Emotions run high on the topic of the monkey virus SV40 and its relationship to human cancer.
Some scientists believe that the virus likely plays a role in development of some human cancers, while others are skeptical. And underlying these divisions are more fundamental disagreements about whether SV40 even infects humans and whether laboratory tests can discriminate between an infection by SV40 and those by similar viruses.
The discovery of SV40 in the earliest polio vaccines in the 1950s set off several decades of research, most of which was reassuring. While SV40 causes cancer in hamsters and abnormalities in human cells, 40 years of epidemiological studies in hundreds of millions of people have found no evidence that those exposed to the virus have an increased risk of cancer (see sidebar, p. 1286).
However, controversy came into the picture in 1990s with the advent of the polymerase chain reaction, an extremely sensitive technique for detecting very small amounts of DNA. Using PCR, traces of the viral DNA were detected in some rare human tumors, including mesotheliomas (a cancer of the lining of the lung, which occurs primarily in men over age 55 who were exposed to asbestos), osteosarcomas, and some rare childhood brain tumors.
Because most of the mesothelioma patients were too old and most of the brain tumor patients were too young to have been vaccinated with the contaminated vaccine, these results suggest that SV40 is circulating in the general population. (People most likely to have received contaminated vaccines were born from 1941 through 1961.)
The PCR findings vary greatly. Some report that as few as 4% of the tumors contain the virus, some find 90% with traces of the virus, and others are in between. The proportion of control (nonmalignant) cells containing the virus varies as well. At this point, more than 40 studies have reported the presence of SV40 in tumor tissue. In contrast, fewer than five laboratories have found either no trace of the virus or have had inconsistent results. These conflicting findings have led investigators to very different conclusions about the link between human cancer and SV40.
The Skeptics
"If you truly found that SV40 was present in tumors and not present in normal tissues, that would be strong evidence that the virus causes cancer because this is a virus that in animals can cause cancer. But that hasnt been demonstrated," said Eric Engels, M.D., an investigator in the Division of Cancer Epidemiology and Genetics at the National Cancer Institute.
Keerti V. Shah, M.D., Dr.P.H., from the Johns Hopkins Bloomberg School of Public Health, Baltimore, also feels uncomfortable with the connection. In addition to the failure of the epidemiologic data to show a link and the inconsistent results in tumor cells, Shah said that even in tumor samples where the virus is found, not every tumor cell contains the virus, and that the virus is lost when tumor cells are cultured. "This is a very odd behavior for an virus-derived tumor." He pointed out that human papilloma virus, for example, which is a strong risk factor for cervical cancer, is found in every cervical cancer cell and is present in cell lines derived from those cancers.
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"Which came first, the tumor or the virus?" Weiss asked. "We know that tumor tissue is more permissive for viral replication." As an example, he noted that parvoviruses preferentially grow in tumor cells.
Almost Believers and Believers
And even though Robert L. Garcea, M.D., at the University of Colorado Health Sciences Center in Denver, has found traces of SV40 in osterosarcomas, ependymomas, choroid plexus tumors, and recently in tumors associated with Li-Fraumeni syndrome in children, he still is skeptical.
"Whether theres a cause and effect relationship with any kind of cancer is far from established," he said. "But Im coming to the view that SV40 might be associated with some cancers."
Janet Butel, Ph.D., from Baylor College of Medicine in Houston, whose laboratory has been working on the biology of SV40 for many years, seems convinced of the connection. "It is very likely that the virus is involved in the initiation of some tumors," she said.
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Several investigators are proceeding full-speed ahead on the conviction that the virus is a cofactor, along with asbestos, for mesotheliomas. One of them is Adi Gazdar, M.D., from the University of Texas Southwestern Medical Center in Dallas, who detected SV40 in more than half of his samples of malignant mesotheliomas, but not in normal lung tissue or lung carcinomas, using laser-capture microdissection techniques. The work was published last year in the International Journal of Cancer.
"We looked at 40 lung tumorsadenocarcinomasand didnt find the virus, but found it in 50% of 150 mesothelioma cases from all of North America and Germany. I went from a total skeptic to a zealot," Gazdar said.
Now he is collaborating with Michele Carbone, M.D., from Loyola University Medical Center in Maywood, Ill. In 1994, Carbone and his colleagues were the first to find pieces of SV40 in 60% of mesotheliomas they tested. Since then, he and others have shown that an SV40 protein, large T-antigen, binds to human tumor-suppressor proteins in mesotheliomas and brain tumors and causes chromosome rearrangements in mesothelioma cells, suggesting possible mechanisms that may be involved in the development of cancer.
Is Human Infection Possible?
Underlying these differences is a more fundamental question: Can humans be infected with a monkey virus? As evidence of infection, scientists look for the presence of SV40 antibodies in the blood.
