© 2003 by Oxford University Press
Journal of the National Cancer Institute, Vol. 95, No. 13, 1001-1003,
July 2, 2003
© 2003 Oxford University Press
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Association Between Simian Virus 40 DNA and Lymphoma in the United Kingdom
Affiliation of authors: Leukaemia Research Fund Virus Centre, Institute of Comparative Medicine, University of Glasgow, Glasgow, U.K.
Correspondence to: Ruth F. Jarrett, M.B., Ch.B., F.R.C. Path., F.R.C.P. (Glasg.) Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Rd., Glasgow G61 1QH, U.K. (e-mail: r.f.jarrett{at}vet.gla.ac.uk).
ABSTRACT
Recent studies have reported the presence of simian virus 40 (SV40) DNA sequences in approximately 40% of tumor samples from non-Hodgkin’s lymphoma (NHL) patients from the United States. We examined a series of 259 tumor and blood samples, including 152 NHL samples, from patients in the U.K. with lymphadenopathy and lymphoid leukemia for the presence of SV40 DNA using a highly sensitive quantitative polymerase chain reaction (PCR) assay and a consensus PCR assay capable of detecting the polyomaviruses SV40, BK, and JC. SV40 DNA sequences were not detected in any sample using either assay. Because the incidence of NHL is similar in the U.K. and the United States, this finding suggests that SV40 is unlikely to have an etiologic role in NHL.
Two recent publications (1,2) reporting the presence of simian virus 40 (SV40) DNA sequences in a high proportion (42% and 43%, respectively) of non-Hodgkin’s lymphoma (NHL) samples have generated great interest. SV40 is a macaque polyomavirus that is potently oncogenic in laboratory animals (3). SV40 does not naturally infect humans. However, inadvertent human exposure to SV40 occurred during the 1950s and 1960s, when batches of inactivated poliovirus vaccine that were contaminated with SV40 were administered in the United States and elsewhere (4,5). Although information on SV40 contamination of vaccines used in the U.K. is limited, vaccine from the United States was administered in the U.K. during this period (Minor P: personal communication). Estimates suggest that 98 million people in the United States were exposed to contaminated vaccine and, of these, 10–30 million were potentially exposed to live, non-inactivated SV40 (4,5). These estimates led to the search for, and detection of, SV40 genomic DNA in human malignancies, particularly in mesotheliomas and brain and bone tumors (3). These tumor types are frequently found in animals experimentally infected with SV40; however, experimentally infected animals also frequently develop lymphomas and lymphocytic leukemias (3). Previous studies that examined the association between SV40 and human lymphoma and leukemia reported that low-level SV40 infection occurred in a smaller proportion of samples (6,7) than that described in the recent studies (1,2).
To determine whether SV40 is detectable in lymphoma samples from the U.K., we examined 232 lymph node biopsy samples and 27 blood samples obtained from patients with lymphadenopathy and lymphoid leukemia, with the use of a sensitive, quantitative, real-time polymerase chain reaction (PCR) assay for SV40 (TaqMan; Applied Biosystems, Warrington, U.K.). Our case series included 152 NHLs, 35 Hodgkin’s lymphomas, and 27 B-cell chronic lymphocytic leukemias; we also analyzed samples from 27 benign lymphadenopathies and 18 miscellaneous tumors (Table 1
). Among the 250 patients whose birth dates were known, 210 were born before 1963, the last year that batches of poliovaccine known to be contaminated with SV40 were administered (4,5). Patients’ residential histories were not available. However, 250 samples were obtained from hospitals located within Scotland and nine samples were obtained from hospitals located within England, suggesting that the vast majority of samples were from Scottish patients. This study was approved by our local institutional review board. Patients either gave informed consent or samples were anonymized (the latter were from an archival collection).
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DNA was extracted from fresh or viably-frozen lymph node biopsy samples and from blood samples obtained from patients with B-cell chronic lymphocytic leukemia by using either proteinase K digestion followed by organic solvent extraction (n = 194) or a QIAamp DNA Blood Mini Kit (Qiagen, Crawley, U.K.). Both methods have been used to successfully extract SV40 DNA from tissue specimens (8,9). However, because concern has been raised about the efficiency of extraction of low-molecular-weight genomes using commercial kits (8), we investigated whether circular SV40 genomes were efficiently extracted from a cell suspension derived from an SV40 DNA–negative reactive lymph node (from a patient with lymphadenopathy) to which we had added known amounts of purified SV40 DNA, using both of the methodologies described above. Efficient recovery of the added SV40 DNA was confirmed using the quantitative PCR assays for
-globin and SV40 described below.
