Science article "False Positive" chronicles XMRV research controversy

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Science article "False Positive" chronicles XMRV research controversy

  1. 1. NEWSFOCUS Downloaded from www.sciencemag.org on September 22, 2011 A report in Science 2 years ago that linked a mouse retrovirus, XMRV, to chronic fatigue syndrome astonished scientists and patients alike. But the theory soon began to take hits, and now, to all but a few researchers, it has completely unraveled Done. Case closed. Finito, lights off, The End. must have occurred. The leader of the team that conducted the study, For the past 2 years, a controversy has roiled around the purported Judy Mikovits of the Whittemore Peterson Institute for Neuro-Immune link between a mouse retrovirus, XMRV and chronic fatigue syndrome , Disease (WPI) in Reno, Nevada, resolutely maintained that her lab had (CFS), a baffling, debilitating disease with no known origin. Many no evidence of contamination and that it could repeatedly find the virus researchers who have followed this saga closely thought that a definitive with its techniques. Millions of dollars have gone into clarifying the study, published online this week by Science (http://scim.ag/xmrv-cfs) question, which has had far-reaching consequences for people with CFS and conducted by nine labs—including the main proponents of the the- and, if the virus lurked in the blood supply, the public at large. CREDIT: M. HICKS/SCIENCE sis—would finally bring a halt to the impassioned debate. The study just published found that none of the nine labs could repro- Think again. ducibly detect XMRV or relatives of the virus in blood samples distrib- The uproar began with an October 2009 paper in Science that found uted under a blinded code. Pounding another nail into the coffin, Science XMRV in the blood of two-thirds of the CFS patients examined. A is also running a partial retraction (http://scim.ag/R-H-S) of the origi- steady assault on the report soon began, with more than a dozen labs nal paper, as a contributing lab found that it in fact had a contamination. failing to replicate it to date and several asserting that contamination In an unexpected twist to this operatic saga, Mikovits co-authored1694 23 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org Published by AAAS
  2. 2. NEWSFOCUS the Science Express paper and has no quarrel with the results. Her col- ciation of America, a patient group in Charlotte, North Carolina. laborator, Francis Ruscetti, a retrovirologist at the U.S. National Cancer Many retrovirologists wish the entire controversy, which has Institute (NCI) in Frederick, Maryland, who is a co-author of both the ensnarled dozens of labs and cost millions of dollars, would simply original Science report and the new one, concurs. “Where there is dis- disappear. “All of it’s a waste of money and it’s wrong,” says Robert agreement is in the interpretation,” Ruscetti says. Gallo, head of the Institute of Human Virology in Baltimore, Mary- By their lights, the new study—conducted by the Blood XMRV land. “It’s like a bad dream.” Scientific Research Working Group sponsored by the U.S. National Heart, Lung and Blood Institute—does not rule out the possibility that mouse retroviruses infect people with CFS. “The conclusion of the Blood Working Group was that X MRV owes its discovery to a little-known enzyme called RNase L that helps the body battle viruses—and has ties to both prostate cancer and CFS. we don’t have a reproducible In the early 1990s, a few CFS assay to detect XMRVs in the researchers reported that their blood—not that they weren’t patients had higher levels of RNase in the patients at all,” Mikov- “ I don’t care if L than healthy people had, sug- its says. Ruscetti adds that the nobody else in gesting that this natural viricide working group analyzed their signaled an undiagnosed infec- the world wants Downloaded from www.sciencemag.org on September 22, 2011 original patients but used samples tion causing the disease. RNase L taken a few years later. to work on it. levels, they contended, provided a If this seems like wordsmith- long-sought “biomarker” for CFS, ing and splitting hairs, welcome to Fine, leave us which has caused much confusion the confusing, maddening world alone! ” because clinicians use varying cri- of XMRV Mikovits and Ruscetti, . —JUDY MIKOVITS, teria to make a diagnosis. It also who have become increasingly WHITTEMORE PETERSON could potentially help researchers isolated from the broader scien- INSTITUTE gauge the effectiveness of treat- tific community, now say their ments, as a drop in the enzyme original paper erred by focusing would mark a decrease in under- on a single XMRV isolate that lying, undetected infections. This turned out to be a contaminant. idea became central to tests using They say that isolate is but one Ampligen, an immune modulator of many XMRVs, which belong made by Hemispherx Biopharma to a still larger family of gamma- in Philadelphia, Pennsylvania, to retroviruses. They also contend treat CFS. that the virus may lurk in tissues, One of the clinicians testing only traveling to the blood occa- Ampligen was Daniel Peterson sionally. “We still stand by our of Incline Village, a town on the data that we isolated gamma- northern shore of Lake Tahoe, retroviruses from patients with the popular tourist destination on CFS and also from healthy con- Nevada’s border with California. trols,” says Mikovits, who has taken a more public role than Ruscetti in Peterson had investigated a mysterious outbreak of CFS that began in battling critics and reaching out to supporters. Incline Village in 1984 and had become one of the world’s best-known Mikovits has become something of a savior in the community of CFS clinicians. His work drew the attention of Harvey Whittemore, a people with CFS (also known as myalgic encephalomyelitis, or