A simian virus known as SV40 has been associated with a number of rare
human cancers. This same virus contaminated the polio vaccine
administered to 98 million Americans from 1955 to 1963. Federal health
officials see little reason for concern. A growing cadre of medical
researchers disagree
by Debbie Bookchin and Jim Schumacher
http://www.theatlantic.com/issues/2000/02/002bookchin2.htm
HARVEY Pass, the chief of thoracic surgery at the National Cancer
Institute, in Bethesda, Maryland, was sitting in his laboratory one
spring afternoon in 1993 when Michele Carbone, a wiry young Italian
pathologist who was working as a researcher at the NCI, strode in with
an unusual request. Pass had never before met Carbone, and had talked to
him for the first time, on the telephone, only a few hours before. Now
Carbone was asking Pass for his help in proving a controversial theory
he had developed about the origins of mesothelioma, a deadly cancer that
afflicts the mesothelial cells in the lining of the chest and the lung.
Mesothelioma was virtually unheard of prior to 1950, but the incidence
of the disease has risen steadily since then. Though it is considered
rare -- accounting for the deaths of about 3,000 Americans a year, or
about one half of one percent of all domestic cancer deaths -- the
disease is particularly pernicious. Most patients die within eighteen
months of diagnosis.
Pass, one of the world's leading mesothelioma surgeons, knew, like other
scientists, that the disease was caused by asbestos exposure. But
Carbone had a hunch he wanted to explore. He told Pass that he wondered
if the cancer might also be caused by a virus -- a monkey virus, known
as simian virus 40, or SV40, that had widely contaminated early doses of
the polio vaccine, but that had long been presumed to be harmless to
people.
Pass listened as Carbone described for him the history of the early
polio vaccine. A breakthrough in the war against polio had come in the
early 1950s, when Jonas Salk took advantage of a new discovery: monkey
kidneys could be used to culture the abundant quantities of polio virus
necessary to mass-produce a vaccine. But there were problems with the
monkey kidneys. In 1960 Bernice Eddy, a government researcher,
discovered that when she injected hamsters with the kidney mixture on
which the vaccine was cultured, they developed tumors. Eddy's superiors
tried to keep the discovery quiet, but Eddy presented her data at a
cancer conference in New York. She was eventually demoted, and lost her
laboratory. The cancer-causing virus was soon isolated by other
scientists and dubbed SV40, because it was the fortieth simian virus
discovered. Alarm spread through the scientific community as researchers
realized that nearly every dose of the vaccine had been contaminated. In
1961 federal health officials ordered vaccine manufacturers to screen
for the virus and eliminate it from the vaccine. Worried about creating
a panic, they kept the discovery of SV40 under wraps and never recalled
existing stocks. For two more years millions of additional people were
needlessly exposed -- bringing the total to 98 million Americans from
1955 to 1963. But after a flurry of quick studies, health officials
decided that the virus, thankfully, did not cause cancer in human
beings.
After that the story of SV40 ceased to be anything more than a medical
curiosity. Even though the virus became a widely used cancer-research
tool, because it caused a variety of tumors so easily in laboratory
animals, for the better part of four decades there was virtually no
research on what SV40 might do to people.
Carbone had reviewed some old research papers on the contamination and
some of the early tests on SV40. He had even reviewed the notes from a
crucial 1963 epidemiological study, by Joseph Fraumeni, an NCI
researcher, which had concluded that children inoculated with
contaminated vaccine did not show increased mortality rates. The studies
did not impress Carbone: no one had systematically searched for evidence
of the virus in tumors, and, as Fraumeni himself noted, the
epidemiological study was too short to have detected certain
slow-developing cancers. (Mesothelioma can take twenty to forty years to
develop.)
Carbone had just finished a series of experiments in which he had
injected the virus into dozens of hamsters. Every one of them developed
mesothelioma and died within three to seven months. The results made
Carbone wonder if SV40 might also play a role in human mesothelioma. He
had come to see Pass because he had heard that the senior surgeon had
meticulously saved tumor tissue from every one of the dozens of
mesothelioma surgeries he had performed, and now had one of the largest
collections of mesothelioma biopsies in the world. Carbone asked Pass if
he could look for SV40 DNA in Pass's tumor-tissue samples, using a
sophisticated molecular technique, known as polymerase chain reaction,
or PCR, to extract tiny fragments of DNA from the frozen tissue and then
amplify and characterize them.
