SV40-Cancer paper

from http://www.nature.com/cgi-taf/DynaPage.taf?file=/onc/journal/v21/n8/full/1205173a.html.

14 February 2002, Volume 21, Number 8, Pages 1141-1149
Table of contents ]
Meeting Report
Association of SV40 with human tumors
George Klein1, Amy Powers2 and Carlo Croce3
1Microbiology and Tumor Biology Center, Karolinska Institut, S 171-77, Stockholm, Sweden
2Cardinal Bernardin Cancer Center, Loyola University, Chicago, Maywood, Illinois, IL 60153, USA
3Kimmel Cancer Center, Jefferson Medical School, Philadelphia, Pennsylvania, PA 19107, USA
Correspondence to: A Powers, Cardinal Bernardin Cancer Center, Room 205, 2160 South First Avenue, Maywood, Illinois, IL 60153, USA; E-mail: apower@lumc.edu
Abstract
In 1994, PCR and protein studies suggested that SV40 DNA sequences and proteins were present in 29/48 (60%) USA human mesothelioma samples. Sequence analysis confirmed that the sequences were homologous to SV40. One year later, SV40 was also found in 5/9 human mesotheliomas, and in 1996 SV40 was also reported to be present in 1/3 of the tumor specimens examined. These reports, in combination with an earlier study in 1992 which had detected SV40 in human brain tumors, raised concerns that SV40 was associated with certain types of human tumors, specifically mesothelioma, bone, and brain tumors. These findings raised concerns, because these tumor types are the same malignancies that had been observed in animals injected with SV40. However, a study in 1996 and a presentation made at the International Mesothelioma Interest Group, IMIG in 1997 failed to detect SV40 in mesotheliomas, suggesting the possibility that laboratory artifacts, such as PCR contamination, had caused the previous positive findings. In 1997, the FDA, the NIH, and the CDC organized an international conference in Bethesda to review the literature and to address the possibility that SV40 was present in, and was possibly the cause of, some human tumors. The results of that conference were reported the same year in a meeting review in Oncogene by Carbone and colleagues. Briefly, the consensus was that before accepting the possibility that SV40 was present in human tumors, a multi-laboratory study needed to be conducted. It was recommended that a blinded multi-laboratory study be directed by an independent scientist not previously associated with the controversial reports of SV40 in human specimens. It was also recommended that this study include laboratories that had reported positive findings as well as laboratories that had failed to detect SV40 in human specimens. Since 1997, about 30 independent reports have been published on this topic, including the multi-laboratory study. Evidence in favor and against a possible association of SV40 with human cancer was reviewed at an international concensus meeting at the University of Chicago on 20, 21 April 2001, entitled 'Malignant Mesothelioma: Therapeutic Options and the Role of SV40, 2001'. The main focus was the association of SV40 with mesothelioma and other human tumors. At the end of the meeting, a panel discussion, which included independent experts who had not published on this topic, critically reviewed the evidence presented at the meeting. The results of the meeting and of the final panel discussion are outlined below.
Oncogene (2002) 21, 1141-1149 DOI: 10.1038/sj/onc/1205173
Keywords
SV40; mesothelioma; brain tumors; bone tumors
SV40 and human mesothelioma
Studies conducted by the various investigators were presented addressing the association of SV40 with human mesothelioma. Joseph Testa (Fox Chase Cancer Center, Philadelphia, PA, USA) reported the conclusions of a multi-laboratory study he had directed following the recommendation of the 1997 NIH conference (see Carbone et al. (1997b) for meeting review of 1997 NIH conference) and upon the request of the International Mesothelioma Interest Group (IMIG). This study included the laboratory of Michele Carbone (Loyola University Chicago, USA) who had reported positive findings for SV40 in human tumors, and that of Kaija Linnainmaa (Finnish Institute of Occupational Health, Finland) who had reported negative findings in Finnish mesotheliomas. In addition the laboratory of Kamel Kalily (MCP Hahnemann University, PA, USA), a JC expert, was included to verify that the sequences, if detected, did not belong to another human papovavirus. The result of this multi-laboratory study confirmed that SV40 sequences were present in human mesotheliomas, as DNA and protein analysis showed that 10/12 (83%) USA mesotheliomas contained SV40 DNA sequences and expressed Tag (Testa et al., 1998). Testa said he was very skeptical about the possible presence of SV40 in mesotheliomas when he organized the study and was surprised by the results. To verify his own results, Testa organized a second study. This time he blindly tested 11 USA mesothelioma biopsies that had not been previously studied for SV40 and nine Turkish mesotheliomas in parallel. The latter were chosen, because in Turkey the poliovaccine campaign started in the 1970s, well after poliovaccines had been cleared of SV40. To minimize the risk of contamination, the DNA's were extracted and analysed in parallel by a post-doctoral fellow who had not previously tested mesotheliomas for SV40. Furthermore, the analyses were performed in a new laboratory where SV40 or plasmids containing SV40 had not been used. Testa found SV40 sequences in 0/9 Turkish mesotheliomas. However, he did detect SV40 in 4/11 US mesotheliomas, confirming the previous findings of his multi-laboratory study. Salih Emri from the University of Hacettepe in Ankara, Turkey, reported similar findings. In his lab, SV40 sequences were detected in 0/29 Turkish human mesotheliomas and in 3/5 Italian mesotheliomas studied in parallel (Emri et al., 1999). This discrepancy in results between US and Turkish mesotheliomas prompted a critical discussion in which the various experts agreed that SV40 prevalence appears to vary from country to country. Furthermore, it was agreed that such results were inconsistent with lab contamination. A report about the geographical differences (Hirvonen et al., 1999) in SV40 prevalence was presented in the poster session at the Chicago Conference. The data by Hirvonen et al. (1999) suggested that the absence of SV40 in Finnish samples may be due to the fact that SV40 contaminated vaccines were not distributed in Finland, similarly to Turkey. Whether these differences in prevalence should be attributed to poliovaccine contamination or other unknown factors remains to be demonstrated.
