[Cancer Research 63, 7606-7608, November 15, 2003]
© 2003 American Association for Cancer Research
Simian Virus 40 Sequences in Malignant Lymphomas in Japan
Shin-ichi Nakatsuka1,
Angen Liu1,
Zhiming Dong1,
Shintaro Nomura1,
Tetsuya Takakuwa1,
Hajime Miyazato2 and
Katsuyuki Aozasa1 Osaka Lymphoma Study Group
1 Department of Pathology, Osaka University Graduate School of Medicine,
2 Department of Hematology, Nephrology and Clinical Immunology, Kinki University School of Medicine, Osaka, Japan
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ABSTRACT
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Recent studies showed that SV40 is detected in >40% of non-Hodgkin’s lymphoma (NHL) in United States, suggesting SV40-contaminated poliovaccines widely used during the period 1955–1963 to be a major source of SV40 in NHL. We examined the presence of SV40 sequences in 122 cases with NHL and 3 with Hodgkin’s lymphoma from Japan. The detection rate of SV40 sequences in diffuse large B-cell lymphoma (19%) was higher than that in peripheral blood cells of normal healthy volunteers in Japan (4.7%; P < 0.05) reported previously as controls for comparison with the study results from cancer patients, suggesting a role for SV40 in the development of diffuse large B-cell lymphoma. In contrast, the frequency of SV40 sequences in NHL cases born between 1951 and 1963 (12%), during which SV40-contaminated poliovaccines might have been inoculated, is not significantly different from that in cases born before 1950 (11%) or after 1964 (15%). SV40 is a new candidate etiologic factor for malignant lymphoma not only in the United States but also in Japan.
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Introduction
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SV40, a small double-strand DNA virus, belongs to polyomaviruses. Genomes of polyomaviruses encode large T antigens, which are nuclear phosphoproteins with multiple biochemical functions essential for virus replication and transformation of rodent cells (1)
. Large T antigens form a complex with the products of tumor suppressor genes, such as p53 and pRb, and inactivate their functions (1
, 2)
. In addition, they have mutagenic and clastogenic properties, and can, thus, induce gene mutations, and numerical and structural aberration of chromosomes. These properties of large T antigens are responsible for transformation of rodent and human cells (1
, 3)
.
Specific monkeys are the natural host of SV40, which is believed not to infect humans directly. However, SV40 could infect humans through inoculation of poliovaccines accidentally contaminated with SV40, when they were prepared using monkey kidneys (4
, 5) . It is speculated that at least 98 million people in the United States and many more worldwide were inoculated with potentially contaminated poliovaccines during the period 1955–1963. SV40 is known to induce several kinds of malignant neoplasms in humans, with malignant mesothelioma and brain tumors being the most common (6
, 7)
. In 2002, Vilchez et al. (8)
and Shivapurkar et al. (9)
showed the presence of SV40 sequences in a large series of NHLs3
but not in nonmalignant lymphoid samples and control cases from the United States using a highly sensitive PCR-based method. They suggested that SV40-contaminated poliovaccines are a major source of SV40 for NHL. Also in Japan, 
90% of children in the age range from 3 months to 10 years old were inoculated with oral poliovaccines that were highly suspected of being contaminated with SV40 during the period from 1961 to 1963 (10)
. Since 1964, SV40-free domestic vaccines have been used in Japan. The purpose of this study is to examine whether the detection rate of SV40 in malignant lymphomas in Japanese individuals born between 1951 and 1963 is higher than that in individuals born before and after this period.
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Materials and Methods
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Case Selection.
The presence of SV40 sequences was evaluated in 122 Japanese cases with NHL and 3 with Hodgkin’s lymphoma for whom frozen tissues were available; they were admitted to hospitals from 1987 to 2002. There were 71 males and 54 females with age at the first diagnosis ranging from 1 to 85 (median, 61) years old. They received biopsy from the lymph nodes in 76 cases and extranodal organs in 49. The biopsied tissues were fixed in 10% formalin and routinely processed for paraffin embedding. Histological sections, cut at 5 µm, were stained with H&E and immunoperoxidase procedures (avidin-biotin complex method).
Immunohistochemistry.