ME), prominent Reno attorney who lobbied on behalf of the gaming industry who for decades have endured charges that the disease is psycho- and later became a major real estate developer, and his wife, Annette, a somatic. The 2009 Science paper shouted out that CFS may well teacher of children with cognitive disorders. The Whittemores’ daugh- have a clear biological cause, and, in turn, raised hopes of effec- ter, Andrea, had developed CFS at age 12, and at Peterson’s suggestion tive treatments and even a cure. The new she began receiving Ampligen. findings give her “great pause,” yet she sus- pects they’re but a speed bump. “I haven’t Online sciencemag.org With the Whittemores’ financial support, Kenny De Meirleir, a Belgian physician who had an RNase testing lab in his own country, changed my thinking at all,” she says. And helped them open RedLabs USA in Reno to monitor the enzyme in she worries that the Blood Working Group Podcast interview U.S. patients taking Ampligen. Vincent Lombardi, who would soon with co-author conclusions will confuse people with CFS, Jon Cohen. play a prominent role in the XMRV saga, co-founded the new lab and some of whom got wind of the results early served as its director of operations. in the blogosphere and contacted her in a panic. “I had 15 suicidal Lombardi was a late bloomer in science. In 1990, while he worked patients call me last week,” she says. as a securities trader in Lake Tahoe, he decided to pursue an under- In scientific circles, Mikovits has developed a less flattering reputa- graduate degree at a local school. As part of a biostatistics class, heCREDIT: J. COHEN/SCIENCE tion. Critics have accused her and her backers of stubbornly wedding worked with Peterson, analyzing immune parameters of CFS patients. themselves to a thesis and moving the goalposts with each study that After his graduation in 1995, he ran investment companies and also challenges their conclusions. Even disease advocates who welcome the worked as a marketing director for a pawnbroker business. He went attention XMRV has brought to CFS believe the time has come to put on to pursue a Ph.D., first studying RNase L but switching to peptides this line of research to rest. “It’s hard to say that this has not received a in the tobacco hornworm, which he completed at the University of fair appraisal,” says Kimberly McCleary, president of the CFIDS Asso- Nevada, Reno, 2 years after starting RedLabs USA. www.sciencemag.org SCIENCE VOL 333 23 SEPTEMBER 2011 1695 Published by AAAS
  3. 3. NEWSFOCUS In 2002, meanwhile, a team of prostate cancer researchers had discovered that a muta- tion in the RNase L gene frequently occurs in families in which men are prone to the early- “ I began comparing onset form of that disease. A few years later, one of those investigators, Robert Silverman Judy Mikovits to Joan of the Cleveland Clinic in Ohio, began probing of Arc. The scientists for a viral link: If RNase L fights viruses, he will burn her at the reasoned, then perhaps having a crippled form of the enzyme opened the door for a cancer- stake, but her faithful causing infection. following will have Silverman teamed up with Joseph DeRisi and Don Ganem, two veteran virus hunters at her canonized. ” the University of California, San Francisco —JOHN COFFIN, (UCSF), who had developed a microarray TUFTS UNIVERSITY called ViroChip that had proved its mettle Downloaded from www.sciencemag.org on September 22, 2011 in identifying new viruses. The assay con- firmed Silverman’s hunch: In the prostate cancer tumors, ViroChip found a novel retro- virus, apparently a cousin of a known mouse virus and therefore given the ungainly name of xenotropic murine leukemia virus–related virus (XMRV). Silverman first reported the existence of XMRV and its prostate cancer link in April 2005 at an HIV meeting in a mountain resort in Banff, Canada. “My talk was well received, people were clearly while the Whittemores’ alma mater, the University of Nevada, Reno, interested, but it did not receive that much attention,” Silverman wrote offered about 1400 square meters of lab space within a new, gleam- to Science in an e-mail delivered through a public affairs manager— ing $78 million Center for Molecular Medicine. It would take 4 years the only form of communication the Cleveland Clinic would allow. for that building to open, so the nascent Whittemore Peterson Insti- (XMRV has become such a radioactive topic that several institutions tute (WPI) set up shop in borrowed space on campus in 2006. Annette, restricted what their researchers could discuss, and both Ganem and WPI’s president, hired Mikovits to run the lab. DeRisi declined to talk to Science about their XMRV work.) Mikovits had come into the CFS world by a circuitous route. She had In March 2006, Silverman, DeRisi, Ganem, and colleagues pub- spent more than 20 years at NCI, first as a technician studying HTLV-I lished their report in PLoS Pathogens, a respected but decidedly low- and HIV with Ruscetti, who served as her Ph.D. adviser. In 2001, she key choice for the description of a new retrovirus that appeared to married and moved to southern California, becoming chief scientific infect humans—the only three others are HIV and HTLV-I and -II— officer at EpiGenX Biosciences in Santa Barbara, which aimed to use a and had ties to a serious disease. Although they wrote that there was a new epigenetic approach to develop drugs and diagnostics. “strong association” between XMRV and the RNase L mutation, the In 2006, Mikovits became a consultant to a CFS-related nonprofit researchers still had misgivings about whether the virus contributed to foundation that Annette Whittemore co-founded, which explored prostate cancer. the link between the disease and another virus, human herpesvirus 6 In a commentary in the 30 January 2007 issue of the Proceedings of (HHV-6), that had been discovered