New developments about the association of SV40 with human mesothelioma

(2003) 22, 5173 5180 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc M Carbone*,1, HI Pass 2, L Miele 3 and M Bocchetta 1 1 Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Chicago, Cancer Immunology Program, Loyola University Chicago, Maywood, IL, USA; 2 Karmanos Cancer Center, Wayne State University School of Medicine, Detroit, MI, USA; 3 Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois, Chicago, IL, USA Simian virus 40 (SV40) has been detected in human tumors in over 40 different laboratories. Many of these reports linked SV40 to human mesotheliomas. The Vaccine Safety Committee of the Institute of Medicine (IOM), National Academy of Sciences, USA, recently reviewed the evidence associating polio vaccines and/or SV40 with human tumors. The IOM conclusions about polio vaccines and human cancer were: (1) the evidence is inadequate to accept or reject a causal relation between SV40-containing polio vaccines and cancer because the epidemiological studies are sufficiently flawed ; (2) the biological evidence is of moderate strength that SV40 exposure from the polio vaccines is related to SV40 infection in humans. The epidemiological studies were considered flawed because it was not possible to distinguish reliably among exposed and nonexposed cohorts. Concerning SV40, the IOM concluded that (1) the evidence is strong that SV40 is a transforming virus; (2) the evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions (IOM, 2002). Similar conclusions were reached at an International consensus meeting on SV40 and human tumors held at the University of Chicago in 2001. G Klein and C Croce, who chaired the final panel that reviewed all the published evidence linking SV40 to human tumors, stated that the presence of SV40 in human tumors has been convincingly demonstrated (Klein et al., 2002). In addition, a workshop organized by the Biological Carcinogenesis Branch of the National Cancer Institute, Bethesda, MD, chaired by J Pagano, has reached similar conclusions (Wong et al., 2002). Therefore, three independent scientific panels have all agreed that there is compelling evidence that SV40 is present in some human cancers and that SV40 could contribute to the pathogenesis of some of them. It should be noted that the presence of SV40 in mesothelioma and other human tumor types has been challenged by a research team that has consistently reported negative findings (Strickler et al., 2001). However, a member of this research team has recently acknowledged in sworn testimony sensitivity problems and possible irregularities that raise concerns about these negative reports (MacLachlan, 2002). These revelations, together with *Correspondence: M Carbone, Department of Pathology, Cardinal Bernardin Cancer Center, Room 205, Loyola University Chicago, Cancer Immunology Program, 2160 S First Avenue, Maywood, IL 60153, USA; E-mail: mcarbon@orion.it.luc.edu the conclusions of the three independent panels mentioned above, appear to bring to an end the apparent controversy about the presence of SV40 in human mesotheliomas and brain tumors. (2003) 22, 5173 5180. doi:10.1038/sj.onc.1206552 Keywords: SV40; mesothelioma; human cancer Mesothelioma Mesothelioma, a malignancy of the pleural and peritoneal surfaces, is the tumor type in which the evidence causally linking SV40 to tumor development is most convincing. Here, we review the literature about SV40 in human mesothelioma and some of the most important recent developments in this new and exciting research field. These tumors were almost unknown until the second half of the 20th century. A retrospective analysis using current methodology of all lung cancer deaths for over a 100-year period at the Massachusetts General Hospital in Boston revealed no mesotheliomas until 1947 and a steep increase after 1960. The authors suggested that mesothelioma is a new disease (Mark and Yokoi, 1991). Presently, mesotheliomas account for 2500 deaths/year, and in the past the increased incidence has been attributed solely to asbestos. Yet, 20% or more mesotheliomas occur in nonexposed individuals, and among heavily exposed individuals, including crocidolite miners, less than 5% developed mesothelioma (reviewed in Carbone et al., 2002). Thus, additional factors cause mesothelioma, alone or in conjunction with asbestos (Carbone et al., 2002). It appears that SV40 is one of these factors (Carbone et al., 1994, 2002; Gazdar et al., 2002), and asbestos and SV40 have been shown to be cocarcinogens in vitro (Bocchetta et al., 2000). SV40 carcinogenesis SV40 and other DNA tumor viruses are seldom oncogenic in their natural host, but may become more oncogenic when they cross species. For example, human adenoviruses cause tumors in hamsters and rodents,