Primary antibodies used and their dilutions were CD3 (Dakopatts, Glostrup, Denmark; 1:100), CD43 (Bioscience, Emmenbrucke, Switzerland; 1:50), CD20 (Kyowa Medex, Tokyo, Japan; 1:200), CD45RO (Dakopatts; 1:100), CD79a (Dakopatts; 1:50), TIA-1 (Coulter, Hialeah, Florida; 1:500), and CD56 (Zymed Laboratories, San Francisco, CA; 1:40). Ninety-two cases showing CD20+, CD79a+, CD3-, CD43-/+, and CD45RO-/+ were judged as B-cell lymphoma. Eleven cases were T-cell (CD20-, CD79a-, CD3+, CD43+/-, and CD45RO+/-) and 19 were natural killer/T-cell lymphomas (CD20-, CD79a-, CD3+, CD43-/+, CD45RO-/+, CD56+, and TIA-1+). The distribution of cases according to the WHO classification is shown in Table 1
. The remaining biopsied samples were snap-frozen in liquid nitrogen and stored at -80°C until use.
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Table 1 Frequency of SV40 sequences by histologic type of malignant lymphoma in the present and the reported cases
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PCR, Southern Blot, and Sequencing for SV40 Large T Antigen.
Genomic DNA was extracted from the frozen tissues with standard phenol-chloroform procedures and stored at -25°C until use. PCR amplification of SV40 large T-antigen sequences was performed in a total volume of 10 µl consisting of 1 µl of DNA template, 0.5 units of AmpliTaq Gold (Applied Biosystems, Foster City, CA), 1.5 mM of MgCl2, 0.2 mM of deoxynucleoside triphosphate, and 0.2 µM of each primer. Two sets of primer pairs [SVTAGP1 (4388–4413, GenBank accession no. J02400), SVTAGP2 (4517–4543), and SVTAGP1-SVTAGP3 (4496–4513)] were used as described previously (11)
. The PCR conditions were as follows: 95°C for 10 min, 45 cycles of denaturation at 95°C for 1 min, annealing at 53°C (for primer set SVTAGP1-SVTAGP2) or 49°C (for primer set SVTAGP1-SVTAGP3) for 1 min, extension at 72°C for 1 min, and a final extension of 7 min at 72°C. Eight µl of PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. PCR was repeated at least four times for all of the samples, and samples showing a clear band in at least three independent PCR reactions were judged as positive. To confirm the specificity of the PCR, the products were transferred to a nylon membrane, and hybridized overnight at 65°C with a 32P-labeled oligonucleotide probe [SVPROBE (4452–4473)]. In addition, the products were extracted from the gel and subcloned, followed by sequencing with the dye termination method using a DNA sequencing kit (Applied Biosystems). To assess the sensitivity of our method, serially diluted control plasmids containing SV40 sequences were tested, and about 10–50 genome copies of SV40 per one reaction could be detected on the gel stained with ethidium bromide. As positive control, diluted control samples containing SV40 sequences were tested in parallel with samples of malignant lymphomas. Fisher’s exact test was performed to evaluate the significance of differences in the frequencies of SV40 sequences among each disease.
In Situ Hybridization for SV40.
A 541-bp fragment (2667–3208), a digest of SV40 strain 776 by PstI and HincII, was subcloned into pBluescript plasmid. In vitro transcription was performed using a DIG RNA labeling kit (SP6/T7; Roche Diagnostics, Mannheim, Germany). Briefly, deparaffinized sections were treated with proteinase K (1 mg/ml) for 30 min, acetylated in 0.1 M triethanolamine for 10 min, and incubated with hybridization buffer containing 1 mg/ml digoxigenin-labeled probe at 50°C overnight. After treatment with the fragment-crystallizable fragment of antidigoxigenin antibody, color development was performed with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate solution.
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Results and Discussion
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This is the first report showing the detection rate of SV40 in malignant lymphoma in Japan. Our results and those of previous reports (8
, 9)
are summarized in Table 1
.