in Gallo’s lab. At a meeting in Bar- the National Academy of Sciences (PNAS), mouse retrovirologist Hung celona, Spain, that year, Mikovits spoke to Whittemore for the first time Fan of UC Irvine called it an “exciting” discovery. But others were and heard Peterson give a talk. Peterson focused on a non-Hodgkin’s skeptical. Gallo, whose lab played a central role in the discovery of lymphoma in some of his CFS patients, and Mikovits smelled a virus. all three known human retroviruses, contacted PNAS’s editor to com- She offered to work with him, and Whittemore helped set up a collabo- plain. The commentary was “substantially over the top,” says Gallo, ration. Later that year, Mikovits joined WPI. who saw no compelling evidence that this supposedly new human She soon enlisted Ruscetti, who had worked in Gallo’s lab when it retrovirus caused disease and doubted that a mouse retrovirus could discovered HTLV-I, to screen blood samples from Peterson’s patients even infect humans. “Once claims of etiology were made, I just gasped for viruses. Intrigued by the RNase L link to XMRV Mikovits and , for breath,” Gallo says. “My own experience argued to me that it’s best Lombardi—who by then had joined WPI as well—met Silverman in to stay away from this one.” October 2007 at a prostate cancer conference in Lake Tahoe, where they discussed the possible role of XMRV in CFS. Silverman was A CREDIT: KELVIN MA/TUFTS UNIVERSITY round the time Silverman first publicly described XMRV Annette happy to collaborate and sent WPI a clone of the virus, known as VP62. , Whittemore took on one of the most ambitious pursuits of her The institute could use it as a reference to start hunting for the virus in career: using her family’s wealth and powerful contacts to build a full- CFS patient blood samples that Peterson had stored. fledged research and clinical institute devoted to CFS. Peterson would A little over a year later, on 18 November 2008, Mikovits had the head a medical research team to elucidate the causes of the disease and first evidence of XMRV in CFS patients. Working with Ruscetti and provide state-of-the-art care. Nevada Governor Kenny Guinn and the Silverman, the group amassed evidence that the virus occurred in 67% current U.S. Senate Majority Leader, Harry Reid (D–NV)—both fam- of 101 CFS patients and 3.7% of 218 healthy controls. If the latter ily friends of the Whittemores—helped arrange government funding, number was representative of the general population, up to 10 million1696 23 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org Published by AAAS
  4. 4. NEWSFOCUS healthy people in the United States alone might Seattle, who had studied XMRV left the meet- , be infected, and the virus might be spreading ing convinced that the link to CFS was real. through blood donations and organ transplan- The group had not only detected the virus using tation—a silent epidemic of frightening pro- PCR but also grown it from patients’ cells and portions. Mikovits, Ruscetti, Silverman, and found antibodies to it. “It sounded really good their co-authors submitted a paper to Science to me because they had all these different lines in May 2009. The paper, of which Lombardi of evidence,” Miller says. The fact that an NCI was first author, did not claim that XMRV veteran such as Ruscetti endorsed it helped caused CFS, noting that the disease might sim- convince him. “Frank said, ‘I grew it with my ply make patients more vulnerable to infection. own hands,’ ” Miller recalls. “At the time, it Causality “is probable but not definitive at this sounded really exciting.” time,” Lombardi et al. stated. Coffin, who chaired the meeting, thought But what they were asserting was stunning Mikovits and her colleagues had persuasive enough: WPI, NCI, and the Cleveland Clinic evidence, especially because XMRV formed had all found evidence of XMRV in the same its own branch on a genetic family tree of CFS patients. Some of these people had par- mouse retroviruses, strengthening the case that Downloaded from www.sciencemag.org on September 22, 2011 ticipated in a 2007 NCI drug study, too, and it was not a contaminant. their blood samples, stored separately, also The next day, as Mikovits sat at Washing- tested positive, “ruling out the possibility of ton, D.C.’s Dulles International Airport wait- lab contamination as a source,” the authors ing for her flight back home, she received a wrote. When the team calculated the so-called phone call from Science, indicating for the first p-value—which generally needs to be under time serious interest in publishing the paper. 0.05 for a finding to be considered signifi- Science accepted the Lombardi paper on cant—they arrived at the astonishingly 31 August and published it online on 8 October low number of 8.1 × 10–35. 2009, accompanied by a supportive commen- Oddly, Peterson, who had supplied “Here was this tary that Coffin co-authored. “There are several the patient samples, was not one of the lines of evidence that transmission happened authors. His name was left off—and he was mysterious disease, … in the outside world and was not a laboratory kept out of the loop on the study’s results— and along comes a new contaminant,” the commentary declared. The because of worries that he might prematurely publication catapulted Mikovits into the spot- tell his patients, Mikovits says. virus. … It seemed to … light, where she has remained ever since. have the makings of T he manuscript didn’t convince Science. After reviews by three referees and members of the Board of Reviewing Editors a medical breakthrough.” “H ere we go again.” That’s what crossed Kimberly McCleary’s mind when she read the first headlines (provided for this article by Mikovits), the —ROBERT SILVERMAN, about the paper that week in her office editors rejected the paper. “Although the ref- CLEVELAND CLINIC in Charlotte. In her 20-plus years at erees