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HE presence of SV40 in human tumors has been reported on in more than forty independent research papers. But one molecular study that has had an enormous impact on the direction of SV40 research and funding was performed not by a virologist, like Butel, or a molecular pathologist, like Carbone, but by an epidemiologist named Howard Strickler. Strickler served as a senior clinical investigator in the NCI's Viral Epidemiology Branch for many years before he joined the Albert Einstein College of Medicine, in New York, last winter. He has been persistently skeptical of any association between the vaccine contaminant and tumors. Though he is no longer at the NCI, he remains instrumental in the government response.
In June of 1996 Strickler published a paper with Keerti Shah, of the School of Public Health at Johns Hopkins University, in Baltimore, in the journal Cancer Epidemiology, Biomarkers and Prevention. Strickler and Shah reported that they had come up empty-handed in their search for SV40 in fifty mesothelioma samples. Their study and a 1999 British study are the only two published SV40 studies with negative results. These two papers, particularly Strickler's, are cited again and again by federal health officials as proof that the dozens of peer-reviewed papers reporting SV40's presence in human tumors are unpersuasive and that a major research effort on SV40 is unnecessary.
Several SV40 researchers have criticized Strickler's 1996 study and the more recent British one, saying that they treated specimens in a manner that would not result in the efficient extraction of SV40 DNA. Bharat Jasani, the director of the molecular diagnostic unit at the University of Wales, in Cardiff, has found SV40 in British mesothelioma samples. He recently wrote a lengthy critique of the two studies that has not yet been published. In this critique Jasani concludes that the negative results "are explainable by the paucity of the diagnostic biopsy material used and/or insufficient sensitivity of the overall PCR methodology used." Jasani says that Strickler's PCR technique would have missed low levels of SV40.
Federal health officials are understandably concerned that any link between SV40 and human cancers could frighten people away from the polio vaccine and vaccination in general. They stress that before SV40 in the polio vaccine can be linked definitively to cancer, the proposition must clear important scientific hurdles. Carbone and others must prove that the SV40 they have found is not a laboratory contaminant. They must demonstrate that SV40 is responsible for the cellular damage that leads to cancer and is not just a benign "passenger" in human tumors. And they must show that it was introduced into human beings through the polio vaccine.
In assessing the research to date, Strickler is perplexed that the virus has been found in so many kinds of tumors. In addition to the confirmed research reporting the virus in more than a half dozen kinds of brain tumors and a similar number of bone tumors, researchers in new, isolated studies have reported finding the virus in Wilms tumors, which afflict the kidney, and adenosarcomas, rare cancers of the uterus. "It's not likely that a single virus causes ten thousand different diseases," Strickler says. "That's not how it works."
These anomalies have fueled Strickler's suspicion that many of the SV40 findings in human tumors may really be false positives resulting from laboratory contamination. He points out that SV40 is used for cancer research in so many laboratories around the world that almost any lab involved with tumor assays could conceivably harbor it. "Is it possible that SV40 is in human tumors and that SV40 is at some level circulating in the human population?" Strickler asks. "Could it be true? I can't exclude the possibility, but the studies to demonstrate it haven't really been done, and the data in our hands have been negative." Strickler's former boss, James Goedert, the chief of the NCI's Viral Epidemiology Branch, agrees. Although he says he has an open mind about SV40, he believes that contamination may lie behind the findings of Carbone, Butel, and others.
In 1997, largely in response to Strickler's study, the International Mesothelioma Interest Group set out to determine once and for all if the virus was present in human mesothelioma samples. The organization asked an internationally known molecular geneticist, Joseph R. Testa, the director of the Human Genetics Program at the Fox Chase Cancer Center, in Philadelphia, to oversee a study. Testa, who specializes in mesothelioma research, confesses that initially he doubted the idea that SV40 could be found in human mesotheliomas, because he believed it was well established that asbestos was the cause of the disease. "I'm a very careful person," Testa says. "I had a fair amount of skepticism about it." But the results of the investigation he led changed his mind. Four laboratories participated in the tightly controlled study, including Carbone's. All four found SV40 in at least nine out of the twelve mesothelioma samples they tested. Each laboratory's control samples tested negative, suggesting that the positive SV40 samples were not the result of laboratory contamination. The results were published in the journal Cancer Research in 1998.
Strickler believes that Testa's study "did not really move the ball forward" in determining whether contamination lies behind findings of SV40 in human tumors. He questions Testa's conclusions. "They are trying to make a large point out of the fact that results were reproduced," he says. But according to Strickler, that such a high percentage of tumors tested positive actually casts doubt on the study's reliability and raises the possibility that the labs merely exchanged contaminated samples. "The prevalence [of SV40-positive samples] was so high ... that you have no way to make the distinction between [contamination] and a true positive result," he says.