Ironically, one of the most convincing papers in support of the presence of SV40 in mesotheliomas was presented by Adi Gazdar (University of Texas, USA) who was in the past one of the most skeptical critics of the association of SV40 with malignant mesothelioma. Gazdar micro-dissected malignant mesothelioma cells and nearby stromal cells from the same paraffin sections. Using PCR analysis and DNA sequencing he found SV40 sequences in 57% of samples of pleural and peritoneal epithelial mesothelioma tumor cells. However, these sequences were absent in matched adjacent lung tissue microdissected in parallel, making contamination an unlikely explanation. Furthermore, the SV40 sequences were present in both the pre-invasive and invasive component of the tumor cells, which indicated that SV40 is present beginning in the early stages of the neoplastic process (Shivapurkar et al., 1999).
In another study using the novel primed in situ (PRINS) method and immunohistochemistry, Mark Ramael (St. Elizabeth General Hospital, Belgium) tested mesotheliomas, non-neoplastic mesothelium, and pleural carcinoma metastases for the presence of SV40 (Ramael et al., 1999). He reported the presence and the expression of the SV40 genome in 14/25 (60%) of the mesothelioma biopsies. In contrast, none of the 30 non-neoplastic mesothelium samples or 30 pleural carcinoma metastases contained SV40. Furthermore, in the mesothelioma biopsies, SV40 was detected only in malignant cells and not in nearby stromal cells, supporting the specificity of the results. Ramael underscored the fact that on the same paraffin section, in situ hybridization detected SV40 in malignant cells and not in nearby stromal cells, which ruled out any possible question of contamination.
Bharat Jasani (University of Wales, UK) further examined the validity of the contamination theory. Using PCR, coded DNA aliquots obtained from York University (UK) of 32 matched blood samples and prostatic biopsies were tested for SV40 DNA. 9/64 (14%) samples were positive, and in 31/32 cases, prostatic and blood samples gave the same results (Jasani et al., 2001). The low level of SV40 detection as well as the concordance between the matched samples were inconsistent with the possibility of laboratory contamination. Instead, he proposed that the positive results were caused by SV40-positive circulating mononuclear phagocytes present in the blood and thus in the prostate. Jasani cautioned about interpreting positive results for SV40, because in SV40 infected patients circulating blood cells rather than malignant cells may contain SV40. This underscores the importance of confirming the PCR DNA analyses with additional techniques, such as immunohistochemistry, in situ hybridization, or microdissection before linking SV40 to a particular tumor type.
Frederick Mayall (Waikato Hospital, New Zealand) reported that SV40 was present in human mesotheliomas in New Zealand, and also showed a relationship between the presence of SV40 and asbestos exposure in mesotheliomas. 11 mesotheliomas analysed for the presence of SV40 and asbestos exposure showed that 7/11 tumors were associated with abnormally high levels of asbestos exposure. Of these 7, five were positive for SV40. None of the non-asbestos associated mesotheliomas were positive for SV40. Mayall suggested the possibility of a synergistic relationship between SV40 and asbestos in the development of malignant mesothelioma (Mayall et al., 1999).
Alfonso Cristaudo (University of Pisa, Italy) said that because of the doubt surrounding the presence of SV40 in mesotheliomas by some scientists, he was concerned that his previous positive findings (Cristaudo et al., 1995) might have been caused by some type of laboratory contamination. Therefore, he set up a new experiment with new specimens in a separate laboratory situated in a new building where experiments with viruses or plasmids (which may contain SV40 sequences) had not been performed. His group found that 10/18 (55.5%) mesotheliomas contained SV40 regulatory region sequences. 8/10 of these samples also contained SV40 Tag sequences, thus confirming that SV40 is present in some human mesotheliomas (Cristaudo et al., 2000).