were intrigued by your findings, they CFIDS, McCleary had seen many infec- had a number of serious reservations,” read a 4 June letter, which tious agents fingered as a potential cause of included excerpts from reviewers. CFS. HHV-6. Epstein-Barr virus. Mycoplasma. Adenovirus. Cyto- The rejection letter noted that Science would re-review the paper megalovirus. HTLV-I and -II. And on and on. Each time, hopes were if the authors could both retain the “novelty of its main message” and dashed as scientists closely evaluated the suspects. “address the referees’ concerns with new data rather than with counter- The string of disappointments had made McCleary cautious, arguments.” But the criticisms were substantial. “Chronic Fatigue Syn- and CFIDS urged patients not to get carried away. “We tried to tem- drome is full of false alarms,” wrote one advisory board member, “and per things early on, and we were criticized heavily for raining on the the detection of XMRV could be false positive PCR.” An otherwise parade,” she says. Still, many patients went wild with joy. “ME/CFS enthusiastic referee wrote that the “one major caveat I have is that the patients have never seen anything like this,” wrote Cort Johnson in an issue of potential contamination has not been completely dealt with.” A item, “Game Changer,” posted on his popular blog Phoenix Rising. second referee found it odd that the genetic sequence of XMRV derived Lombardi et al. had made XMRV a superstar. “Here was this myste- from CFS patients and the virus earlier discovered in prostate cancer rious disease (CFS-ME) with no known cause, all of these patients suf- were 99% similar. This “seems very unlikely and may indicate con- fering, and along comes a new virus that associates with the disease,” tamination, despite the evidence presented to the contrary,” the referee Silverman says. “It seemed to some at the time to have the makings of warned. One also wondered why they omitted Peterson as a co-author. a medical breakthrough.” Mikovits and her co-workers addressed many of the critiques, and But many scientists were skeptical. Simon Wessely, a psychiatricCREDIT: LISA DEJONG/LANDOV on 14 July, they resubmitted the paper, this time with Peterson’s name researcher at King’s College London who has long studied CFS, says on it. The next week, a committee—which included John Coffin, a the virology went over his head, but the fact that fully two-thirds of CFS prominent retrovirologist at Tufts Sackler School of Graduate Biomed- patients harbored the virus was an alarm bell. CFS, whose definition ical Sciences in Boston, and Silverman—organized a workshop at NCI has been the subject of years of debate, is far too heterogeneous a phe- with Mikovits and Ruscetti to help the institute better understand the nomenon for that, he says. unanswered scientific questions and the potential public health ramifi- With a few U.K. colleagues, Wessely started penning a letter to cations. Dusty Miller, a retrovirologist at the University of Washington, Science to address what they saw as methodological flaws in the paper. www.sciencemag.org SCIENCE VOL 333 23 SEPTEMBER 2011 1697 Published by AAAS
  5. 5. NEWSFOCUS One problem was the unclear patient selection, they wrote. If all patients hope,” Lombardi said. “We’re not the doctors ordering these tests for came from Nevada and healthy controls were from elsewhere, then per- our patients.” Whittemore stressed that all profits would go back into haps XMRV wasn’t a CFS-related virus, Wessely says, but something the institute. “This wasn’t set up to make money, and it never has,” she that happened to be more prevalent in the gambling state. said. Whittemore also dismissed critics—a group that included Peter- Wessely also contacted Jonathan Weber, a retrovirologist at Imperial son—who cringed at CFS patients taking antiretrovirals. “How many College London, to set up a study of British CFS patients. “Our reac- years does this patient population have to be impacted and their lives tion was, ‘It’s probably wrong, but if it’s true, it’s a pretty big advance. destroyed?” she asked. So it’s worth testing,’ ” he says. Andrea Whittemore was one of the CFS patients who tested posi- The first published critique appeared a few weeks later—and it tive for XMRV . would prove to be prophetic. On 18 November, Patrick Moore, a cancer virologist at the University of Pittsburgh in Pennsylvania, and his post- doc Masahiro Shuda published a blistering commentary on Faculty of T he flood of negative data started in January 2010, when Wessely, Weber, and their colleagues reported in PLoS ONE that 1000, a Web site that evaluates reports in what it calls postpublication they couldn’t find any trace of XMRV in 186 British CFS patients. By the peer review. end of February, two more negative reports “Unfortunately, in my field, there’s a were published, one of them by Jonathan tendency with any new virus to hope that it Stoye, Coffin’s co-author on the favorable Downloaded from www.sciencemag.org on September 22, 2011 is causing a disease,” says Moore, who co- Science commentary that accompanied the discovered HHV-8 and helped prove that it Lombardi paper. causes Kaposi’s sarcoma. “One has to be so Back in Reno, Mikovits and Lombardi cautious about that. A lot of stories sound began feeling besieged. “After the first neg- good, but they’re built on a house of cards.” ative study, it was a dog pile,” Lombardi As Moore and Shuda noted, Mikov- says. “Let’s be honest: A number of peo- its’s group used a technique called “nested ple in the mainstream medical community PCR” to identify XMRV infections, which, heard chronic fatigue, and they rolled their they wrote, “is inherently prone to inter- eyes and laughed.” mittent false positivity that has occurred The problem, as they saw it, was that in our lab and many others.” What’s more, nobody was following their recipe