5174 human polyomavirus JC cause tumors in rodents and in owl monkeys, and monkey SV40 polyomavirus cause tumors in hamsters, mastomys and certain strains of mice. Thus, when studying SV40 oncogenicity, one has to consider the species in which oncogenicity is being studied and the cell type, because certain cell types are very susceptible to malignant transformation and others are not (see below). Finally, the individual susceptibility and immune status always play an important role in any type of viral infection. Viral dose is critical in nonpermissive animals where all the viral load is the input virus. Viral dose is less important in humans because millions of new SV40 particles are produced upon infection of a few cells. Keeping these differences in mind, it is intriguing that in humans SV40 has been detected in the same tumor types that SV40 specifically causes in hamsters, namely, mesotheliomas, brain and bone tumors, and recently lymphomas (reviewed in Gazdar et al., 2002). It should be noted, however, that while there is sufficient evidence to indicate that SV40 is a pathogen in mesotheliomas and in certain forms of brain cancer, the evidence linking SV40 to lymphomas and bone tumors is still preliminary (Gazdar et al., 2002). It remains to be shown that in the latter two tumor types SV40 is a pathogen rather than a passenger. Lymph node biopsies are known to harbor many different viruses, because they contain mononuclear phagocytes that clear viruses from the body and recirculate them through the lymph and the lymph nodes. Thus, the mere detection of SV40 in lymph node biopsies does not establish pathogenesis. Functional studies are in progress to verify that the virus is present in the lymphoma cells rather than in the mononuclear phagocytes, and that SV40 is biologically active (Adi Gazdar personal communication). In addition, sporadic reports have also linked SV40 to other human tumor types and tissues (reviewed in Jasani et al., 2001). However the biological significance of these reports is dubious because of the small percent of these tissues reported to contain SV40 (usually less than 5%). Moreover, the presence of SV40 in tissues other than mesothelium, brain, lymphoid and bone, in animals injected with SV40 does not lead to tumor development. Thus, the presence of SV40 in a given tumor type does not establish causation, because only specific cell types are susceptible to SV40 carcinogenesis. SV40 infection in different species and cell types SV40 has different effects in different cell types (Figure 1). When SV40 infects rodent and hamster cells, SV40 cannot replicate (nonpermissive cells); however, the SV40 genome is expressed and causes morphological transformation and cell division. Since SV40 cannot replicate, the cell progeny does not contain SV40 and these nonpermissive cells revert to the normal phenotype within a few passages in cell culture (abortive transformation). Malignant transformation can occur only if SV40 becomes integrated into the host genome. This Figure 1 Possible outcomes of SV40 infection. (Top) SV40 infection of nonpermissive rodent cells, no viral particles are produced, malignant transformationis rare; (middle) infection of permissive monkey or human fibroblasts, many viral particles are produced and the cells are lysed, malignant transformation is very rare; (bottom) infection of HM cells leads to limited viral production compared to fibroblasts, limited cell lysis, and frequent malignant transformation. See text for a critical analysis of these different outcomes rare event, which occur in less than 1/10 7 infected cells, assures that the SV40 genome is propagated and expressed in the cell progeny, thus maintaining the malignant phenotype (Figure 1, top). Monkey cells, human fibroblasts, and epithelial cells are permissive to SV40 infection: upon infection, millions of viral particles are produced and the cells are lysed (Figure 1, middle). SV40 infects only a fraction of human fibroblasts (B20%) compared to 100% of monkey cells; thus, human cell infection has been sometimes referred to as semipermissive. Once infected, monkey and human cells are similarly permissive to viral replication, which causes cell lysis. Because human fibroblasts are lysed, malignant transformation cannot occur, except when cells are infected at low multiplicity of infection (MOI) (1 virus/cell) and SV40 becomes integrated into the host cell genome with disruption of the late gene coding sequences preventing viral assembly. This event is very rare (1/10 8 cells), thus it was thought that the likelihood for SV40 to cause human cancer was minimal (Carbone et al., 1997b). Until recently, these (Figure 1, top and middle) were the two only models of SV40 transformations that had been studied. When we and others reported the presence of episomal (nonintegrated) SV40 in certain types of human tumors, we were puzzled by the paradox that,