Keerty Shah (Johns Hopkins, Baltimore, MD, USA) presented studies conducted in his laboratory upon request of Drs Howard Strickler and Jim Goedert, who failed to detect SV40 in mesotheliomas (Strickler et al., 1996, 2001). A number of possibilities were formulated to account for the possible negative data, including limited sensitivity, different technical approach etc., but it remained unclear why his analyses appeared to be at odds with those presented by the other participants in the meeting. Shah appeared less skeptical about the possible association of SV40 with human tumors compared to the 1997 NIH meeting, however, he cautioned that until we understand how the virus is spread in humans, we should be very careful in linking the virus to human disease.
Jeffrey Kopp (NIH, Bethesda, MD, USA) provided the first experimental evidence to address how SV40 is spread in humans. He found infectious SV40 in the urine of immunocompromised patients and in the urine of patients with collapsing segmental glomerulosclerosis. He also demonstrated SV40 DNA and RNA in the tubular kidney cells by in situ hybridization. He suggested that immunocompromised patients and patients with segmental glomerulosclerosis may shed the virus and thus infect other people. Of interest, segmental glomerulosclerosis is a disease that was first described in the early 1960s and collapsing segmental glomerulosclerosis is a variant that first appeared in the 1980s. Thus, there is an intriguing temporal coincidence between the appearance of these new diseases and exposure to the SV40 contaminated polio vaccines. Kopp, however, cautioned against prematurely causally linking collapsing segmental glomerulosclerosis to SV40 infection.
Riccardo Puntoni (National Cancer Research Institute, Genoa, Italy) presented data underscoring the possible importance of SV40 detection in human mesotheliomas when he revealed data showing that the presence of SV40 in these tumors is a negative prognostic marker for survival outcomes. Biopsy specimens from 83 malignant mesothelioma patients were analysed for the presence of SV40. Using Cox's proportional hazards regression model, the study revealed that histology independently predicts outcome in malignant mesothelioma, with epithelial variants having a more favorable prognosis than both the biphasic and sarcomatoid variants. When histology was associated with SV40 status Cox's regression model showed that SV40 positivity has an additive effect on the predictive value of histology. For example, SV40 positive epithelial variant malignant mesothelioma patients had a 54% greater chance of dying within 12 months from the time of diagnosis than SV40 negative patients with this histotype. The effect of SV40 positivity was associated with an even greater risk of death when combined with biphasic and sarcomatoid variants compared to SV40 negative cases with the same histology. Puntoni suggested that the presence of SV40 in these tumors is associated with a more aggressive clinical course, making the ability to accurately determine its presence essential (Procopio et al., 2000).SV40 pathogenesis and human mesothelioma
The SV40 genome is a double-stranded circular DNA molecule that can be divided into early and late regions by the order in which they are transcribed (reviewed by Pipas and Levine, 2001). The early region encodes the large and small tumor antigens (Tag and tag) and 17kT, while the late region encodes viral coat proteins. Tag is a 90 kDa protein found mostly in the nuclei of transformed cells, and is largely responsible for SV40 mediated transformation (reviewed by Testa and Giordano, 2001; Pipas and Levine, 2001; Ali and DeCaprio, 2001). Tag binds and inactivates tumor suppressor gene products, including p53 and the Rb protein family, and allows for unregulated cell division. By inhibiting p53, Tag also inactivates an apoptotic checkpoint. Tag has also been shown to cause structural chromosomal alterations, point mutations, and aneuploidy in infected cells. The small t antigen (tag) is a 19 KDa protein found in the cytoplasm of infected cells which has been shown to enhance the transforming capacity of the large T antigen (reviewed in Rundell and Parakati, 2001). It increases production of Tag, contributes to p53 inactivation, and stimulates mitosis in quiescent cells through stimulation of MAP kinase and AP-1 activity. The small t antigen specifically inhibits phosphatase 2A (PP2A) which alters the phosphorylation state of several cell cycle regulatory proteins including p53.
The carcinogenic effect of SV40 infection in animal models is well established. In a study by Cicala et al. (1993), intrapleural injection of wild type SV40 produced mesotheliomas in 100% of hamsters in 3-6 months. Sixty per cent of hamsters injected intracardially with the virus developed pleural mesotheliomas (Carbone et al., 1991), and 67% of hamsters injected intraperitoneally developed the tumor. In hamsters, subcutaneous injection of the virus has also been shown to produce sarcomas (Eddy et al., 1961), while intravenous injection caused lymphomas and osteosarcomas (Diamandopoulous, 1972). Ependymomas, and choroid plexus tumors developed in hamsters following intracranial injection (Kirschstein and Gerber, 1962). Interestingly, in humans SV40 sequences have been demonstrated in many of these same tumor types, including ependymomas, choroid plexus tumors, mesotheliomas, and osteosarcomas (reviewed in Jasani et al., 2001).