exactly. the researchers had not randomized and Many labs failed to find XMRV using PCR blinded patient and control samples, a because the virus exists in scant amounts, standard way to protect against bias and to they said—so much so that a patient can detect errors. Together, Moore and Shuda test positive on one bleed and negative wrote, this was “a recipe for uncontrolled on the next. Mikovits said they overcame PCR contamination.” this problem by first mixing patient blood The next month, a study co-authored samples with an uninfected cell line that by Silverman and published online by is especially permissive to the virus and Virology showed that AZT, an anti- HIV drug, worked against XMRV in “ We tried to temper coculturing for 8 weeks. Other researchers, including XMRV discoverer Silverman, test tubes. Again, CFS patients were things early on, and we questioned the need for this step. overjoyed. Not only had a likely culprit of were criticized heavily for Seven months after Lombardi et al., their suffering been found, drugs already on the market might treat it. raining on the parade. ” Science published Wessely’s critique and two others, which discussed patient Of course, no one had proved that the —KIMBERLY MCCLEARY, selection problems and the growing virus actually caused CFS. But by the THE CFIDS ASSOCIATION OF AMERICA list of negative studies, including three start of 2010, three commercial labs, one that failed to find XMRV in prostate of which was WPI’s VIP Dx (formerly RedLabs USA), offered an cancer. None of the Technical Comments, XMRV test. however, mentioned the possibility of contamination. Peterson, sitting in one of his practice’s waiting rooms in Incline Despite a vigorous defense by Mikovits and Ruscetti in the same Village in June, explained that his patients split into two groups. One issue of Science, the three comments further eroded confidence in the had ridden the CFS roller coaster so many times that these patients findings. But then, 2 weeks later, a research group unconnected to WPI wanted validation and replication before buying the XMRV story. would rekindle the fading hope that the link between XMRV and CFS “There’s another group that, for whatever reason, has made this ill- was real. It would also spark a peculiar new controversy. ness into a religion and becomes polarized into who believes and who CREDIT: COURTESY OF KIMBERLY MCCLEARY doesn’t believe,” he said. Increasing numbers of believers began taking XMRV tests, which cost $500 or more, and, if they were positive, ask- T hat study originated with Shyh-Ching Lo, an infectious-disease researcher at the U.S. Food and Drug Administration (FDA) who ing their physicians to prescribe anti-HIV drugs, says Peterson, who by took a keen interest in CFS. In the early 1990s, Lo had tested a theory then had severed ties with WPI over a contractual issue. that a microbe called Mycoplasma was involved in CFS. For that study, When a Science reporter visited WPI the same month, Annette he had received blood samples from Anthony Komaroff, a physician Whittemore and Lombardi both strongly defended the decision to sell who had treated hundreds of CFS patients at Brigham and Women’s an XMRV test before evidence existed of a causal link. (The group has Hospital in Boston. Like so many others, the lead had proved spurious. filed related patent applications, too.) “Every physician who requests After XMRV surfaced as a new candidate, Lo suggested to patient testing is aware of contamination and the potential of false Komaroff they test the same patient samples, stored at –80°C and1698 23 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org Published by AAAS
  6. 6. NEWSFOCUS aggressive, ACT UP–style pro- “Once claims of tests at blood banks to demand publication: “I believe we need etiology were to act quickly, before the FDA/ made, I just NIH paper is killed.” Some scien- gasped for breath. tists thought the federally ordered delay impinged on scientific My own freedom. Why not publish the experience competing papers and let other researchers scrutinize them? “It argued to me was very strange business,” Cof- that it’s best to fin says. Alter says nothing sinis- stay away from ter was afoot peril. Retrovirology were never in and that the papers this one. ” published the CDC study on —ROBERT GALLO, 1 July 2010, and the Lo- Downloaded from www.sciencemag.org on September 22, 2011 INSTITUTE OF Alter study ran in PNAS on HUMAN VIROLOGY 23 August. Once they saw it, many researchers concluded that untouched for almost 2 decades. Komaroff was enthusiastic. Lo also the Lo-Alter data didn’t confirm the Lombardi paper at all. They had contacted Harvey Alter, an infectious-disease specialist who works on found that 86.5% of samples from CFS patients harbored DNA from transfusion medicine at the U.S. National Institutes of Health’s Clinical mouse retroviruses as did 6.8% of healthy controls, but it was not Center in Bethesda, Maryland. Alter—who won the prestigious Lasker XMRV; the sequences were more closely related to a different, well- Award for his role in the discovery of hepatitis C—provided Lo with known group, the murine leukemia viruses (MLVs). It was as if a new blood samples from healthy people to serve as controls. suspect suddenly had been nabbed. Miller says the findings actually It was Alter who accidentally dropped the bombshell news. At a argued against Lombardi et al. “If XMRV is everywhere and these blood bank meeting in the Croatian capital Zagreb in late May 2010, guys are doing the same experiment, why didn’t they find XMRV?” he presented an overview of current blood safety issues, and his Power- he asks. Alter now says he regrets asserting that the paper confirmed Point presentation had one slide on what he called the “Agent du jour,” the XMRV results and that it was “naïve” to show the slide in Zagreb. XMRV Although Alter didn’t present