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F E B R U A R Y 2 0 0 0
ESPITE the accumulating evidence of SV40's association with human tumors, the NCI has been preoccupied with determining whether the virus is even present in human tumors. For more than two years the NCI's chief focus with respect to SV40 was the design and administration of a multi-laboratory study whose stated purpose was to assess whether PCR was a reliable tool for identifying the presence of SV40 in human tissue. Critics of the study, including scientists at some of the participating labs, worried that other agendas were involved. The study was directed by Howard Strickler and overseen by James Goedert. Nine labs participated in the study, including those of Keerti Shah, at Johns Hopkins; Bharat Jasani, at the University of Wales; and Janet Butel, at Baylor, but not Carbone's. The study, which was planned and administered by the NCI's Viral Epidemiology Branch, had a fairly unusual design. Instead of just seeing whether different labs could replicate one another's work, as is usually done, the labs were asked to prove that they could replicate their own work. Each lab was given a variety of samples from unidentified human mesothelioma tissues and asked to see if it could find SV40 DNA. Then it was asked to find SV40 DNA again in masked samples from the same tumor tissue.
We asked Richard Klausner, the director of the NCI, about his views on SV40 and about the design of the experiment. Klausner said that the research to date hadn't quelled his doubts that SV40 is present in human tumor tissue, and he questioned the reliability of the techniques that Carbone and others have been using. "These sorts of molecular technologies are wonderful tools but very complicated and sometimes misleading to use," Klausner said. "I think there is very good reason to question whether there has been the development of adequate standards or probes, PCR probes," for detecting the virus.
Like Strickler and James Goedert, Klausner raised the possibility of contamination to explain the positive findings of dozens of laboratories. "I do not see any compelling molecular data" to support the association of SV40 with human tumors, he told us. "In the absence of compelling clinical or epidemiological data, it's very difficult to say this looks like a pressing problem." We asked him about the many molecular studies, from numerous independent laboratories around the world, that had identified SV40 in human tumors. "There's too much irreproducibility and too many good explanations for artifact," he said. Klausner told us that the NCI has taken "an open approach but a critical one" to the notion that SV40 is associated with human tumors, and he insisted that it is seriously studying the issue. Michele Carbone's work, for instance, has been funded by the NCI. (Carbone is also funded by the American Cancer Society.)
We asked Klausner to explain why the Viral Epidemiology Branch had directed the multi-laboratory molecular-biology study, especially given that neither Strickler nor the head of the branch, Goedert, has a strong background in the field. Why hadn't he tapped an NCI division with more expertise in DNA extraction, sequencing, and characterization? "Their expertise in viruses and virus-associated disease makes [the Viral Epidemiology Branch] really the right place to do it .... As an expert in doing this sort of work, I feel that I can make that decision and I feel very comfortable with the decision," Klausner said. "What we are trying to do is establish some agreed-upon probes and standards that independent laboratories could utilize to provide ways of either validating or not validating molecular findings."
On another issue, Klausner referred to an epidemiological study that Strickler had done to determine whether SV40 was linked to human cancer. That study appeared in 1998 in the Journal of the American Medical Association, and received extensive publicity upon its release. It concluded that the NCI's database on cancer incidence shows no statistically significant correlation between exposure to SV40-contaminated vaccine and rates of cancer, including rarer cancers such as mesotheliomas, ependymomas, and osteosarcomas.
Strickler did find elevated cancer rates among those exposed to SV40, including a threefold increase in mesothelioma. Susan Fisher, an associate professor of epidemiology and biostatistics at Loyola, says that although the correlation Strickler found did not achieve statistical significance, it was at least "scientifically interesting." Strickler's study was "technically correct," Fisher says, but "it's hard to look at these numbers and turn around and say there is no evidence to suggest an association."
Moreover, Fisher says, standard epidemiological techniques may be useless in determining whether SV40 exposure is linked to higher cancer rates. If the research of Janet Butel and others is correct and SV40 is now spreading among human beings, it may be impossible to assemble an experimental group that has never been exposed to SV40.
The multi-lab NCI study concluded with six of the nine laboratories detecting SV40 in some samples. However, only two of the labs got the same positive results on samples from the same tissues. Although the multi-lab study was completed at the end of 1998, at the time this article was written it had yet to be submitted for publication.
Memos sent to Strickler by some of the participating laboratories show that from its inception the study was plagued by considerable internal strife. (Participating laboratories we approached declined to share the memos or discuss them. We obtained them independently.) Two laboratories suggested that poor DNA-extraction techniques by the outside laboratory Strickler had chosen to provide the DNA samples were to blame for the largely negative results obtained. Their concerns were heightened when it was learned that the contractor had contaminated some of the negative controls.