based on the current dogma, episomal SV40 did not cause human cell transformation, rather it caused cell lysis (Carbone et al., 1997b). Mesotheliomas, malignancies often associated with SV40, derive from mesothelial cells. These undifferentiated cells are the last remnants of human mesoderm and have unique biological characteristics that distinguish them from epithelial cells and fibroblasts (Carbone et al., 2002). SV40 infection of human mesothelial (HM) cells led to the discovery that most cells are infected, compared to B20% of fibroblasts, and that most SV40- infected HM survive infection (Figure 1, bottom) (Bocchetta et al., 2000; Cacciotti et al., 2001; Yu et al., 2001). When SV40 infects HM, it replicates; however, fewer viral particles are produced than in human fibroblasts (Bocchetta et al., 2000) and, therefore, cell lysis is infrequent. Because there is no pressure for viral integration, SV40 remains episomal in the HM nucleus (Bocchetta et al., 2000). Expression of the SV40 tumor antigens (the large T antigen, Tag; and the small t antigen, tag) in 100% of the infected cells, with minimal cell lysis, causes a very high rate of malignant transformation (B1/10 3 cells) (Figure 1, bottom). SV40-transformed HM are immortal from the very early passages, because SV40 directly induces telomerase activity (Foddis et al., 2002). This recent finding explained why 87.5% (14/16) of morphologically transformed HM clones were established as cell lines (Bocchetta et al., 2000), compared to less than B5% success when trying to establish in culture other SV40- transformed human cell types. To summarize, when considering the possible biological effects of SV40 upon detection in a given human tissue, it is important to consider the cell type in which SV40 has been detected. SV40 is unlikely to cause malignant transformation of human fibroblasts and epithelial cells because these cells are lysed. HM instead, and possibly other cell types, are very susceptible to SV40 infection. In these cells SV40 replicates without causing cell lysis. The prolonged expression of SV40 in 100% of HM causes a high rate of malignant transformation. Detection of SV40 in human tumors: specificity SV40 has been specifically detected in mesothelioma cells, and not in nearby stromal cells using a variety of techniques in different laboratories: immunostaining and mrna in situ hybridization (Carbone et al., 1994, 1997a), in situ PCR (Ramael et al., 1999). Moreover, Shivapurkar et al. (1999) detected SV40 in tumor cells from 57 of 118mesotheliomas and in 1 of 75 nearby stromal cell areas microdissected from the same sections. This finding (which was independently confirmed by one of us, M Carbone, unpublished results) is of particular relevance because it is virtually impossible to produce a laboratory artifact of this kind. Presently, there are a total of at least 61 reports from 41 different laboratories worldwide that have detected SV40 DNA, RNA, and/or proteins in human mesothelioma, lymphoma, brain and bone tumors (reviewed in Jasani et al., 2001; Carbone et al., 2002; Gazdar et al., 2002). Different techniques have been used to detect and to confirm that true SV40 was present in these tumors, including: Southern blot hybridization of genomic DNA, viral rescue, Tag immunofluorescence and immunohistochemistry, PCR, DNA sequencing, mrna in situ hybridization, Tag immunoprecipitation and Western blot analysis, antisense Tag, PCR in situ, microdissection followed by PCR, coprecipitation of Tag with cellular p53 and Rb, Electron Microscopic (EM) demonstration of SV40 in human mesothelioma biopsies (the same biopsies showed SV40 positivity by PCR, Cacciotti et al., 2001), specific induction of cellular oncogenes in SV40-positive tumors and inactivation of tumor suppressors. The overwhelming number of positive reports, from different laboratories, using different techniques proves that SV40 is present in some human tumors. This conclusion does not rule out that some papers may have erroneously reported laboratory artifacts, including PCR contamination in some of the laboratories that relied only on this technical approach without performing rigorous controls. Moreover, a multilaboratory study to verify the presence of SV40 in human mesothelioma was conducted upon recommendation of a consensus conference organized by the NIH, FDA and CDC in 1997 (Carbone et al., 1997b) and by further recommendation of the International Mesothelioma Interest Group (IMIG). This study directed by an investigator (JR Testa) not previously associated with this research a requirement set by the 1997 consensus conference confirmed the presence of SV40 in mesotheliomas from the USA (Testa et al., 1998). Geographical and technical differences Different research teams have reported different incidences of SV40 positivity from different regions. True geographical differences, technical discrepancies, or both may cause this. Hirvonen et al. (1999) failed to detect SV40 in 49 Finnish mesotheliomas but detected SV40 in three of five mesotheliomas from the USA. Emri et al. (2000) and De Rienzo et al. (2002) did not detect SV40 in 29 and nine Turkish mesotheliomas, respectively, but they detected SV40 in two of two Italian and four of 11 American mesothelioma biopsies analysed in parallel. Of particular interest is the study of De Rienzo et al. (2002), because of the rigorous precautions taken to rule out laboratory artifacts, and to verify the reproducibility of the results in three separate specimens from each tumor biopsy. Leithner et al. (2002) found SV40 in three of three tumors from the USA and Italy but did not detect SV40 in eight mesotheliomas and 24 bone tumors from Austria. The authors of these publications attributed the negative results they obtained in Finland, Turkey, and Austria, respectively, to the fact that SV40-contaminated polio vaccines were not used in those countries. 5175

