest BamHI fragment with one TR unit will contain 8 Kb of EBV DNA, and depending upon the number of TR units incorporated during circularization, the fragments will vary in size with increments of 500 b.p. each. Independent infection events can thus be visualized on Southern blots as single bands more than 8 Kb in size, each corresponding to one fused terminal restriction fragment. All the biopsy samples we analyzed by this method displayed a single band that was greater than 8 Kb (Fig. 1), indicating a monoclonal population of EBV episomes. These data are consistent with an earlier report by Neri et al. [7], and demonstrate that the monoclonal proliferation of these tumors occurred after EBV infection. However, we have also been able to detect a ladder of fragments (Fig. 2, lanes 3 and 5), hybridizing to the NJ-het termini probe in 8 tumors (Table 1). The presence of the ladder was also confirmed by using a combination of B a m H I and BglII restriction enzymes (Fig. 2, lanes 9, 11, 14, 16 and 17). The smallest fragment in this ladder range is approximately 4.0 Kb (Fig. 2) and the bands increase in size by increments of 500 b.p. Similar fragments were also observed in D N A from the cell line P3HR1 (Fig. 2, lane 9). This cell line is known to make virus particles. The presence of multiple fragments in the biopsies, forming a ladder, many having a size incompatible with circular molecules, indicates the presence of linear EBV genomes. It is known that during virus production, EBV terminal repeat sequences from linear concatamors, which include a variable number of TRs [15]. Thus, the presence of multiple linear fragments varying in size by 500 b.p. in these samples demonstrates the presence of replicative viral DNA. In latently infected cells, the EBV genome is subjected to extensive methylation and nucleosomal organization [16]. Both these processes probably prevent lytic cycle reactivation. Drugs that reduce methylation and cause D N A damage increase the likelihood of lytic cycle reactivation [17]. However these biopsy samples were obtained from patients prior to chemotherapy, and the presence of lytic EBV cannot therefore be attributed to drug effect. It has been shown that in the cell line P 3 H R 1 - particularly in the subclone 5A---defective, self contained replicons are formed which have a high propensity to induce normal replication [18]. Defective EBV has also been correlated with replicative forms in r i v e in oral hairy leukoplakia [19]. The ability to induce supernormal replication maps to a 2700 b.p.

Lytic EBV in Burkitt's lymphoma

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TABLE 1. DISTRIBUTIONOF REPLICATIVEEBV IN A SPECTRUMOF EBV-POSIT1VE BL

AIDS lymphomas Sporadic lymphomas North America South America Endemic lymphomas

Biopsies tested

Biopsies with replicative EBV

2

1

9 ]20 24

2 2 3

FIG. 2. Southern blot analysis of the EBV termini in Burkitt's lymphoma biopsies. Cellular DNA obtained from various biopsy material was digested with either BamHI (lanes 1-8, lanes 1 and 6 are EBV-negative tumors) and probed with the BamHI NJ het probe (BamHI-EcoRI 5.2 Kb in Fig. 1), or BamHI and BgI II (lanes 9-17) and probed with~a P32-radio-labelled 3.5 Kb EcoRI-BamHI fragment illustrated in Fig. 1. Lane 17 contains DNA from the cell line~P3HR1 and serves as the positive control for replicative forms of EBV DNA. Lane 15 contains DNA from peripheral blood lymphocytes used as a negative control. Lanes 2, 4, 7, 10 and 12 contain DNA from EBVassociated BLs, carrying only the episomal form of EBV. Lanes 3, 5, 9, 11, 13, 14 and 16 contain DNA from tumors carrying replicative forms of EBV.

WZ-het fragment resulting from recombination and deletion of D N A between the B a m H I Z and W regions of the E B V genome. To determine whether the replicative forms seen in the Burkitt's lymphoma biopsies result from such defective D N A , we used PCR amplification of the W Z region as described previously [19]. No genomic rearrangement was observed in any of these samples, except in the control HRI-clone 5A (data not shown). However, identification of the defective virion D N A may not always be possible since the recombination that gives rise to such heterogeneous D N A may be associated with variability of the W-Z junction sequences, such that the set of primers used in this reaction may not always lie within the available recombinant product. Nevertheless, although it is still possible that some of these biopsies contain the defective virion, our inability to amplify the recombinant products from any of the samples makes it highly unlikely that the replicative forms of E B V D N A we observed can be

attributed solely to the presence of defective virions in these samples. Although the biopsy samples were unsuitable for the performance of immunofluorescent assays for viral antigens known to be associated with replication (e.g. EA, VCA) we did attempt to detect such antigens by Western blots analysis. Our attempts were unsuccessful. This could be due to the lability of these proteins in the biopsy samples. It is also, however, highly probable that we were unable to detect these antigens because only a small percentage of the population of Burkitt's lymphoma cells within the biopsy samples contained the replicative forms of the E B V virion. Replication is associated with cell lysis, and hence a high fraction of cells containing virus would be incompatible with tumor growth. It is also possible that rapid destruction of cells which contain virus particles leads to an inability to detect cells containing viral replicative antigens, although linear viral D N A molecules may persist in cyto-

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plasmic fragments, or simply be more amenable to detection because of the techniques used. The presence of such replicative viral antigens is also inferred from well-established observations that antibodies to replicative antigens ( E A and VCA) are present in higher titre in patients with EBV associated Burkitt's lymphoma [20]. Moreover, the primary presentation or relapse of BL appears to chronologically follow an elevation in these antibody titres [21, 22]. Although this observation appears to be in direct contrast to the obligate requirement of EBV latency in the development and maintenance of BL, it is noteworthy that cell kinetic studies have demonstrated that the rate of cell loss in African Burkitt's lymphoma is about 70% that of cell renewal accounting for the difference in theoretical and actual doubling time [23]. EBV-induced cell lysis probably accounts for only a small fraction of this cell loss. Our findings in conjunction with the serological data suggest that EBV-associated Burkitt's lymphomas contain a small and variable proportion of cells in which virus replication is occurring. In the majority of tumors, however, this process is below the level of detection--perhaps because of the resultant rapid cell lysis--but is reflected by, and almost certainly explains, the increased titres of replicative cycle antigens that are associated with tumor growth. Moreover, this is entirely consistent with previous observations [24] that the titre of anti-EA at presentation correlates with prognosis in African EBVassociated tumors. The antigen load hence and antibody titre probably simply reflect tumor burden. One cannot, however, exclude the possibility that in some cases the increased antibody titre partly reflects replication in a non-neoplastic cell population as a result of altered immunity associated with tumor development. The presence of replicative E B V in Burkitt's lymphomas raises the possibility that methods could be devised to increase this process, thus leading to tumor regression, as occurs naturally in the frog adenocarcinoma induced by the Luck6 herpes virus [25]. Acknowledgement--MIG was supported by a Fellowship Award by the Cancer Research Foundation of America. This study was partly supported by World Laboratories.

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