Carbone and some of the other scientists we have interviewed say that Strickler's contamination theory is a red herring. "We've documented that it is the case that this virus is present and is expressed in these tumors," Testa says. "I think the onus is on [federal health officials] to take this new research into consideration." Carbone, not surprisingly, is even more adamant. "The idea that these tumor samples, tested in laboratories all over the world, were all contaminated, while all the controls remained negative, is ridiculous," he says. "There is no scientific evidence in support of contamination, and plenty of evidence to the contrary. Moreover, many labs have demonstrated SV40 using techniques other than PCR."
Recently we asked several prominent scientists to evaluate the SV40 studies. George Klein, at the Karolinska Institute, in Stockholm, who chaired the Nobel Assembly, and is a longtime expert on SV40, read Testa's study. His conclusion was different from Strickler's. According to Klein, the Testa study is "quite convincing concerning the association between SV40 and mesothelioma," and "the evidence suggests that SV40 may contribute to the genesis of some human tumors, mesothelioma in particular."
Carlo Croce, the editor of Cancer Research and a member of the National Academy of Sciences, agreed. Not only is it indisputable that SV40 is present in human tumor samples, he told us, but "it looks like the presence of the virus contributes to the cause of mesothelioma."
Janet Rowley, the editor of the journal Genes, Chromosomes and Cancer and a professor of molecular genetics and cell biology at the University of Chicago, is a pioneer in the study of chromosome abnormalities in cancer. Rowley's groundbreaking research was itself called into question for years. "People didn't believe that chromosome abnormalities had anything to do with leukemia," she recalled. "It took a long time to break down that prejudice." She told us that Carbone had faced the same kind of doubts that first greeted her. "Everybody had assumed that mesothelioma was associated with asbestos. One of the important things in medicine is not to let your assumptions and those generally accepted paradigms obscure the fact that maybe there's more." Rowley believes that Carbone and Testa's work strongly implicates SV40 as a causal factor in some mesotheliomas.
ARBONE'S office is tucked into a quiet second-floor corner of the glass-and-concrete Cardinal Bernardin Cancer Center, at Loyola University, in Maywood, Illinois. The center is just a few miles west of Chicago and about ten minutes by car from Oak Park, where Carbone lives in a stately Frank Lloyd Wright house, with his wife and two daughters. Carbone came to Loyola in 1996 after a two-year stint at the University of Chicago. Now an associate professor of pathology, he works with Paola Rizzo, his senior scientist and closest collaborator, and a handful of post-docs and lab assistants in a tidy laboratory just down the hall from his office.
The lab is lively. Carbone has recruited compatriots as some of his research assistants, and the whir of high-tech machinery is punctuated by good-natured banter in Italian. This afternoon Carbone is examining an SV40-infected cell-culture plate under a microscope. He speaks almost fondly of the virus he has studied for most of the past decade. SV40 is "the smallest perfect war machine ever," Carbone murmurs. "He's so small. But he's got everything he needs."
Magnified 50,000 times under an electron microscope, SV40 doesn't seem particularly menacing. It looks almost pretty -- bluish snowflakes, against a field of white. The virus consists of six proteins, three of which make up the twenty-sided triangular scaffolding that is the virus's protein skin. But one of the remaining proteins, called large T-antigen (for "tumor antigen"), is, according to Carbone, the most oncogenic protein ever discovered. It is unique, he says, in its ability to cause cancer when it is set loose inside a cell.
In 1997, in Nature Medicine, Carbone published the first in a series of papers that outlined how large T-antigen blocks crucial tumor-suppressor pathways in human mesothelial cells. Whenever a cell begins to divide, in the process known as mitosis, a small army of quality-control agents goes to work. Running up and down the cell's DNA, these genes and proteins work together to scrutinize the DNA's integrity. If at any stage of cell division they detect DNA abnormalities that cannot be repaired, mitosis is halted and the cell undergoes apoptosis, or cellular suicide. The principal in this elaborate regulatory dance is a gene called p53. Arnold Levine, the president of The Rockefeller University, in New York City, and the discoverer of p53, says that 60 percent of all cancers involve some sort of p53 damage, mutation, or inactivation. "The p53 gene is central to human cancers," he says, describing it as "the first line of defense against cancer formation."
Carbone's experiments have shown that in human mesotheliomas large T-antigen attacks p53, binding to it so that it cannot function properly. Large T-antigen also strangles a series of proteins called Rbs, which together serve as some of the final gatekeepers in cellular division.
No other cancer-causing virus uses just one protein to knock out two different regulatory pathways simultaneously. For example, human papilloma virus must produce two proteins, E6 and E7, to inactivate p53 and the Rbs respectively; SV40 does its damage in one stroke. Levine calls large T-antigen "a remarkable protein."
Large T-antigen's cancer-causing havoc isn't limited to disabling a cell's most important tumor suppressors. It can also damage chromosomes by adding or deleting whole sections of DNA or reshuffling the genes. Once the virus is finished with a cell, Joseph Testa says, "it looks like somebody set off a bomb inside the cell's nucleus, because of all these chromosome rearrangements." Carbone says that because SV40 binds to tumor-suppressor genes and also causes genetic damage, it "is one of the strongest carcinogens we know of."