They also complained that Strickler was wrongly using the study to imply that previous positive findings were caused by contamination. "It cannot be that all of these laboratories are contaminated and that contamination always happens in mesotheliomas, osteosarcomas and brain tumors, while the negative controls are always negative," a scientist from one of the laboratories wrote Strickler. "Contamination is a random event .... [The] flaws and unresolved scientific issues ... have become so cumulative as to outweigh any positive scientific benefit which might be derived from the publication of this study." From another laboratory came this objection: "We feel that our comments about data interpretation are being dismissed and ignored. Your intransigence about the interpretation of the data and the conclusions of the study have forced us to admit that the collegiality and the scientific collaboration that was the basis of this study is very strained." Both laboratories maintained that Strickler's draft manuscript summarizing the study results was wrong in asserting that contamination was the cause of previous SV40 findings.
An unlikely ally in the laboratories' cause has been William Egan, the acting head of the Food and Drug Administration's Office of Vaccines Research and Review. Egan believes there is no strong epidemiological proof that SV40 is associated with human cancers and emphasizes that the current polio vaccine is free of SV40. However, he says, there is evidence that the virus may well be present in some tumor samples. After he had reviewed Strickler's draft manuscript, last February, Egan wrote a lengthy letter to Strickler criticizing it. "I think that this paragraph, and the following paragraph, imply, unintentionally so, that the positive results [of SV40 in tumors] that have been reported are due to laboratory contamination; I do not think that this should be implied." Strickler responded, "This study would not have been conducted if there was not some doubt. That point must be made and made clearly."
Later Egan chided Strickler about another section of his draft, which stated, "This multi-institutional study failed to demonstrate the reproducible detection of SV40 in human mesotheliomas." Egan wrote,
More exactly, it failed to demonstrate SV40 sequences in this set of mesotheliomas. This is not inconsistent with SV40 being found by others previously. Indeed, the fact that laboratories that previously found SV40 in their samples do not now find SV40 in these samples (and get the study controls correct) only lends credence to their previous findings .... These laboratories are able to find SV40 when it is there, and do not find it when it is not there.Frustrated by continuing objections, Goedert and Strickler considered publishing the study without the approval of the dissenting labs, but that plan was dropped. Last September an independent arbitrator was called in to rewrite Strickler's manuscript. The dissenters apparently gained some ground. The arbitrator made major changes in its tone and conclusions. The study now states that "laboratory contamination was unlikely to have been the source of SV40 DNA"found in human tumors in previous experiments (by Butel, Jasani, and the other participating labs).
HIRTY miles north of Venice, in the seaside resort town of Lignano Sabbiadoro, 200 clinicians and researchers are gathered at the international Conference on Malignant Pleural Mesothelioma. At a similar conference in Paris five years ago Carbone startled his audience when he presented his first SV40 paper.
Today a significant portion of the conference is devoted to SV40's association with mesothelioma -- testament to a sea change among researchers regarding the simian virus. Brooke Mossman, the director of the environmental-pathology program at the University of Vermont, was the first scientist to tease out the complex molecular pathways by which asbestos disrupts cellular regulatory mechanisms and causes mesothelioma. She has been impressed by Carbone's work. At Lignano she and Carbone are co-chairing a panel on the molecular pathways employed by asbestos and SV40 which lead to tumor development. In another presentation Luciano Mutti, a researcher at the Salvatore Maugeri Foundation's Institute for Research and Care, in Pavia, will report that mesothelioma patients who test positive for SV40 have a shorter life-span than those who test negative.
At the moment the floor belongs to David Schrump, the new chief of thoracic surgery at the NCI. Schrump matter-of-factly announces the results of a series of experiments he has just completed. When he "turned off" SV40 large T-antigen, he says, human-mesothelioma cell cultures that contained the virus stopped proliferating and started to die. Schrump explains that he undertook the experiment partly because he was skeptical of SV40's role in the development of mesothelioma. He and his team assembled human mesotheliomas that tested positive for SV40 and then devised a genetic bullet, a strand of RNA called an "antisense," which would prevent the expression of SV40 large T-antigen.
Within days after the antisense was administered to the cancer cultures, Schrump found, the growth rates of mesotheliomas with SV40 in them dropped dramatically; the negative controls were unaffected. One important finding was that even very low levels of SV40 appeared to be biologically important -- a discovery that speaks to Strickler's objection about the low levels of SV40 often found in tumor tissue. Schrump's study was published late last year in Cancer Research.
Another study in that same issue also supports the idea that SV40 is actively involved in mesothelioma. Adi Gazdar is a professor of pathology and the deputy director of the Hamon Cancer Center, at the University of Texas Southwestern Medical Center. He originally doubted Carbone's work on SV40. "Here's a monkey virus suddenly popping up in a rare tumor -- I was skeptical of the data," he told us. So Gazdar devised an experiment that could determine at one stroke whether the SV40 found in tumors was a lab contaminant and whether the virus is involved in tumor formation. Gazdar used a technique called laser microdissection to separate cancerous cells from nearby noncancerous ones. He found SV40 in more than half of the mesothelioma tumors. He also found the virus in some adjacent pre-cancerous cells. Significantly, 98 percent of nearby noncancerous cells tested negative for SV40. "That rules out any contamination," Gazdar says, "because if a specimen was contaminated, the SV40 would be in all parts of the specimen -- it wouldn't be localized to the mesothelium alone." Moreover, Gazdar says, his study "suggests that the virus is in the right type of cells many years before they become malignant" -- evidence that SV40 contributes to the development of cancer. Gazdar says of Carbone's work, "I feel everything he's said, I've been able to confirm, and more."