any evidence, the slide was blunt: . “That is something I shouldn’t have said because we really hadn’t “The data in the Lombardi et al. Science manuscript are extremely found it,” he says. “I somehow got to be the spokesperson for this. I strong and likely true, despite the controversy,” one bullet point said. had no idea what it would mean.” Lo agrees that the team didn’t find “We (FDA & NIH) have independently confirmed the Lombardi group the virus that Mikovits and Lombardi had reported. findings,” read another. Mikovits to this day contends that the Lo-Alter paper confirms After the editor of a Dutch newsletter discovered the slide on Lombardi et al. and insists that from the beginning, she viewed XMRV the meeting’s Web site and sent out a press release, the CFS world as one of many gammaretroviruses, which includes the MLVs, involved exploded, again. “Is this it, then, the big one???? Holy ****!!!!!” one with CFS. In the Lombardi study, some patients tested negative in PCR blogger wrote on the Phoenix Rising forum. tests for XMRV and yet produced MLV-related proteins, she claims, Some researchers had doubts. True, Alter had a stellar reputa- but they decided to count them as negatives. She has another serious tion, but he wasn’t a retrovirologist. And Lo in the 1990s had pushed regret about the paper. “I’d not put XMRV in the title,” Mikovits says. the widely discredited theory that a Mycoplasma infection played an “We never considered that it would be a single sequence.” important role in HIV causing AIDS. Yet news of the impending confirmation had a big impact on a spe- cial XMRV panel at the AABB, an international association focused on transfusion medicine. The panel, which had heard about the Lo-Alter A s more negative data poured in, Mikovits and Lombardi became ever more ardent. Improved techniques now found the virus in almost every CFS patient, they said. study before the news broke, recommended on 18 June that AABB Patient groups began to see Mikovits as a martyr—a Galileo-like members discourage CFS patients from donating blood. figure fighting an all-powerful scientific establishment to expose the Patients and scientists alike were eager to see the data. Lo and Alter truth. But many of her initial supporters had joined the growing camp had written a paper accepted by PNAS, but they ran headfirst into an of skeptics. “I began comparing Judy Mikovits to Joan of Arc,” Coffin institutional roadblock: Retrovirologists at the U.S. Centers for Disease says. “The scientists will burn her at the stake, but her faithful follow- Control and Prevention (CDC) had their own paper in preparation that ing will have her canonized.” showed no evidence of XMRV antibodies, proteins, or DNA in well- Mikovits has her own theory about when Coffin changed his mind. characterized CFS patients. Faced with contradictory results from two She and Lombardi had found evidence, not included in the ScienceCREDIT: GAIL BURTON/AP IMAGES teams of researchers under its purview, the U.S. Department of Health paper, that XMRV was also linked to autism. On 11 November 2009, and Human Services (HHS) ordered the groups to delay publishing Lombardi presented those data at a meeting at the Cleveland Clinic. until each could review the other’s data. “You don’t talk about autism in the U.S.—it’s too politically charged,” Patients cried foul. Many had long distrusted the federal govern- Mikovits claims Coffin told him. She believes Coffin turned against her ment, and especially CDC, for not taking CFS seriously and suppress- that very day. Coffin confirms he was upset that Lombardi presented ing research results. Now, they said, CDC was trying to bury another such preliminary data on such a fraught topic but says, “I did not ‘turn theory it didn’t like. The author of a blog, CFS Central, called for against’ Judy at that or any other point.” www.sciencemag.org SCIENCE VOL 333 23 SEPTEMBER 2011 1699 Published by AAAS
  7. 7. NEWSFOCUS Mikovits further lost credibility with her contribution to an early Retroviruses and Opportunistic Infections in Boston. Working with study organized by the Blood XMRV Scientific Research Working Oya Cingöz in Coffin’s lab, Pathak had analyzed the 22Rv1 cell line Group, formed by HHS 1 month after the publication of Lombardi all the way back to its origins, a prostate cancer patient at Case West- et al. It included safety experts such as Susan Stramer of the Ameri- ern Reserve University in Cleveland, Ohio. To create the cell line, can Red Cross, as well as Mikovits, Alter, Coffin, and other players researchers had used a common technique called passaging in which in the XMRV saga. The group had devised a four-stage program that human tumor cells are grown repeatedly in mice and then harvested. included sending blinded samples to various labs to see whether they Early versions of the cell line, still stored in university freezers, had could detect XMRV At a meeting on 14 December 2010, the work- no XMRV But two different strains of mice used in the later experi- . . ing group discussed the results of the second stage of its study, which ments each harbored DNA that matched half of the virus. This sug- again did not support Mikovits’s findings. Her lab had found XMRV gested XMRV was created in the 1990s when two viruses combined in a sample from a healthy person who all labs agreed beforehand was in a lab culture, and the widely used cell line spread the virus in many negative for the virus. labs, infecting other cell lines, too. The finding furthered doubts that Mikovits had an explanation. The false positive was caused by a XMRV had ever infected a human being—let alone had a role in either postdoc who mistakenly used the same needle twice to lyse cells and CFS or prostate cancer. shear DNA, contaminating the sample. She dismisses the error as triv- The accidental-origin evidence hasn’t convinced Mikovits. “Yes, ial, the