The association of SV40 with mesothelioma development in hamsters and the presence of SV40 in human mesotheliomas established a strong association between the virus and mesotheliomas. However, the question of causality remained. Experiments to investigate if Tag was biologically active in human mesotheliomas were presented by Dr Pier Paolo Claudio (Thomas Jefferson University, Philadelphia, PA, USA). In collaboration with Giordano and Carbone, Claudio and De Luca found that Tag and p53 were often co-expressed in mesotheliomas. Immunoprecipitation experiments showed that Tag binds and stabilizes p53, allowing its detection. Tumor cells expressing Tag also failed to induce p21, suggesting that p53 was inactivated through its interaction with Tag (Carbone et al., 1997a). When SV40 positive mesothelioma cell lines were treated with Tag antisense, the p53 pathway was restored, and p21 expression was resumed, which led to eventual growth arrest and apoptosis of these cells (Schrump and Waheed, 2001). Aside from p53, Tag was also shown to interact in a similar manner with the retinoblastoma tumor suppressor family pRb, p107 and pRB2/p130 (De Luca et al., 1997). Thus, Tag appears biologically active in human mesothelioma, suggesting that it may play a role in human mesothelioma development.
Adi Gazdar (University of Texas, USA) reported experiments he conducted in collaboration with the laboratory of John Minna (Toyooka et al., 2001) which revealed that SV40 infection of mesothelial cells may affect methylation patterns and gene expression. DNA methylation at CpG sites located in promoter regions normally leads to gene silencing. Aberrant methylation of tumor suppressor genes is seen in some human mesotheliomas, but it is infrequent in mesotheliomas in comparison to lung adenocarcinomas. Gazdar found that the frequency of aberrant methylation of RASSF1A, a tumor suppressor gene, was significantly higher in SV40 positive mesotheliomas compared to SV40 negative mesotheliomas. Thus, he suggested that SV40 pathogenesis in the development of malignant mesothelioma may involve methylation and silencing of RASSF1A and other tumor suppressor genes.
Giovanni Gaudino (University of Piemonte Orientale, Italy) reported that SV40 induces Hepatocyte growth factor receptor, or Met, activation in mesotheliomas (Cacciotti et al., 2001). In human mesothelial cells transfected with full-length SV40 cDNA, Met activation led to progression into the S phase, a fibroblast morphology, and assembly of viral particles. Co-cultures of SV40 infected mesothelial cells with CV1 cells, human mesothelial cells, and murine BNL CL cells lead to subsequent infection of these cells. CV-1 cells were lysed, but infection of the human mesothelial cells and murine BNL cells lead to Met activation, Met tyrosine phosphorylation, and S phase entry. These cells, when co-cultured with uninfected CV1 cells, lead to CV1 infection and cell lysis. Treatment with HGF blocking antibodies inhibited Met tyrosine activation in all SV40 positive cells, indicating that Met activation and its subsequent effects were mediated by an autocrine pathway. This autocrine loop was found to be induced by Tag, as transfection experiments with the early region alone induced Met activation. Overall, these findings suggest that SV40 infection of mesothelial cells leads to Met activation via an autocrine loop. They also show that SV40 replicates in mesothelial cells leading to infection of adjacent cells which induces a human growth factor dependent Met activation and cell cycle progression into S phase. Gaudino suggested that this may explain how a limited number of SV40 positive cells may be sufficient to direct noninfected mesothelial cells towards malignant transformation. Furthermore, Met activation was found only in SV40 positive mesotheliomas, while Met was not activated in SV40 negative human mesotheliomas. This suggested that while Met activation may contribute to the development of some SV40 positive mesotheliomas, Met activation is not an indispensable step towards all mesothelioma development (Cacciotti et al., 2001).
Maurizio Bocchetta (Loyola University, Chicago, USA) reported data developed in Michele Carbone's laboratory showing that human mesothelial cells are uniquely susceptible to SV40 transformation. In experiments comparing SV40 infection in human mesothelial cells and fibroblasts, Bocchetta et al. (2000) found that mesothelial cells are uniformly (nearly 100%) infected by SV40 and express Tag. In contrast, only a minority (5-20%) of fibroblasts are infected by SV40 and express Tag. Furthermore, mesothelial cells were found to express 4-5 times more wild-type p53 than fibroblasts. While SV40 Tag normally binds and inhibits p53, this interaction also inhibits the replicase activity of Tag. Thus, the higher levels of p53 in human mesothelial cells leads to low levels of both SV40 replication and subsequent cell lysis in comparison to human fibroblasts. Mesothelial cells treated with antisense p53 reduced p53 levels to that of fibroblasts, and thus led to high rates of viral production and cell lysis that were similar to those seen in fibroblasts. In infected mesothelial cells, prolonged exposure to the mutagenic effects of Tag without cell lysis probably accounted for the high rate of transformation observed in comparison to fibroblasts, in whom the rate of transformation was 1000 times less than mesothelial cells. Thus, human mesothelial cells are unusually susceptible to SV40 infection and malignant transformation in comparison to fibroblasts or other cell types (Bocchetta et al., 2000). This may explain the ability of SV40 to preferentially induce mesotheliomas in hamsters and its presence in most human mesotheliomas. Lucio Miele (Loyola University, Chicago, USA) reported that in recent experiments done in conjunction with Michele Carbone, SV40 has also been shown to increase Notch 1 expression in primary human mesothelial cells, an effect that was partially mediated by the SV40 small t antigen. Activation of Notch 1 by SV40 may promote cell division and prevent apoptosis in SV40 infected cells.