As they talked, Pass became more and more impressed with Carbone. The
young scientist was energetic and extremely self-confident -- something
Pass attributed to Carbone's surgical patrimony. (Carbone's father is a
well-known orthopedic surgeon in Italy.) When Carbone had finished
describing his proposed experiment, Pass realized that the implications
were potentially significant. Only a handful of viruses have been
directly associated with human cancers, and none of them are simian in
origin. If SV40 was linked to mesothelioma in people, might it also
cause bone and brain cancers in human beings, as it had done in
hamsters? What if the monkey virus could spread from person to person?
And if the virus was cancer-causing, or oncogenic, what was one to make
of the fact that millions of Americans had been exposed to it as part of
a government-sponsored vaccination program?
"I thought to myself, He's got this wild-assed idea," Pass recalls. "If
it's true, it's unbelievable. Even if it's not, I'm going to get a hell
of an education in state-of-the-art molecular biology."
Others at the National Institutes of Health -- including some of the
scientists who had been around at the time of the contamination scare --
were less receptive to the novel theory. They told Carbone that the last
thing anyone wanted to hear was that the exalted polio vaccine was
linked to cancer. Too much was at stake. Implicating a vaccine
contaminant in cancer -- even if the contamination occurred some forty
years ago -- might easily shake public confidence in vaccines in
general. And besides, everyone knew that asbestos was the cause of
mesothelioma.
Carbone sought the advice of two renowned pathologists, Umberto
Saffiotti, the chief of the NCI's Laboratory of Experimental Pathology,
and Harold L. Stewart, a former director of pathology at the NCI who was
once the head of the American Association for Cancer Research. Both
urged Carbone to follow his intuition. "Forget what people tell you,"
Stewart told Carbone. "They told me I was wrong all my life. If you want
to do it, you should, or you will regret it." That spring afternoon in
1993, with Pass's mesothelioma samples in hand, Carbone called an old
friend, Antonio Procopio, a professor of experimental pathology in Italy
who had worked for three years at the NIH. "I asked him if he was
willing to do this crazy project with me," Carbone says. "I told him I
could not pay him or his expenses." A month later Procopio arrived in
Bethesda. "We had no money," Carbone recalls. "He slept in my house for
six months, and we worked day and night."
It turned out that Pass's samples were loaded with the monkey virus: 60
percent of the mesothelioma samples contained SV40 DNA; the nontumor
tissues used as controls were negative. Moreover, Carbone found that in
most of the positive samples he tested, the monkey virus was active,
producing proteins -- suggesting to Carbone that the SV40 was not just
an opportunistic "passenger virus" that had found a convenient hiding
place in the malignant cells but was likely to have been involved in
causing the cancer.
In 1994 Carbone, Pass, and Procopio published the results of their
experiment in Oncogene, one of the world's leading cancer-research
journals. They proposed SV40 as a possible co-carcinogen in human
mesothelioma. It was the first time researchers had put forward hard
evidence that the all-but-forgotten vaccine contaminant might cause
cancer in human beings.
A Solution to an Enigma
MICHELE Carbone is almost stereotypically Italian: generous with his
emotions, outspoken, and jovial. He is strikingly handsome, with large
brown eyes and shoulder-length brown hair. Carbone grew up in a cultured
home in Calabria, on the shores of the Mediterranean in southern Italy.
As a youth he often spent hours poring over medical texts, some of them
300 years old, in the voluminous library started by the first of the
seven generations of Carbone physicians to date. If his father gave him
science, from his mother he may have inherited the strong intuition that
is his distinguishing characteristic as a researcher. She is an
accomplished artist whose work is exhibited widely in Europe.
Carbone graduated in 1984, at the top of his class, from the University
of Rome Medical School, one of the largest in the world, and quickly won
a coveted NIH doctoral fellowship. In 1993 he received a Ph.D. in human
pathology. In less than a decade he has risen to the top of his
profession. Today he is internationally recognized as an expert in
mesothelioma.
Since 1994 Carbone has written more than twenty studies and reviews
investigating SV40's link to human cancer. "There is no doubt that SV40
is a human carcinogen," he says. "SV40 is definitely something you don't
want in your body." Carbone suggests that the virus works in tandem with
asbestos or by itself to transform healthy mesothelial cells into
cancerous ones.
Since he published his first study, scientists at seventeen major
laboratories -- in the United States, Great Britain, France, Belgium,
Italy, and New Zealand -- have confirmed Carbone's research with respect
to the presence of SV40 in human mesothelioma. Their results point to a
solution to an enigma that long puzzled researchers. At least 20 percent
of mesothelioma victims report no asbestos exposure, and only 10 percent
of people who have had heavy exposure to asbestos ever develop
mesothelioma. The experiments suggest that SV40 may be another factor at
work in the tumors.