5176 A low incidence of SV40 has also been reported in Germany (Heinsohn et al., 2000). The overall exposure of Germans to SV40-contaminated polio vaccines is unclear, because East and West Germany received different types and batches of vaccines. Weggen et al., 2000 in the former West Germany, detected SV40 in two of 27 ependymomas, a lower rate than that reported in the USA (Bergsagel et al., 1992). Remarkably, the two positive specimens were from US patients who had been treated in Germany, while the 25 negative biopsies were from German patients. Together these findings argue for true geographical differences, possibly related to the use of SV40-contaminated vaccines. However, contaminated vaccines are not necessarily the only cause, because geographical differences are found also with other DNA tumor viruses. For example, the Epstein Barr virus (EBV) is associated with 90% of nasopharyngeal carcinomas in Asia and with 90% of Burkitt lymphomas in Africa, yet the same tumor types are rarely associated with EBV in the USA. Technical differences may also account for some discrepancies. Gordon et al. (2002) reported that they found SV40 in two of 35 mesothelioma biopsies and three of seven mesothelioma cell lines. The same authors stated that initially they had failed to detect SV40 in the same specimens. When they modified the technical approach they detected SV40. Thus, some negative results may represent false negatives. Negative reports In addition to the negative reports that appear related to geographical differences, there are five published reports that failed to detect SV40 in mesothelioma. These four reports used only PCR to test for SV40. The most recent, Hubner and Van Marck (2002), was a retraction from Dr Van Marck s laboratory (Belgium), which had previously reported SV40 in the same mesothelioma specimens (Dhaene et al., 1999). No explanation was provided for the two contrasting sets of results. Another negative paper was by Mulatero et al. (1999) who failed to detect SV40 in 12 British mesothelioma; however, the technical approach used had limited sensitivity (Jasani, 1999). The other two negative reports about SV40 in mesothelioma, were from the Viral Epidemiology Branch at the National Cancer Institute, USA, and their contracting laboratory, directed by Dr K Shah (Strickler et al., 1996, 2001), and caused a major controversy. Some recent developments have addressed some of the issues of this controversy. As documented in a recent paper by MacLachlan (2002), Shah has acknowledged, in sworn testimony, that the sensitivity of his original experimental approach (used in the study reported in Strickler et al., 1996) was at times very low, because using this approach he failed to detect SV40 in masked controls containing purified SV40 DNA diluted in buffer. Furthermore, he acknowledged irregularities about the purported blinded experimental approach that raise concerns about the negative results of the Strickler et al. (2001) multilaboratory study (MacLachlan, 2002). In addition, the same study was flawed by the fact that half of the negative water controls had turned out to be positive in this investigation (Strickler et al., 2001). More recently, this same research team reported that SV40 was not present in brain tumors from India. A critical analysis of this latter manuscript revealed that the technical approach was flawed because of technical errors that invalidate the conclusions of this paper (Carbone et al., Int. J. Cancer in press). Briefly, Engels et al. (2002) used quantitative PCR (Q-PCR) to test for SV40 and to determine the amount of SV40 actually present in tumors. However, the sensitivity of their assay was so low that they estimated at less than 10 the total number of SV40 genomes present in six mouse tumors formed by the CRL-2162 SV40-positive mouse cell line (ATCC). This is clearly wrong, because all the cells in this cell line express SV40 tumor antigens and contain 3 6 copies of integrated SV40. A seventh SV40-positive control mouse tumor (i.e. formed by the same cell line) tested completely negative for SV40 in their Q-PCR assay. Clearly, one cannot expect to detect SV40 in human tumors when using a methodology with such low sensitivity that fails to detect SV40 in a tumor formed by an SV40 positive cell line. Furthermore, all the calculations, and thus all the results, were