Fourteen cases showed the positive products for SV40 at PCR, which were subsequently sequenced to confirm the specificity. Representative results of PCR-Southern blot and sequencing of PCR products are shown in Fig. 1
. The in situ hybridization studies were conducted in each of 7 cases with positive and negative results at PCR. Three of 7 cases with positive results at PCR demonstrated SV40 sequences in the tumor cells (Fig. 2)
. Seven cases with negative results at PCR uniformly showed negative results. The detection rate of SV40 sequences in the present cases with DLBL (19%) was higher than that in peripheral blood cells of normal healthy volunteers in Japan (Ref. 12
; 4.7%; P < 0.05), suggesting a role of SV40 in the development of DLBL. In contrast, the detection rate in NHL as a total (11%) was significantly lower than that in recent reports from the United States (Refs. 8
, 9
; 43%; P < 0.05). Meanwhile, relatively similar incidences of SV40 in lymphomas (10–14%) to those in Japan were reported in the United States (13
, 14)
. The authors of those reports interpreted their results as indicating a lack of pathological association between SV40 and malignant lymphomas.
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Fig. 1. A, PCR-Southern blot for SV40 large T-antigen sequences. Top panel shows PCR products stained with ethidium bromide in representative 4 cases with DLBL and control plasmids including JCV (JC virus), BKV (BK virus), and SV40. Bottom panel shows Southern blot using SVPROBE labeled with 32P in the same cases. Case 1, 2, and 3 clearly showed a positive band of 126-bp amplified with SVTAGP1 and SVTAGP3. B, 14 cases showed the positive products for SV40 at PCR, which were subsequently sequenced to confirm the specificity. The region of each product corresponding to 4418 to 4513 of SV40 large T antigen is illustrated. Electrophoregram in DLBL case 2 is shown.
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Fig. 2. A, in situ hybridization studies were conducted in each of seven cases with positive and negative results at PCR: representative results of in situ hybridization for SV40 in a case of DLBL with SV40 sequences detectable by PCR-Southern blot were illustrated. Approximately 60% of lymphoma cells show positive signals in the nucleus. B, lymphoma cells never show positive signals in a case without products at PCR-Southern blot.
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The distribution of the year of birth of patients was not mentioned in the reports by Vilchez et al. (8)
and Shivapurkar et al. (9)
. The detection rate of SV40 sequences in the present patients with NHL born between 1951 and 1963 (n = 17; 12%), during which poliovaccines possibly contaminated with SV40 might have been inoculated, is not significantly different from that in patients born before 1950 (n = 95; 11%) or after 1964 (n = 13; 15%). These findings suggest that inoculation of oral poliovaccines used nationwide in Japan during the period from 1961 to 1963 did not influence the overall prevalence of SV40 in humans, or that SV40 was transmitted from monkey to humans through the contaminated vaccines, and it is now spreading in the human population, as most infectious viruses. Because the virus spreads, it infects randomly people of different ages. The detection rate of SV40 in peripheral blood cells from normal healthy volunteers in Japan was 4.7% (12)
. In the United States, the detection rate of SV40 in blood cells was reported to be 0% in some reports (8
, 9)
but 16% in another (14)
. It is significant that 15% of patients with lymphomas born after 1964 in Osaka, Japan were SV40 positive, suggesting the possibility that SV40 is being transmitted from human-to-human among the Japanese population by an unknown route.
The known causative factors of malignant lymphomas include viruses, bacteria, environmental factors, and genetic factors. In particular, information on the association between viruses and malignant lymphoma has been accumulating: human T-cell lymphotropic virus type 1 and adult T-cell leukemia/lymphoma; EBV and Burkitt’s lymphoma, Hodgkin lymphoma, immunodeficiency-related lymphoproliferative disorders, and pyothorax-associated lymphoma; human herpesvirus-8 and primary effusion lymphoma; and hepatitis C virus and lymphoplasmacytic lymphoma. In conclusion, SV40 is a new candidate etiologic factor for malignant lymphoma not only in United States but also in Japan.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Katsuyuki Aozasa, Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3710; Fax: 81-6-6879-3713; E-mail: aozasa{at}molpath.med.osaka-u.ac.jp
3 The abbreviations used are: NHL, non-Hodgkin’s lymphoma; DLBL, diffuse large B-cell lymphoma. 
Received 1/ 8/03.
Revised 9/24/03.
Accepted 10/ 3/03.
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ABSTRACT
Introduction