Yet he emphasizes that most people who carry SV40 in their cells won't develop cancer, because a healthy immune system generally seeks out and destroys invading viruses. He points out that large T-antigen normally provokes a particularly strong immune response, unless a person has been exposed to asbestos, a known immunosuppressant. "Human beings," Carbone says, "have devised many mechanisms to defend themselves against cancer. This is one of the reasons that human beings live so long compared with other animals. Human cancer is usually the result of a number of unfortunate events that together cause a malignant cell to emerge."
But SV40 may have evolved other strategies to elude the immune system. In a recently published article Carbone writes that sometimes SV40 produces such small amounts of large T-antigen that the virus escapes detection. Paradoxically, in this hypothesis small amounts of the virus are even more dangerous than large amounts.
Other scientists suspect that SV40 can inflict damage and then disappear completely, in what is described as a "hit-and-run" attack. This analogy is lent credence by a recent German study in which rat cells were infected with SV40 and transformed into cancer cells. When scientists searched for large T-antigen, it was no longer present in some of the cells. Further, these cells appeared to be even more malignant than those that were still expressing the protein, because the immune system could no longer recognize them as a threat.
The new theory may explain how SV40 and perhaps other viruses can induce cancer and yet not be readily detectable once tumors start proliferating rapidly. But that notion runs counter to traditional scientific thinking about cancer. "As a geneticist, I would like to see every single cell have evidence of the virus," Testa says, noting that the hit-and-run theory must still be proved. But, Testa observes, "This is an area that's going to perhaps establish a new paradigm."
Although Carbone's T-antigen research has bolstered his contention that the SV40 found in human tumors is not simply a passenger virus, until recently he had no answer to a criticism commonly voiced by those skeptical that the polio vaccine could be linked to cancer: some of the SV40 he and others have isolated in human tumors has a crucial genetic difference from the virus that contaminated the polio vaccine. The SV40 that its discoverers isolated from the polio vaccine in 1960 had a genetic feature that allowed it to replicate more quickly than the SV40 subsequently found in human bone and brain cancers and in most monkeys. That led some to question the idea that the SV40 that researchers were finding in these tumors was related to the SV40 in the polio vaccine.
To settle the issue Carbone sought to examine old vaccine stocks. He was told by government and drug-company officials that they had thrown out all the old lots. Then, two years ago, Carbone found an elderly Chicago-area physician who had an unopened case of polio vaccine from 1955, which he had stored in his refrigerator for more than forty years. "I would have gone all the way to Alaska to find this stuff, and here it was three miles away," Carbone says. Last summer Carbone finally completed tests on the vintage vaccine. He found that the tiny vials contained SV40 genetically identical to the strains found in human bone and brain tumors and in monkeys. "This proves that the SV40 that was present in the polio vaccine is identical to the SV40 we are finding in these human tumors," he says. Why was the SV40 isolated from the 1960 vaccine the faster-growing version? Because, Carbone says, both kinds occurred in the monkey kidneys used to grow the vaccine. Carbone and Janet Butel say that the SV40 that grew more quickly might have had an advantage in cell cultures -- perhaps explaining why it was the strain originally isolated from the vaccine. However, the slower-growing virus would almost certainly have an advantage in tumor formation, because it would be less likely to be detected by the immune system.
Because he believed that the slower-growing SV40 was more likely to induce tumors, Carbone wanted to see if federally mandated vaccine-screening tests for viruses were adequate to detect it. Vaccine manufacturers are not required to use state-of-the-art molecular techniques -- PCR, for example -- for virus detection. Instead they rely on ordinary light-microscope examination to look for evidence of cellular damage by viral contaminants after fourteen-day cycles in tissue culture. Although the current screening protocols -- themselves forty years old -- are, according to Carbone, more than adequate to detect the faster-growing form of SV40, his tests found that the slower-growing SV40 took at least nineteen days to grow out, and thus wouldn't be detected in the fourteen-day screening cycles. Carbone says his experiments suggest that any slow-growing SV40 present in the vaccine after the early 1960s could have gone undetected.
Carbone recently tested six vials of polio vaccine manufactured in 1996, and found that they were negative for SV40. He concludes that the colonies of monkeys used today must be free of the virus, because if slow-growing strains were present, the tests used for routine screening would not detect them. (Today's injected vaccine is produced on monkey cell lines, and is therefore free of any viral contaminants, whereas the oral vaccine is still produced on actual kidneys. Under Centers for Disease Control regulations that went into effect last month, American children should now receive only injected vaccine.) In a paper on his tests of vaccines Carbone recommends conducting extensive molecular testing of polio-vaccine stocks from the 1960s, 1970s, and 1980s to look for the slower-growing SV40. The issue is more than academic: the results would help to establish whether SV40 is present in young children today as a result of continued exposure to contaminated vaccine or as a result of human-to-human transmission based on the original, 1955-1963 exposure.
Copyright © 2000 by The Atlantic Monthly Company. All rights reserved.
Copyright © 2000 by The Atlantic Monthly Company. All rights reserved.