New Research Says XMRV Not Cause of CFS, Prostate Cancer

Findings Disputed by Researchers Who First Reported XMRV Link

Janis C. Kelly

June 07, 2011

June 7, 2011 — Two studies published online in Science dismiss any link between the mouse retrovirus xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome (CFS) or prostate cancer and suggest that XMRV is merely a lab contaminant derived from the recombination of 2 mouse proviruses. However, the new data are being disputed by the researchers who first reported XMRV in blood samples from patients with CFS.

In one new study, Konstance Knox, PhD, from the Wisconsin Virus Research Group in Milwaukee, and colleagues attempted to replicate the landmark 2009 study by Lombardi et al that found XMRV in 67% of patients with CFS vs 3.7% of control patients.

Dr. Knox and colleagues examined blood samples from 61 patients with CFS from the same medical practice that had supplied samples in the older study, including 43 patients who had been diagnosed previously as XMRV-positive. The Knox analysis showed no viral genetic material, infectious virus, or virus-specific antibody evidence of XMRV in any of the samples.

"There was no evidence of XMRV or other [murine-like gammaretroviruses (MLVs)] in the blood samples tested," senior investigator Jay A. Levy, MD, told Medscape Medical News.

Dr. Levy, who is director of the Laboratory of Tumor and AIDS Virus Research at the University of California–San Francisco, had been asked by Daniel L. Peterson, MD, a coauthor of the Lombardi et al paper, to test blood samples from some of the patients in the original CFS paper after several other groups had failed to replicate the earlier findings.

Retraction of XMRV-CFS Paper Refused

The negative outcome of the Knox study was one reason the journal has asked for retraction of the Lombardi et al paper. Lead investigator Judy A. Mikovits, PhD, director of research at the University of Nevada's Whittemore Peterson Institute for Neuro-Immune Disease in Reno, has stoutly refused.

Science editor-in-chief Bruce Alberts, PhD, has weighed in with a rare "Expression of Editorial Concern," about the Lombardi et al study, which was accompanied by an even more unusual " Science Statement on Editorial Expression of Concern" further explicating Dr. Alberts' comments, and now retroactively attached to the Lombardi et al paper.

Dr. Alberts writes that the "validity of the study by Lombardi et al. is now seriously in question" and that the journal "will take appropriate action" when additional National Institutes of Health–sponsored studies of XMRV are completed.

Responding to the Science papers and editorial, Dr. Mikovits contends that Knox et al failed to replicate data from the Lombardi et al study because the researchers used different, less sensitive methodology.

Dr. Mikovits told Medscape Medical News, "There are no data in Knox et al to support the authors' conclusions or those expressed in the Editorial Expression of Concern. In fact, a close look at western blots shows seropositive patients. They did not use our assays for infectious virus. A glaring example of this is that the infectious viral assay used in Knox et al uses [less sensitive] mink lung cells as a target."

She continued, "Mink lung cells do not support the growth of all animal xeno- and polytropic viruses. Metzger et al (J Virol. 2010;84(4):1874-1880) showed that 22RV1 XMRV virus has a titer of 103th - 4th on mink lung cells, while this virus has a titer of 1010th on human LNCaP used by Lombardi et al. This is 6 to 7 logs difference of sensitivity."

Dr. Levy countered, "We used the most sensitive assays for infectious xenotropic and polytropic mouse viruses. We did not observe these differences in sensitivity with the XMRV we obtained from the 22Rvl line."

Second Study Disputes XMRV Link to Prostate Cancer

In the second new study, Tobias Paprotka, PhD, and colleagues suggest that XMRV arose in the 1990s by the recombination of 2 mouse leukemia viruses during laboratory passage of a human prostate tumor in mice and is a contaminant, rather than a cause of cancer. Dr. Paprotka is in the Viral Mutation Section of the National Cancer Institute's HIV Drug Resistance Program in Frederick, Maryland.

Dr. Paprotka and colleagues compared viral loads in both early and later generations of cultured prostate cancer cells. The first 7 generations of passaged cells had 1 to 3 copies of an XMRV-like genetic sequence per 100 cells. This blossomed to 60 to 70 copies in later passages, and to 2000 to 3000 in later passage xenografts. "We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages," the authors write.

"For a retrovirus to cause cancer, almost every cancer cell should harbor the virus. When we found the virus was not in the early passages of the tumor cells, but plentiful in the later ones, it made us suspicious that XMRV was not the cause of the original tumor," said study coauthor Hsing-Jien Kung, PhD, professor and deputy director of the University of California–Davis Cancer Center, in a press statement.

The researchers also found 2 novel proviruses in the mouse lines used to passage the prostate cancer tumors. "When we put [their gene sequences] next to each other, they fit together like a puzzle. We knew immediately that they had generated XMRV. It was one of those eureka moments that comes along only a few times in a career," said senior investigator Vinay K. Pathak, PhD, also of the Viral Mutation Section in the National Cancer Institute's HIV Drug Resistance Program, in a press statement.

The researchers calculate that the odds of generating an identical XMRV sequence a second time are roughly 1 in 1 trillion. Based on the dates that the cell passages occurred, they concluded that the genetic recombination happened between 1993 and 1996, after which XMRV got loose in the world as a lab contaminant.

Dr. Levy said, "These enzymes and antibodies used in research labs were contaminated with MLV sequences that we feel gave the false results reported in the other papers on MLV and CFS. I was surprised at the high degree of MLV contamination of reagents used to measure MLV in the blood and to purify white blood cells from the blood."

Debate Continues

However, in a reply, Dr. Mikovits has described extensive tests her group used "to ensure that the reported data were not as a result of contamination, including the detection of a human antibody response to the virus [and] the screening of all reagents and cell lines for any evidence of gammaretrovirus contamination, human or otherwise.

"None of the negative papers which demonstrated contamination used the enzymes or antibody reagents used and recommended by Lombardi et al," Dr. Mikovits said.

Dr. Pathak told Medscape Medical News, "I think research should focus on the real causes of CFS, and stop wasting time and resources on the laboratory-derived virus known as XMRV."

Dr. Pathak added, "Similar to CFS research, XMRV has nothing to do with human prostate cancer. The association with prostate cancer is also an artifact of laboratory contamination."

The XMRV dispute is of more than academic interest. Concern about the retrovirus had led Canadian health authorities to forbid blood donation by patients with CFS and inspired some patients with CFS to demand antiretroviral therapy usually prescribed for HIV infection.

Dr. Levy said, "Hopefully our paper, as well as others published, will now completely eliminate the concerns for XMRV in the blood supply. We hope it will put more emphasis on looking for a causative agent or, as importantly, helping to improve the immune system in CFS patients so they do not suffer from the illness. CFS patients should not be taking antiretroviral drugs."

Dr. Levy, Dr. Pathak, and Dr. Alberts have disclosed no relevant financial relationships. Dr. Mikovits has applied for a patent on a method of detecting XMRV seroconversion in several diseases including prostate cancer, lymphomas, neuroimmune disease, CFS, fibromyalgia, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and autism.

Science Express. Published online May 31, 2011. Knox study; Paprotka study; Alberts statement; Science statement

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