based on GAPDH analyses to estimate the total cell number. Unfortunately, the GAPDH estimates were incorrect, leading to an error in their calculations of SV40 copy number in the cells. Engels et al. (2002), stated that GAPDH is a single-copy gene. In fact, human cells contain 25 copies of highly homologous GAPDH sequences, 12 of which share extensive nucleotide identity with the universally known cdna sequence (Hanauer and Mandel, 1984). Furthermore, the GAPDH standard curve was produced using the K562 human cell line from ATCC. As stated in the ATCC web site, this is a triploid erythroleukemia cell line; therefore it is likely that this line contains even more than 25 GAPDH gene copies/cell. Additional problems are outlined in Carbone et al., in press. To conclude, the recent information about problems with some of these negative reports (MacLachlan, 2002) together with the technical mistakes of the most recent publication of this research team raise concerns about the validity of their negative reports. Epidemiology No epidemiological data are available to verify whether SV40 causes human cancer, because we cannot clearly identify infected from noninfected cohorts. A few studies, however, investigated if recipients of polio vaccines during the years 1955 1963 had a higher incidence of cancer or of certain types of cancers. These studies assume that contaminated vaccines were the only source of SV40 infection in humans, because in the event

of natural transmission from monkey to human, or human to human, these studies would be mistakenly comparing exposed to exposed cohorts. Furthermore, these studies are limited by the fact that it is unclear who received contaminated vaccines because apparently only about 1/3 of the vaccines were contaminated (Carbone et al., 1997b). Recent data have demonstrated the presence of SV40 in individuals born after 1963 (Butel and Lednicky, 1999). Thus, studies that compared the incidence of certain tumor types in cohorts born before and after 1963 (i.e. all of the epidemiological studies published so far) may have compared two SV40-exposed cohorts (IOM report, 2002). With this background, we will briefly review the epidemiology available. Innis (1968) found a significant association between the immunization with the Salk vaccine and childhood malignancies in the 1- to 14-year-old age group (Po0.005). Heinonen et al. (1973) found an increased incidence of ependymomas in children born during the contaminated years. Studies conducted by the Viral Epidemiology Branch at the NCI, instead, consistently failed to detect an increase in cancer risk (Fraumeni et al., 1963; Strickler et al., 1998; Carrol-Pankurst et al., 2001). Fisher et al. 1999 found that the rate of mesothelioma, ependymoma and bone tumors increased in exposed cohorts, yet the small number of cases did not reach statistical significance. Ependymomas and bone tumors are rarer than mesothelioma, and about half of the patients are cured; therefore, very few cases are captured in cancer mortality data, the only type of data available for these epidemiological analyses. Geissler (1990) compared the overall cancer rates in East Germany prior and after 1962 and found no differences. Arguably, one would not expect rare tumors such as those associated with SV40 to influence the overall cancer rates. However, Geissler found SV40 DNA and proteins in 30 of 110 brain tumors of children born during the exposed years. In Sweden polio vaccines were prepared locally and were not contaminated with SV40. In 1957, however, an American batch of polio vaccine was used. Olin and Gieseke (1998), did not find any increased risk of cancer in children born in 1957 compared to other years. However, there are no data to verify whether the American batch used in Sweden in 1957 was contaminated with SV40. Because it has been estimated that only 1/3 batches were contaminated, the odds are that Sweden never received contaminated vaccines. The Vaccine Safety Review Committee of the IOM, National Academy of Sciences, USA, in 2002 reviewed these epidemiological studies. The IOM concluded that the evidence was inadequate to conclude whether or not the contaminated polio vaccine caused cancer because the epidemiological studies are sufficiently flawed. Can SV40 cause human cancer? Disruption of the intracellular pathways regulated by SV40 Tag and tag, oncogenic ras and telomerase activity suffices to create a human tumor cell (Hahn et al., 2002). In addition, since the publication of this manuscript, SV40 has been shown to induce directly telomerase activity (Foddis et al., 2002). Therefore, SV40 is a human carcinogen. The genetic damage caused by SV40 can be appreciated by karyotype analyses. Figure 2 shows a near-tetraploid metaphase 5177 Figure 2 Giemsa-banded methaphase spread demonstrating the extent of genetic damage caused by SV40 infection of HM cells. (Left) Uninfected HM cell (control); (right) mesothelial cell transformed by SV40 infection (Bocchetta et al., 2000) containing 85 chromosomes. Arrows point to rearranged chromosomes