All DNA samples were assayed for the presence of SV40, the JC and BK human polyomaviruses, and two lymphotropic herpesviruses (Epstein–Barr virus and human herpesvirus 8) with the use of TaqMan PCR and the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Oligonucleotide primers and probes were derived from the large tumor antigen (T-ag) gene of the polyomaviruses and from the polymerase gene of the herpesviruses and designed using Primer Express software (Applied Biosystems). Oligonucleotide primer and probe sequences are shown in Table 2. The DNA fragment amplified in the SV40 PCR assay is located in the central region of the T-ag gene, which is well conserved among different strains of the virus. Each reaction included 100 ng of DNA and oligonucleotide probes and primers at concentrations of 200 nM and 50 nM, respectively, except for reactions to amplify JC and BK polyomaviruses, which contained primers at 300 nM. Amplification reactions were performed using TaqMan Universal PCR Master Mix (Applied Biosystems) in a final volume of 50 µL. A negative control (i.e., a reaction lacking template DNA) was included for every two samples, and a positive control (i.e., a reaction containing 5 fg [8 x 103 copies] of purified SV40 DNA [Life Technologies, Paisley, U.K.]) was included in each assay. DNA from COS-7 African green monkey kidney cells, which contain integrated SV40, was extracted using both methods described above and used as a further positive control in PCRs. All reactions were incubated at 50 °C for 2 minutes and then at 95 °C for 10 minutes, followed by 40 cycles of thermal cycling at 95 °C for 15 seconds and 60 °C for 60 seconds. Samples were scored as positive for SV40 DNA if the real-time amplification plot crossed the threshold value generated using the default parameters of the ABI Prism 7700 Sequence Detection System. Dilution experiments using purified SV40 DNA (strain 776; Invitrogen, Paisley, U.K.) showed that the PCR assay consistently detected 10 copies of the SV40 genome in a background of 100 ng of genomic DNA (data not shown). All samples included in the study were tested in a TaqMan assay for -globin and gave values consistent with the presence of approximately 100 ng of amplifiable (i.e., template) DNA. All samples were tested in a blinded fashion.
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All samples tested with the SV40 TaqMan PCR assay were negative. By contrast, Epstein–Barr virus was detected at variable levels in 62 (29%) of the 214 malignant lymphoid samples and in 41 (27%) of the 152 NHL samples examined. No samples tested positive for the presence of JC or BK polyomaviruses, whereas one NHL sample showed low-level positivity for human herpesvirus 8.
To reconcile our findings with those of Vilchez et al. (1), who detected SV40 DNA sequences in 42% of the NHL samples tested, we re-analyzed samples for which we had sufficient DNA by using the consensus PCR assay described by Vilchez et al., which simultaneously amplifies the genomes of JC and BK polyomaviruses and SV40. Samples analyzed using the consensus PCR assay are detailed in Table 1
and included 128 NHLs, 23 B-cell chronic lymphocytic leukemias, 18 Hodgkin’s lymphomas, 17 benign lymphadenopathies, and 13 other tumors. Of the 191 patients from whom this series of samples were obtained and whose birth dates were known, 169 were born prior to 1963. We used the consensus PCR assay to analyze 1 µg of DNA extracted from each sample; the assay could detect 1000 copies of SV40 DNA in a background of 1 µg of high-molecular-weight DNA (data not shown). We used the same positive and negative control reactions that were described for the TaqMan PCR assays. Amplification products were resolved by electrophoresis on 8% polyacrylamide gels, transferred to Hybond-N membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) by electroblotting, and hybridized with an SV40-specific oligonucleotide probe that was end-labeled with 32P. Hybridization was carried out in 2x standard saline citrate (SSC) (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7) at 42 °C (approximately 5 °C below the melting temperature of the probe) and the membranes were washed in 6x SSC at 60 °C before exposure to autoradiographic film. Although positive control samples were consistently positive, no experimental samples tested positive using this assay.
In contrast to recent studies that have reported the detection of SV40 DNA in a large proportion of NHL samples from patients in the United States (1,2), we failed to detect SV40 DNA in any lymphoid sample examined. This is the largest study to investigate the association between SV40 positivity and lymphoid malignancies by PCR among patients in the U.K. We previously surveyed a panel of 26 Epstein–Barr virus-negative Hodgkin’s lymphoma samples for the presence of SV40 DNA by Southern blot analysis and detected no positive samples (10). The reason for the difference between the results of the present study and those of Vilchez et al. (1) and Shivapurkar et al. (2) is unclear, but obviously needs to be resolved. Data on SV40 contamination of poliovirus vaccines in use in the U.K. before and after 1963 are limited; thus it remains possible that this group of patients, most of whom were Scottish, was not exposed to contaminated vaccine. Although geographic locale could therefore account for differences between the SV40 detections rates reported in our study and those reported in previous studies, the prevalence of NHL is similar in the U.K. and the United States (11) and it is therefore unlikely that SV40 plays an etiologic role in a substantial proportion of NHLs.
NOTES
Supported by the Leukaemia Research Fund (LRF) as part of an LRF Specialist Program (to R. F. Jarrett and J. MacKenzie).
We thank David Onions, Ray Cartwright, and Phil Minor for helpful discussion. We are indebted to the Scotland and Newcastle Lymphoma Group and to the many clinicians and pathologists who contributed samples to this study.
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Manuscript received October 14, 2002; revised April 14, 2003; accepted April 18, 2003.
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