David Schrump (NCI, Bethesda, MD, USA) presented data showing that the introduction of anti-sense to the SV40 early region in Tag positive human mesothelioma cells inhibits Tag expression and induces growth arrest and apoptosis (Waheed et al., 1999). He showed that inhibition of Tag expression coincided with enhanced p21/WAF-1 expression leading to restoration of the p53 pathway. This may be responsible for the growth inhibition and apoptosis observed. Schrump suggested that SV40 Tag may contribute to the pathogenesis of mesotheliomas and that therapeutic interventions aimed at Tag may be an option for treatment of patients with SV40 positive malignant mesotheliomas. Michael Imperiale (University of Michigan, USA) presented data showing that a recombinant vaccinia virus vaccine against SV40 Tag suppresses tumor growth and prolongs survival of mice injected prior to tumor implantation and in mice with pre-existing tumors (Imperiale et al., 2001). In addition, Tag specific MHC restricted T lymphocytes can be isolated from these immunized mice. Imperiale suggested that a vaccinia vaccine may prove effective in SV40 positive mesothelioma patients. Martin Kast (Loyola University, Chicago, USA) argued that immunotherapeutic aproaches aimed at treating tumor patients are likely to fail. Instead, such approaches should target individuals at risk for developing mesothelioma because of prior asbestos exposure and SV40 infection.
While the studies presented at the meeting showed that SV40 sequences and Tag can be found in human mesotheliomas, Yannick Pilatte (Inserm EMI-U 99.09, France) presented protein data arguing that the anti-SV40 Tag antibodies are not specific (Pilatte et al., 2000). In this study, Pilatte investigated the presence of SV40 Tag protein through Western blot, immunoprecipitation, and immunocytochemistry using the Tag specific mouse monoclonal antibody Pab 419 (Ab-1). None of the mesothelioma cell lines expressed Tag that was detectable via Western blot. Pilatte proposed that Ab-1 as well as Pab 101 (another anti Tag monoclonal antibody) are contaminated with a protein similar in size to Tag that can react with HRP-conjugated anti-mouse IgG. Pilatte argued that this protein may be mistaken for Tag and generate false positive results by immunocytochemistry or immunoprecipitation followed by Western blot with the same antibody. Satvir Tevethia (Pennsylvania State University) noted, however, that the specificity of the Tag antibodies has been proven by many studies in the past 20 years. He also noted that the 90 kDa Tag, like protein detected by Pilatte in his immunoprecipitations, appeared to be an artifact caused by the incomplete separation of the heavy (54 kDa) and light (25-30 kDa) chain of the antibody used in Pilatte's immunoprecipitations. He also noted that the lack of co-precipitation of Tag and p53 in the cell lines, in the absence of data demonstrating wild-type p53, should not be a surprise since p53 is mutated in many cell lines and mutated p53 does not bind Tag.Genetics and Mesothelioma
Michele Carbone (Loyola University, Chicago, USA) provided evidence for a genetic susceptibility factor in the development of malignant mesothelioma, in the absence of SV40 or asbestos exposure (Roushdy-Hammady et al., 2001). In the Turkish villages of Karain and Tuzkoy approximately 50% of deaths are due to malignant mesothelioma. The high incidence of the malignancy was originally attributed to the presence of erionite in the stones used to build the villages, since it was detected in the lungs of several villagers with mesothelioma, and injection of the mineral into hamsters produced mesotheliomas. Carbone found that erionite is not the predominant cause of mesothelioma in these villages. Notably, mesotheliomas developed in only certain households in the villages, despite the fact that all houses contain similar amounts of erionite. Furthermore in Karlik, a nearby village also built with similar types and amounts of erionite, only one case of mesothelioma had been reported. This occurred in a woman from Karain who moved to Karlik because of marriage. The incidence of mesothelioma in immigrants from these two villages living in Sweden and Germany was also found to be similar to or higher than that found in Karian and Tuzkoy. These immigrants included individuals who left the villages as young children and adolescents. These findings suggested that genetics may have played a strong role in tumor development. When the pedigrees of six affected families in Karain and Tuzkoy were analysed, it appeared that mesothelioma was genetically transmitted in an autosomal dominant pattern. It is possible that erionite is a co-factor that produces mesothelioma in genetically predisposed individuals. Isolation of the putative mesothelioma susceptibility gene may lead to the development of possible therapeutic approaches for these families. Such a gene may also be the target of asbestos and SV40 carcinogenesis. Thus, Carbone proposed that mesothelioma is a cancer in which environmental factors (asbestos and/or erionite), viruses (SV40), and genetics may act independently or together to cause malignancy (Roushdy-Hammady et al., 2001).SV40 and human bone tumors
Piero Picci (Rizzoli Institute, Bologna, Italy) presented a study which confirmed the association of SV40 with human bone tumors, as previously found by Carbone et al. (1996) and Lednicky et al. (1997). He reported that 30/107 giant cell tumors, moderately benign bone tumors with malignant potential, contained SV40 sequences (Gamberi et al., 2000). Of these 30 samples, 22 also contained Tag, and 15/30 over-expressed the fos oncoprotein. Interestingly, all 77 of the SV40 negative giant cell tumors did not over-express fos. SV40 is able to induce fos in cell culture. In these giant cell tumors SV40 may also induce fos activity, giving SV40 a potential role in the development of giant cell tumors (Gamberi et al., 2000).