Gazdar and other scientists believe that the time has come for a major federal funding initiative on SV40 to better understand who is infected, how the virus works, and what might be done to prevent disease. "There's still a lot we don't know about the basic biology of this virus in human infections, including what tissues it infects, how it is transmitted, and when people become infected with it," Janet Butel says. "Until more studies are done, we don't know if we're looking at the only types of cancers that have an association with SV40," she says of the lung, bone, and brain cancers with which SV40 has been associated most often. "Maybe these are just the ones we've recognized so far. There may be others people haven't run across." Gazdar says, "It's such a crucial issue. Possibly millions of people are sitting with this virus in their mesothelium or other tissues and are at risk for developing cancers." Cancers that were once rare "may suddenly become not so rare," he says. "I think it's an enormous potential health problem."
Arnold Levine, of The Rockefeller University, is not convinced that the virus causes cancer in human beings, but he, too, believes that the discovery of SV40 in human tumors warrants a serious federal response. "If it's part of the cause of a disease," he says, "it has a significance in public health and I think we ought to find that out. That's a good reason to spend taxpayers' money: to do science to find out whether the public health is really monitored here properly. I think that maybe there's enough evidence in the literature now that the National Cancer Institute ought to put out an RFA." The reference is to a Request for Applications, the formal process by which the federal agency identifies a major health-research initiative and invites scientists to apply for research funds. "That would stimulate people to come in and design experiments and replicate these things." Carbone made the same suggestion to federal health officials in 1997 but was rebuffed.
Like the NCI, the Atlanta-based Centers for Disease Control maintains a stance of neutrality with undertones of skepticism. In a four-page fact sheet called "Questions and Answers on Simian Virus 40 (SV40) and Polio Vaccine" the CDC notes that SV40 has been found in some tumors and adds that "more research is needed" to confirm a causative link with human disease. It also raises the possibility of contamination as an explanation. It cites Strickler's work by name but not that of Carbone, Butel, or Testa.
Some researchers plan to conduct screenings for the virus. Joseph Testa hopes to initiate a screening program at Fox Chase's new cancer-prevention pavilion that focuses on asbestos exposure. He is collaborating with officials from the Asbestos Workers Local 14, in Philadelphia, to identify people who are particularly at risk. Carbone applauds that effort. "If you test positive for this virus, you should not be anywhere near asbestos," he says. Bharat Jasani, who has found SV40 DNA in a high percentage of the British mesotheliomas he examined, has begun testing British and Canadian mesothelioma patients, at their request. He hopes they may be candidates for future SV40-targeted therapy.
Last year scientists reported that a vaccine they had developed targeting large T-antigen appeared to help prevent and reverse tumors expressing large T-antigen in mice. Carbone and Harvey Pass, who is now the chief of thoracic oncology at the Karmanos Cancer Institute, at Wayne State University, in Detroit, are collaborating with Martin Sanda and Michael Imperiale, of the University of Michigan at Ann Arbor, who are among the vaccine's developers. They hope soon to bring the experimental vaccine to Phase I clinical trials, in which it will be tested for its safety in human beings, though not yet for whether it works. Even if the vaccine eventually proves effective in human beings, years may well pass before it is widely available.
In an age of uncontrolled AIDS in Asia and Africa, rampant tuberculosis in Russia, and antibiotic-resistant microbes in American hospitals, does SV40 really warrant a significant public-health response? There is no doubt, Carbone says, that the virus is linked to some cancers. What's more, millions of Americans now have been exposed to the virus. Studying SV40 may teach us something about the dangers of cross-species infection at a time when the use of animal tissue for medical purposes is gaining acceptance.
Good science is ultimately about the exchange of ideas unfettered by presuppositions. Sometimes great breakthroughs come out of theories that at first seemed heretical or even nonsensical. "Can you think of anything more different on earth than asbestos and a monkey virus?" Carbone says. "Yet you stick them together and they work together to be more deadly than either one of them is alone." He goes on, "This research is important in so many different ways. It's not just about SV40 and mesothelioma. It helps us understand the whole picture of how viruses interact with environmental carcinogens. This research can help us understand how completely unrelated carcinogens can work together in causing disease -- a mystery we have barely begun to unravel."
Copyright © 2000 by The Atlantic Monthly Company. All rights reserved.
Copyright © 2000 by The Atlantic Monthly Company. All rights reserved.