result of working late at night on a weekend because of repeated that can be an origin of an XMRV she says. “But it could have arisen ,” Downloaded from www.sciencemag.org on September 22, 2011 power failures in a new building, coupled with intense pressure from multiple times. It’s not one sequence.” Pathak contended the chances the working group to get results quickly. “People make mistakes, and that the exact same virus would arise twice were the same as dropping we reported it as a mistake,” she says. a quarter from a helicopter flying over the Grand Canyon and having it But many researchers threw up their hands: How could WPI botch land on a quarter on the ground. a study essential to the survival of its theory? The University of Wash- “It’s all contamination,” Coffin concluded, which outraged Miko- ington’s Miller was astonished that Mikovits vits. “How can John Coffin shut down research like that?” Mikov- didn’t do the critical tests herself and that its shouted during one interview, her blue-gray they were rushed. eyes shooting fire. “He’s not God!” She specu- Ruscetti is not bothered that most of “ The virus is real. … lates that perhaps the U.S. government, afraid his peers think he’s wrong. “I’ve been there before,” he says. But he thinks Mikovits has I have isolated it from of the huge consequences oftoadiscredit her XMRV outbreak, was trying widespread been treated unfairly. “I’ve been in science for patients. I know it’s work. “We can’t afford another public health 35 years, and she’s as honest a scientist as I’ve ever met,” he says. there. Believe me. ” crisis,” she said. Eventually, two studies that looked for More setbacks followed. A week after the —JUDY MIKOVITS, XMRV in fresh blood samples taken from WHITTEMORE PETERSON INSTITUTE working group meeting, Retrovirology pub- patients in the Lombardi paper failed to lished four devastating papers online that find it. Ila Singh, a virologist at the Univer- together made a compelling case that a contaminant marred both the sity of Utah in Salt Lake City, consulted with Lombardi et al. and Lo-Alter studies. Two reports co-authored by Mikovits and Lombardi to copy their protocols precisely, and among Coffin looked for XMRV and MLV in both CFS and prostate cancer 100 CFS patients in her study, 14 had tested XMRV-positive by WPI. patients; every positive sample they found also harbored mouse DNA, Singh and co-workers found 5% of both patients and controls to be suggesting that a reagent had become contaminated. Another report XMRV-positive—a finding they traced back to a contaminated enzyme put a finer point on it: Researchers discovered MLV—at least 97% sim- used in a PCR reaction. ilar to the Lo-Alter viruses—in a commercially available enzyme used With help from Peterson, UCSF virologist Jay Levy examined 43 in a PCR kit. Some speculated that perhaps the patient samples were patients who tested XMRV-positive at WPI. Peterson says he wanted to handled more than the controls. help his former colleagues at the institute. “I went to Jay Levy to prove The fourth study, led by Greg Towers at University College Lon- them right,” he says. All samples tested negative. Again, Mikovits said don, was even more damning. It was a follow-up to an earlier report, the study was flawed. published in the April 2009 issue of the Journal of Virology by “They didn’t do one thing we did,” Mikovits says. Miller’s group, that described the discovery of XMRV in a cell line “We did it exactly the way they did it,” Levy says. called 22Rv1, which was derived from a human prostate cancer patient. On 31 May, Science published the Levy report and the Coffin- At the time, Miller thought the finding supported the theory that the Pathak origins study online, along with a so-called Editorial Expres- virus infects humans, as he assumed the original prostate tumor used sion of Concern by Science Editor-in-Chief Bruce Alberts. “The study to make the cell line harbored XMRV Now Towers’s team had com- by Lombardi et al. attracted considerable attention, and its publication . pared the genetic sequence of XMRV from different 22Rv1 lines with in Science has had a far-reaching impact on the community of CFS reported sequences from patients. patients and beyond,” he wrote, before stating that “the validity of the The cell line viruses proved ancestral to those in patients. There study … is now seriously in question.” “What Science giveth, Science was also more genetic diversity in viruses from different 22Rv1 lines taketh away,” Johnson blogged on Phoenix Rising. than different patients, precisely the opposite of what should happen if Alberts and Science Executive Editor Monica Bradford had first the virus infected humans: Immune system pressure typically forces a suggested that Mikovits and her co-authors retract the paper volun- virus to diversify as a means of escaping attack. Towers concluded that tarily. “Science feels it would be in the best interest of the scientific XMRV never infected humans and that the cell line virus had somehow community,” they wrote in a 26 May letter. Mikovits was livid and contaminated the patient samples. questioned Alberts’s motives. “Who wrote that letter? I don’t think it In March 2011, Coffin and NCI virologist Vinay Pathak delivered was Science,” she says. The co-authors thought the retraction request what many thought was the final blow to XMRV at the Conference on was premature, too. “What if we walk away from this based on con-1700 23 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org Published by AAAS