Junya Toguchida (Kyoto University, Japan) reported that SV40 sequences were present in 25/54 (46.3%) of osteosarcomas (Yamamoto et al., 2000). SV40 DNA was also found in peripheral blood cells of 43.3% of osteosarcomas patients, but in only 4.7% of normal healthy controls. Toguchida suggested that SV40 may play a role in osteosarcoma pathogenesis.SV40 and human brain tumors
Barbara Krynska (MCP Hannemann University, Philadelphia, USA) reported that JC virus was present in 11/23 medulloblastomas tested (Krynska et al., 1999) and 5/11 JC containing medulloblastomas also contained SV40 sequences. Production of the JC virus tumor antigen was detected in the nuclei of four of these tumors. Krynska suggested that these findings may provide evidence for an association between JC virus and SV40 in the development of some medulloblastomas.
Hai-Ning Zhen (Fourth Military Medical University, China) showed that SV40 Tag is present in a variety of brain tumors, specifically in 8/8 ependymomas, 2/2 choroid plexus tumors, 9/10 pituitary adenomas, 11/15 astrocytomas, 7/10 meningiomas, 4/8 glioblastomas multiformes, and 2/6 medulloblasomtas. 8/8 normal brain tissue specimens did not contain Tag. In addition, Zhen reported that 18/18 Tag positive tumors tested contained Tag-p53 complexes and 15/15 contained Tag-pRb complexes by immunoprecipitation and Western blot analysis. Zhen stated that the expression of Tag and its ability to complex with these tumor suppressor proteins suggests that it may be involved in tumor pathogenesis (Zhen et al., 1999).
David Malkin (University of Toronto, Canada) reported that SV40 was present in tumors from two patients with Li-Fraumeni syndrome (Malkin et al., 2001). In a Li-Frameni family proband with both an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), a mutant p53 allele was reduced to homozygosity in the RMS. However, in the CPC, a mutant and normal p53 allele was found, and SV40 Tag was detected by both PCR and immunostaining. Malkin suggested that SV40 Tag-induced inactivation of the remaining normal p53 allele may have contributed to CPC development in this patient. In a second Li-Fraumeni proband patient who developed CPC, osteosarcoma, and renal cell carcinoma (RCC), normal and mutated p53 alleles were retained in both the CPC and RCC. PCR and immunostaining detected SV40 Tag in both of these samples, further supporting a role for SV40 in tumor formation in these genetically susceptible individuals.
Sascha Weggen (University of Bonn Medical Center, Germany) reported that SV40 was present in human medulloblastomas, meningiomas, and ependymomas, but at a very low frequency (Weggen et al., 2000). SV40 like sequences were detected in 2/116 medulloblastomas, 1/131 medulloblastomas, and 1/25 ependymomas. Tag expression was not detectable in the tumor cells. Out of all of the samples, only one meningioma contained JC sequences, and no tumors were found to contain BK. Weggen suggested that the low frequency of viral sequences and the absence of Tag expression in these tumor types argues against a major role for the viruses in the pathogenesis. Interestingly, all of the SV40 positive tumor samples were from the USA, and all negative samples were from Germany, supporting the suggestion that there are epidemiological differences in SV40 infection.SV40 epidemiology
Susan Fisher (Rochester University, New York, USA) reviewed the literature and presented her analysis of a possible link between SV40 contaminated polio vaccines and the presence of SV40 in some humans (Fisher et al., 1999). SV40 is endogenous to the rhesus monkey. However, polio vaccines and adenovaccines distributed from 1955-1963 were prepared in cell cultures grown on monolayers of infected rhesus monkey kidney cells. Because the virus produces no cytopathic effects in these cells, SV40 contamination of the cultures went unrecognized until 1960, when high titers of the virus were found in some batches of the vaccine. It is now estimated that 98 million people in the USA alone may have been exposed to infectious SV40 through contaminated polio vaccines. Following the finding that SV40 induced tumors when injected into hamsters, initial epidemiological studies investigating if the administration of SV40 contaminated polio vaccines were associated with increased cancer incidence were undertaken. No increase in cancer incidence in children was detected in the years immediately following vaccine distribution. However, only one long-term follow-up of vaccinated children in the USA was conducted (Mortimer et al., 1981). One thousand and seventy-three children born between 1960-62 who received either oral or inactivated polio vaccine shortly after birth were followed for 17-19 years. Overall, the group showed no increased cancer incidence, as only one child developed a malignancy. Fisher suggested that the 17-19 year follow-up period, however, may have been inadequate, since other carcinogens can take 20-40 years to cause cancer. In addition, the small number of children studied may not have been sufficient to detect increases in the rare SV40 associated tumors. Aside from these USA reports, epidemiological studies undertaken in both Sweden and Germany have concluded that exposure to SV40 contaminated vaccines are associated with no increase in the overall incidence of cancer. However, Fisher noted that mesotheliomas, brain, and bone tumors are quite rare, and it would be impossible to detect an increase the overall cancer incidence because of changes in these rare cancers. Of interest, a German study (Geissler, 1990) detected an increase in the incidence of certain types of brain tumors in SV40 exposed cohorts compared to non-exposed cohorts. Furthermore, SV40 was detected by Southern blot hybridization in some of the brain tumors in the exposed cohorts, and not in the brain tumors of the unexposed cohorts.