5178 from a HM cell infected and transformed by SV40 in vitro. Similar extensive genetic damage is seen consistently in SV40-infected HM cells (Testa and Carbone, unpublished results). It is obvious that such extensive genetic damage cannot be harmless to HM cells. Additional strong evidence in favor of a carcinogenic effect of SV40 comes from early studies that demonstrated that human cells from healthy human volunteers infected by SV40 in tissue culture grew as tumor nodules when reinjected subcutaneously into these individuals (Jensen et al., 1964). Recent antisense Tag experiments demonstrated that even low levels of Tag expression were required for the maintenance of the transformed phenotype of SV40-positive mesothelioma cell cultures derived from SV40-positive tumors (Waheed et al., 1999). Antisense Tag treatment caused p21 expression, an effect mediated by p53 now free from the inhibitory effect of Tag, growth arrest and apoptosis (Waheed et al., 1999). Together, these data indicate that SV40 is a potent human carcinogen, not a passenger, when detected in those human cell types that are susceptible to its carcinogenicity. However, viruses are seldom complete carcinogens, and cancer development is not an inevitable outcome of virus infection in any viral system (Butel, 2000). Thus, we believe that SV40 carcinogenesis must be studied together with the environmental and genetic factors associated with SV40-positive tumors. Molecular mechanisms of SV40 carcinogenesis There are multiple lines of evidence, in transgenic mice (Ewald et al., 1996; Tzeng et al., 1998), in rat glioblastoma (Salewski et al., 1999) and in human cells in vitro (Moorwood et al., 1996), indicating that SV40 can transform with a hit and run type of mechanism. The genetic damage caused by SV40 (Figure 2) can be sufficient to drive the growth of the malignant cells independent of continuous Tag expression. Actually, when cells lose the SV40 DNA they may become even more oncogenic, a phenomenon attributed to the inability of the immune system to react to viral antigens expressed by these tumor cells (Salewski et al., 1999). However, SV40 presence and expression is often required for the maintenance of the transformed phenotype, and this allows us to study the mechanisms of SV40 carcinogenesis. Figure 3 uses mesothelioma as a model and summarizes some of the most important mechanism that have been linked to SV40 oncogenesis in this system. It can be seen that SV40 inactivates the tumor suppressors p53 (Carbone et al., 1997a), Rb- and related Rb family proteins p107 and Rb2-p130 (De Luca et al., 1997), and RASSF1A (Toyooka et al., 2001, 2002). At the same time SV40 induces telomerase activity (Foddis et al., 2002) and induces met (Cacciotti et al., 2001), Notch-1 (Bocchetta et al., 2003) and IGF- 1R (Pass et al., 1996) activation on the cell membrane. The activation of these oncogenes and growth factor receptors leads to the activation of the ERK-kinase pathway and to AP-1 activity that promotes cell division. The only known molecular effect of crocidolite asbestos is the activation of the EGF receptor, which through the ERK-kinases induces AP-1 activity (Robledo and Mossman, 1999). Phosphatase 2 A (PP2A) naturally downregulates the ERK-kinases; however, in the presence of SV40 small tag, PP2A is inhibited and the effects of SV40 large Tag and asbestos are enhanced (co-carcinogenesis) (reviewed in Carbone et al., 2002). Moreover, because asbestos impairs the local and systemic immune response (Carbone et al., 2002), SV40-transformed mesothelial cells may have a better chance to evade immune detection and grow as malignancies if asbestos is present. Figure 3 Molecular pathways altered by SV40 infection. The expression of SV40 Tag and tag induces or represses specific key regulatory cellular genes. See text for a critical discussion. Crossed bar: inhibition; arrow; induction

Conclusions The Hill and Hill criteria are often used to establish human carcinogens. Except for the epidemiological evidence which is not available, SV40 satisfy the Hill criteria for causation (Gazdar et al., 2002). The advances in molecular pathology, which is the cellular equivalent of forensic pathology, in the past 20 years, allow us to study the damage caused by a virus and other carcinogens in a given tumor and to establish whether the virus was a passenger or a pathogen. Epidemiology does not address causation in a specific patient, molecular pathology does. Epidemiology, however, is required to establish the percent of tumors linked to a given carcinogen, and to establish the effects of a given carcinogen in the exposed population. Therefore, presently using molecular pathology, we can study if SV40 contributed to the development of a given tumor in a given patient, but we cannot study the effects that SV40 may or may not have had on cancer incidence in a given cohort. 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