"No one has shown that healthy people can be infected with SV40," said Engels. "Some people who took the oral [Sabin] polio vaccine excreted virus in their stool for several weeks, suggesting that SV40 was reproducing locally. But they didnt develop serum antibodies. There may not have been a systemic infection."
On the other hand, Engels said that people who were injected with the inactivated Salk polio vaccines did develop antibodies to the virus. "But that could have been because of exposure to the killed virus," he said. "They might not have actually been infected," he said.
Butel has few doubts about SV40 infecting humans. She found SV40-reactive antibodies in the serum of 2% to 12% of people who were not exposed to the contaminated vaccine, which suggests that the virus is circulating in the general population. "The virus has infected humans, and SV40 is present in brain tumors, mesotheliomas, bone tumors, and possibly non-Hodgkins lymphomas," she said.
Carl W. Miller, Ph.D., of the Cedar-Sinai Research Institute in Los Angeles, who (along with two other laboratories) found SV40 in normal human lymphocytes, agrees. "SV40 is in the human population. Its being passed on somehow. Its clearly a human virus at this point."
Miller pointed out that in laboratory studies human cells coexist very happily with SV40. "They get infected. They get high copy numbers and they continue to grow. I dont think a virus like that would necessarily be cleared from people. And why couldnt it go on and infect mothers who could then pass it on to their children?" he asked. "I think it could pass on very easily that way."
Howard Strickler, M.D., of the Albert Einstein College of Medicine, New York, has a different view. "Mesotheliomas are developing in people who are too old to be vaccinated, and brain tumors [are developing] in children that are too young to have been vaccinated," he said. "To accept the presence of SV40 in these tumors, you have to hypothesize that SV40 is circulating in the general population. But our studies did not detect evidence of SV40 antibodies in the general public or in people with mesotheliomas, ependymomas, or osteosarcomas."
Proving Presence of SV40
A potential problem in the field arises from the fact that the DNA sequence of SV40 is nearly 70% identical to its two closest relatives, JC and BK, viruses that commonly infect humans as children and stay in the body for life but are not associated with any disease in healthy people. The antibodies humans make to all three viruses are similar.
"Most of us have antibodies to BK and JC," said Weiss. "So if someone says that they detect SV40 antibodies, one would want to see the evidence that its not BK or JC. Im not aware of an assay that distinguishes SV40 antibodies from either JC or BK."
Two scientists are trying to correct that failing by developing ELISA (enzyme-linked immunoabsorbant assay) assays that will discriminate among all three viruses. Gene Major, Ph.D., and his colleagues at the National Institute of Neurological Disorders and Stroke in Bethesda, used virion particles as antigens. In collaboration with Hopkins Shah and Dana Rollinson, a doctoral candidate in the Department of Epidemiology at Johns Hopkins Bloomberg School of Public Health, they have tested more than 300 blinded blood samples for the presence of all three viruses. "This will clarify the question as to whether SV40 does in fact circulate in the human population," Major said. The results will be published this year.
Another researcher, Denise Galloway, Ph.D., from Fred Hutchinson Cancer Research Center in Seattle, is developing an ELISA assay using baculoviral-expressed VLPs (virus-like proteins) from each of the three viruses as antigens. "We have banks of sera from lots of studies weve been doing. We will screen the general population first," she said. "If we see no antibodies to SV40, then that should pretty much be the end of it. On the other hand, if we do see a reasonable prevalence in the general population, we will need to be sure that the antibodies are not due to cross-reactivity with JC and BK antibodies. Then we will continue to work on casecontrol studies for cancers that have been associated with the virus."
Another proponent of large population studies is Bharat Jasani, M.D., from the University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom. Rather than looking for antibodies, he developing a real-time PCR assay that will distinguish between BK, JC, and SV40 DNA in blood cells. He is also looking for cytotoxic T-cells in mesothelioma patients that may help explain who is susceptible to SV40 infection.
Shah thinks that specific populations should be evaluated. He believes that if young children with SV40-positive brain tumors acquired an infection in utero from an infected mother, as some people have suggested, the mothers of these children should have high titres of antibodies. "This should be studied," he said. He also thinks that investigators should look at the full signature of "SV40 infection" in patientsnot only for virus in tumor cells but also for virus shedding in the urine, and for B and T cell responses to the infection. "We should study patients, not tumors," said Shah.
Knowing that the ELISA assays are in the pipeline, Garcea is beginning to plan a prospective multi-institutional study of pediatric brain tumors to look for the presence of the viral antibodies in blood samples as well as for viral sequences and p53 status in the tumors.
Everyone agrees that it is important to continue looking for answers. "Its just such an important topic," said Strickler. "Hundreds of millions of people were exposed. If we are ever going to have an important negative in research, this is it."
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