A prospective study of 50 000 pregnant women who did or did not receive polio vaccines between 1959-65 showed a twofold greater rate of malignancies, particularly neural tumors, in the children of mothers who were vaccinated during pregnancy (Heinonen et al., 1973). In addition, a case control study of Australian children hospitalized with malignancies had a higher rate of previous polio-vaccination than matched controls (Innis, 1968).
Fisher commented that overall, the available data from these epidemiological studies are conflicting and inconclusive and limited by a lack of data drawn from large samples of confirmed SV40 exposed and unexposed cohorts. She said that the length of follow-up has also been inadequate for most of these SV40 associated tumors which characteristically have long latency periods. Fisher et al. (1999) using data from the Surveillance Epidemiology and End Results (SEER) data base, analysed the incidence of mesotheliomas, bone, and brain tumors from 1973-93 and the association of these tumors with the administration of potentially contaminated polio vaccines. The SEER Program represents approximately 12% of the USA population and provides population based, tumor specific data on all tumors occurring in specific geographic areas in the USA. Fisher showed that since 1973/74 the age adjusted incidence of ependymomas has increased by 25% and by 49% in the 0-4-year-old age group. The rate of osteogenic sarcomas has increased by 2.4% since 1973/74, and the incidence of other bone and joint tumors has increased by 22.9%. The occurrence of mesothelioma, which was non-existent before 1960, has increased in incidence by 90%. A second, more specific analysis, was also done by Fisher to directly compare cancer incidence in a birth cohort highly likely to have been exposed to SV40 contaminated vaccines (1955-59) versus a cohort unlikely to have been exposed to the contaminated vaccines (196-67). She reported that the overall cancer incidence rate was 11% lower in the exposed cohort than the unexposed cohort, but that the incidence of ependymoma and choroid plexus tumors was almost 20% greater in the exposed versus the unexposed cohort. The incidence of osteogenic sarcoma and other bone malignancies is also higher in the exposed cohort. Interestingly, the incidence of mesothelioma, a highly unlikely finding given the young age of the participants at the time of the analysis, was higher in the exposed (six cases) than the unexposed cohort (two cases) (Fisher et al., 1999).Final panel discussion reviewing the evidence presented
The panel was chaired by Carlo Croce and George Klein who had not been directly included in research to prove or disprove the association of SV40 with human tumors. Panel members included: Nicholas Volgelzang, W Martin Kast, Satvir Tevethia, Hans Schreiber, Harvey Pass, Eva Klein, Joseph Testa, Umberto Saffiotti, David Schrump, Michael Imperiale, Philip Harber, Robet Garcea, Janet Butel, Adi Gazdar, Kathleen Rundell, E Premkumar Reddy and Janet Rowley.
Carlo Croce raised the point that more information regarding the genetics of mesothelioma should be sought. He stated that it might be beneficial to define the genes or chromosomal regions involved in mesothelioma development. Nicholas Vogelzang suggested that one possible approach to this question would be to carefully analyse families with multiple cases of mesothelioma.