Two very recent studies, from Finland and Turkey, found no SV40 in
domestic mesothelioma samples but did find it, respectively, in American
and Italian samples. The authors observe that their negative findings
lend support to the theory that contaminated polio vaccine is associated
with the disease: neither Turkey nor Finland used SV40-contaminated
vaccines. Today Finland has one of the lowest rates of mesothelioma in
the Western world.
The virus has also been located in other kinds of tumors. More than a
dozen laboratories have found SV40 in various kinds of rare brain and
bone tumors. In 1996 Carbone reported that he had found SV40 in a third
of the osteosarcomas (bone cancers of a type that afflicts about 900
Americans a year) and nearly half of the other bone tumors he tested --
research that has since been confirmed by numerous laboratories. The
virus has also been detected in pituitary and thyroid tumors.
The possibility of a link between SV40 and brain tumors is particularly
intriguing. Like mesothelioma, brain tumors have become dramatically
more common in recent years. Brain tumors will be diagnosed in about
3,000 children in the United States alone this year. In 1995 Janet
Butel, the chairman of the department of molecular virology and
microbiology at the Baylor College of Medicine, in Texas, and her chief
collaborator, John Lednicky, also a Baylor virologist, reported that
they had found SV40 in a number of children's brain tumors. Butel and
Lednicky reported that DNA sequencing revealed that the virus was not a
hybrid but rather authentic SV40 -- the same as the SV40 found in
monkeys. In the fall of 1996 an Italian research team, led by Mauro
Tognon, of the University of Ferrara, announced that it had found SV40
DNA in a large percentage of brain and neurological tumors, including
glioblastomas, astrocytomas, ependymomas, and papillomas of the choroid
plexus. The researchers suggested that SV40 may be a "viral cofactor"
involved in the sharp rise in human brain tumors. Late last year an
extensive study undertaken in China reinforced those results. The study
examined sixty-five brain tumors, finding SV40 in each of the eight
ependymomas and two choroid-plexus papillomas, common brain tumors among
children. It also found the virus in 33 to 90 percent of five other
kinds of brain tumor examined. The authors, writing in the November,
1999, issue of Cancer, noted that the virus was actively expressing
proteins.
Recent research also indicates that SV40 has gained a secure foothold in
the human species. In 1996 Tognon and his collaborators reported that
they had also found the virus in 45 percent of the sperm samples and 23
percent of the blood samples they tested from healthy people, suggesting
that the monkey virus could spread through sexual contact or unscreened
blood products. In 1998 the presence of SV40 antibodies in human blood
samples was reported by Butel, who tested several hundred American blood
samples and found antibodies to SV40 in about 10 percent of them.
Butel's laboratory also tested samples from children born from 1980 to
1995 -- decades after the contaminated vaccine was removed from the
market. A surprising six percent tested positive -- offering evidence
that the virus may now be spreading from person to person, including
from mother to child.
THE 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.
Strickler acknowledges that he has never done PCR himself (Shah was
responsible for the PCR work for their 1996 collaboration), but he
challenges the work of other labs that have found SV40 in human tumors.
"I feel that the data are mixed regarding the detection of SV40 DNA in
human tissues," Strickler says, citing his own negative study and the
British study. Strickler also points out that when SV40 is found in
tumor cells, it often occurs only at very low levels. Whereas human
papilloma virus (HPV), which causes cervical cancer, can be detected at
rates of fifty viruses per cancer cell, SV40 is sometimes found at a
rate of one virus per cell. "I find it curious that even the
laboratories that detect SV40 in the cancers report that the virus is
present at such extremely low levels," Strickler says. John Lednicky, of
Baylor, counters that HPV is very different from SV40. Strickler "is
comparing an apple with an orange," he says. "SV40 is known to be far
more tumorigenic than HPV in animals. One copy of SV40 per cell is
enough to transform a cell."
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.
"Like Somebody Set Off a Bomb"
CARBONE'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.
DESPITE 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).
The Search for a Vaccine
THIRTY 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 contaminaŠt 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."
------
Debbie Bookchin specializes in health and political issues. Her articles
have appeared in The New York Times, The Boston Globe, and The Nation.
Jim Schumacher is a freelance writer who lives in Vermont. His articles
have appeared in Boston magazine, The Boston Globe, and Newsday.
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