Simian virus 40 in Japanese patients with lymphoproliferative disorders.

Abstract

Lymphoma and leukaemia are clonal disorders caused by genetic mutations, but in most patients co-factors contributing to their pathogenesis are unknown. Much argument has centred on the possibility that some form of viral infection may directly or indirectly play a causal role in the aetiology of lymphoproliferative disorders. Epstein–Barr virus has been detected in various types of lymphoma/leukaemia, and human herpesvirus 8 is implicated in primary effusion lymphoma and multicentric Castleman's disease. Human herpesvirus 6 has been cited as a possible cause or as a modulating element of several lymphoproliferative disorders (Braun et al, 1997; Daibata et al, 1998). Furthermore, a role of hepatitis C virus in the pathogenesis of a subset of lymphomas has been demonstrated (Hermine et al, 2002). Recently, two groups in the USA independently reported that simian virus 40 (SV40) DNA was detected by polymerase chain reaction (PCR) in approximately 40% of non-Hodgkin's lymphomas, but not in normal lymphoid tissues or blood cells (Shivapurkar et al, 2002; Vilchez et al, 2002). Some other groups also reported a relationship between SV40 and non-Hodgkin's lymphomas (Martini et al, 1998; Rizzo et al, 1999; David et al, 2001). However, all of these reports are based on findings in the USA and Europe, and there has been no report regarding the incidence of SV40 infection in haematological diseases in Asia. In this study, we looked for SV40 DNA sequences in samples from Japanese patients with lymphoproliferative disorders. Two hundred and nine individuals were enrolled (Table I). The histological types of non-Hodgkin's lymphomas were categorized according to the World Health Organization classification. All samples of lymphoma and non-malignant lymphoid tissues were obtained from biopsies, and DNA was extracted from fresh or frozen tissues except for DNA taken from paraffin-embedded specimens of mucosa-associated lymphoid tissue (MALT) lymphoma. For leukaemia samples, DNA was extracted from mononuclear cells of leukaemia cell-enriched peripheral blood or bone marrow. Most specimens were obtained between 1996 and 2002 from residents of Kochi prefecture in southern Japan. DNA was extracted using the phenol–chloroform extraction technique after proteinase K digestion, and 0·1 µg of genomic DNA was amplified by PCR for 45 cycles (each of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C). All DNA samples were amplified in parallel by PCR for human β-globin sequences to show the integrity of the DNA samples. The DNA samples were analysed for the presence of SV40 T-antigen sequences with two sets of the well-characterized primers: 5′-TAGGTGCCAACCTATGGAACAGA-3′ and 5′-G-GAAAGTCTTTAGGGTCTTCTACC-3′; and 5′-TGAGGCTACTGCTGACTCTCAACA-3′ and 5′-GCATGACTCAAAAAACTTAGCAATTCTG-3′. Samples were considered to be positive when both of the sequences were detected. The positive control for the T-antigen sequence was plasmid DNA containing cloned SV40 (pEF321-T) and the negative control was without added DNA template. To confirm the reproducibility of PCR assays and to avoid problems related to contamination during the PCR procedures, the positive samples were examined four times. As indicated in Table I, the SV40 T-antigen sequences were found in one of 39 diffuse large B-cell lymphomas, one of 10 follicular lymphomas and one of 16 B-cell chronic lymphocytic leukaemias. No SV40 DNA was detected in any of other samples tested. These three SV40-positive samples did not carry JC virus DNA sequences, as judged by PCR with three different sets of primers. The SV40-positive rate in this study is lower than that reported in the previous studies (Shivapurkar et al, 2002; Vilchez et al, 2002). This difference might be ascribed to the technical approaches employed to detect viral sequences or geographical variation in the prevalence of the virus. Inoculation with SV40-contaminated polio vaccine in the years around 1960 is thought to be the major route of SV40 exposure in humans. We do not know whether the contaminated polio vaccines were used in Japan and, if so, how long they were administered for. Although the viral ‘hit-and-run’ mechanism can not be fully excluded, our findings suggest that SV40 is unlikely to have played a pathogenetic role in most of the lymphoproliferative disorders in the Japanese population. Worldwide epidemiological surveys are needed to determine a possible association of SV40 infection with the pathogenesis of human haematological malignancies. This work was supported by: Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; the Foundation for Promotion of Cancer Research in Japan; and Mitsubishi Pharma Research Foundation to M. Daibata.