The question of whether SV40 is present in human mesotheliomas was discussed. The panel members agreed that there is now overwhelming evidence proving that SV40 is capable of infecting humans and that SV40 is present in some human mesotheliomas at a rate of 40-50% in the USA and Europe. However, SV40 was not found in Turkish mesotheliomas. This led to a discussion regarding the role of the virus in the pathogenesis of mesothelioma. Carlo Croce said that SV40 may play a role in some mesotheliomas in the USA and in Europe, however, it is clear that mesotheliomas can develop in the absence of SV40. David Schrump demonstrated that treatment with Tag antisense inhibits mesothelioma growth, which Eva Klein said is consistent with an essential proliferation driving role of the protein. Also, George Klein commented that the data presented by Bocchetta (Carbone's laboratory) demonstrating that mesothelial cells were unusually susceptible to mesothelial cell transformation further supports the idea that SV40 may play a role in mesothelioma development. Croce said that the presence of SV40 in mesothelioma tumor cells but not in the surrounding normal cells supports a role for the virus in tumor causation. Exactly how SV40 leads to mesothelioma development remains unknown, but the panel did agree that SV40 is somehow involved in the pathogenesis of some mesotheliomas.
Janet Butel raised the point that there are significant differences in the rates of SV40 positivity in human mesotheliomas. She suggested that this may be due to geographic differences and to differences in PCR techniques. Bharat Jasani stated that the amount of DNA used in the PCR reaction and the number of cycles used may impact the results significantly. For example, Jasani mentioned that SV40 positivity in Germany is low, however, the PCR techniques employed there use lower amounts of DNA per reaction cycle and a lower number of cycles. Thus, it was agreed that there should be some standardization of technique.
Robert Garcea recommended that one of the next steps in SV40 research should be an analysis of the prevalence of SV40 infection in different populations. Serology remains problematic at this time, and, according to Garcea, it may be difficult to discriminate between the different polyoma viruses using this technique. PCR, however, is very accurate if properly conducted. John Lednicky argued that in his own experiments serology reliably distinguished SV40 from other polyoma virus infections.
The possible interaction between asbestos and SV40 was also discussed. The data presented by Maurizio Bocchetta, showing that asbestos and SV40 treated mesothelial cells have a higher rate of transformation than either one alone, suggested that the two may act as co-carcinogens. George Klein pointed out that asbestos may facilitate SV40 mediated transformation through its immunosuppressive effects, since SV40 is a strong inducer of tumors in immunosuppressed animals. Alternatively, or in addition, while SV40 may favor the generation and/or survival of genetically changed cells, asbestos may induce additional changes to tip the cell in favor of malignancy. Elliot Kegan said that the local and systemic immunosuppressive effects of asbestos are well established, because asbestos is phagocytized by mononuclear phagocytes who are then damaged by asbestos. These cells then release a number of cytokines that interfere with the immune response.
The usefulness of a vaccine targeting SV40 to be used as a preventive strategy in asbestos exposed individuals who have not yet developed mesothelioma was also discussed. Harvey Pass was recently awarded a NCI RAID grant to develop an SV40 vaccine for a Phase 1 clinical trial. Carbone raised the question of whether such a vaccine would be a useful product for asbestos exposed individuals. Philip Harber commented that even if such a preventive vaccine is developed, it will be important to test it in a population with a high attack rate, as it would be difficult to determine the efficacy of the vaccine on a rare tumor like mesothelioma. Robert Garcea underscored that for this reason a good SV40 serology test needs to be developed to target individuals at high risk. David Schrump suggested that a preventative vaccine would be a costly endeavour and that much pre-clinical work still had to be done. He stated that first we need to determine if Tag is recognized, processed, and presented in vitro, because without such activity a vaccine would be useless. He suggested that Phase 1 trials aimed at determining if Tag is recognized as immunogenic in mesothelioma patients should be undertaken. He suggested that a therapeutic vaccine used after tumor debulking to control micrometastatic disease may be worth testing. George Klein questioned the utility of a therapeutic vaccine against SV40 in mesothelioma patients, since established tumors are very difficult to influence by immunotherapeutic measures. Therefore, the immune response should be reinforced during the early stage of tumor development. Martin Kast advocated the use of a preventive vaccine directed against SV40, as it would possibly prevent SV40 from transforming the mesothelial cell at the start.
The question of whether titers of antibodies to Tag had any correlation to tumor load was also discussed. Satvir Tevethia discussed experiments done in his lab where hamsters with mesotheliomas were serially bleed and anti-Tag antibody titers were measured and correlated with tumor load. The titers were found to rise with tumor load until high tumor burdens were reached. Titers then began to fall due to the formation of immune complexes in the serum. Thus, Tevethia suggested that measuring serum titers in mesothelioma patients may be a useful way to determine tumor burden.
George Klein summarized his perception of the final panel discussion by saying that the presence of SV40 in some human tumors, especially mesotheliomas, has been convincingly demonstrated in the past four years. Concerning causality, he argued that the possible role of SV40 in the pathogenesis of mesothelioma has been considerably strengthened since the 1997 NCI conference. He said that future studies should investigate the mechanisms of SV40 pathogenesis when present in human cells, and whether the presence of SV40 in human cells can be used to develop new immunotherapeutic or molecular approaches to treat or prevent SV40-associated tumors.
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Received 19 September 2001; revised 2 November 2001; accepted 7 November 2001
14 February 2002, Volume 21, Number 8, Pages 1141-1149
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