  8. 8. NEWSFOCUS tamination and it’s not contamination?” Lombardi asked. “You’ve got to give us time to figure this out.” Alberts stresses that they floated the retrac- “People will tion idea because Science already planned to publish the Expression of Concern. “It wasn’t rather go over a public call for retraction,” he notes, empha- the Niagara in a sizing that the recipients shared it with the media. He also does not think it would have barrel than ever been premature, although he says it’s often a getting involved in tough call whether to retract a paper. “Ulti- mately, it requires expert judgment and a lot of CFS again.” sensitivity to the issues,” he says. “We had lost —SIMON WESSELY, KING’S COLLEGE confidence in the results.” LONDON T he growing rift between XMRV believers Downloaded from www.sciencemag.org on September 22, 2011 and doubters became painfully obvious at a June retrovirology meeting in Leuven, Bel- gium, where the two camps literally kept their distance. In the scientific sessions, questions were mostly polite and informative. During coffee breaks and a poster session with Bel- gian beer and cheese, Mikovits, her old friend Ruscetti, and De Meirleir, who helped open RedLabs USA, stuck together and barely talked to their scientific oppo- But he emphasizes that many scientific unknowns remain about XMRV . nents. The scientific debate was grinding to a halt. He points to a study in the Journal of Virology in May that intentionally “I don’t care if nobody else in the world wants to work on it!” infected macaques with XMRV It shows that XMRV moved out of the . Mikovits exclaimed at one point, rolling her eyes. “Fine, leave us blood of the monkeys but stayed in tissue reservoirs and that antibod- alone!” When Mikovits’s anger subsided, she appeared earnest and ies disappeared. “We know nothing about the viral life cycle,” he says. even confused by all the criticism. “The virus is real. … I have isolated Blood Working Group member Michael Busch, head of the Blood it from patients. I know it’s there,” she said. “Believe me.” Systems Research Institute in San Francisco, California, says Ruscetti, It wasn’t just that scientists were growing tired of the debate, Mikovits, Lo, and Alter deserve kudos for participating fully in the Wessely says. Some were put off by the “appalling, unforgivable study. “I commend them on their scientific integrity and commitment attacks” by some patient advocates on those who criticized WPI’s to the scientific process,” Busch says. “This has been a difficult and findings. Wessely says he has received death threats in recent years. disappointing process for them and for CFS patients, but hopefully we “People will rather go over the Niagara in a barrel than ever getting have all learned lessons that will guide future research and lead to dis- involved in CFS again,” he says. covery of the cause and cure of this disease.” Silverman notified Science and his collaborators on Lombardi et al. The results of yet another multilab study are due early next year. of his discovery that the data he supplied for the paper were wrong. Led by Ian Lipkin of Columbia University, the $2.3 million project Resequencing samples he tested for the study revealed that somewhere plans to test 150 samples from CFS patients and create a repository along the line, VP62 had contaminated them. Without casting blame or of their blood so that future putative causes of the disease can more explaining how the contamination occurred, the partial retraction says easily be tested. Lipkin says the study will continue despite the Blood two figures and a table that reported viral sequences, including one that Working Group’s negative results. “Our study designs are different, our showed XMRV in the family tree, were “spurious.” power is different, our subjects are different, and our assays are differ- In late August, the Blood Working Group completed its roughly ent,” Lipkin says. “Whether our results will differ remains to be seen.” $500,000 study, which conclusively determined that no one need worry Mikovits and Ruscetti are soldiering on. “As long as there are about XMRV or MLVs in the blood supply. The nine labs—which scientific reasons to continue, I think it’s incumbent upon me to do included WPI, Ruscetti, and Lo at FDA—each had received blinded it,” Ruscetti says. “I owe it to both the scientific community and the samples from 15 negative controls and 15 others who had tested posi- patient community.” tive for a gammaretrovirus in Lombardi et al. or Lo-Alter. Different Mikovits says it is irresponsible to dismiss the link between XMRVs teams cultured the virus, looked for antibodies to it, and used PCR to and CFS at this point. “Anyone who says this is a lab contaminant has fish for DNA. All labs could use whatever assays they chose. drawn the wrong conclusion and has done a disservice to the public,” Only WPI and Ruscetti found intermittent evidence of viruses or she says. Okay, maybe not as many CFS patients have XMRVs as they antibodies in patients, but they also reported similar numbers of pos- initially reported, but she’s still convinced that a gammaretrovirus is inCREDIT: KING’S COLLEGE LONDON itive responses in the negative controls. What’s more, there was no at least 20% or 30% of them. “I know of hundreds if not thousands of agreement between the two labs on which patient samples tested posi- people with evidence of this infection, from what we’ve done over the tive. “What this says, at the very minimum, is that we can’t find it reli- last 3 years,” she says. “I don’t know what it means. And I’m gong to ably in the blood of patients we found it in before,” Ruscetti says. keep looking for in vivo reservoirs like the ones seen in the macaques, Ruscetti understands why many of his peers think Lombardi et al. and I’m going to try to figure out mechanisms of pathogenesis, epi- and Lo-Alter have now run their scientific course. “It is quite legitimate genetics, or other things. I’m not going to stop studying it.” for those people to say maybe these two papers were wrong,” he says. –JON COHEN AND MARTIN ENSERINK www.sciencemag.org SCIENCE VOL 333 23 SEPTEMBER 2011 1701 Published by AAAS

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