One thing that resonates from the two-day XMRV conference at the National Institutes of Health is the, at times, testy battle between two camps of scientists. The cohort that believes that mouse retrovirus contamination accounts for the positive XMRV and related murine leukemia virus studies was pitted against the cohort that believes contamination has been ruled out and that the positive studies show a clear link between this family of retroviruses and Chronic Fatigue Syndrome and prostate cancer. Not surprisingly, the discussions around CFS grew more heated than those around prostate cancer. Dr. Mary Kearney of the National Cancer Institute may be the one to resolve this issue, as she reported on a test that discriminates between mouse retroviruses and XMRV with, she said,100 percent accuracy.
Despite several positive retroviral findings, by the time the Q and A began at the end of the second day, the upbeat mood many felt due to the recently published FDA/NIH/Harvard murine leukemia virus study—which found XMRV-related MLV viruses in 86.5 percent of CFS patients—had morphed into uncertainty. Dr. John Coffin of Tufts University, however, reassured the audience that it takes time to grind the sausage—his oft-used phrase—and that the XMRV findings were, in fact, moving along at a brisk clip.
Lyme expert Dr. Joseph Burrascano asked one of the most salient questions of the conference that so far has no definitive answer: Why are some scientists finding XMRV and related MLVs in most of their samples while others aren’t finding it at all? What is responsible? Is it the reagents, the collection, the processing, the methods, the patient cohorts or a combination of the above? The representative of the Blood Working Group suggested that blood collection and processing may hold the key. Dr. Judy Mikovits echoed that sentiment.
Here are some highlights of the conference:
--Dr. Sam Chow of UCLA discussed data from a Chinese study of men with prostate cancer and healthy controls. Of the 117 controls, 17 or 14.5 percent were positive for one of several types of MLVs, including XMRV, polytropic MLVs and modified polytropic MLVs. (Polytropic viruses infect the original host—in this case mice—as well as other species, whereas xenotropic viruses like XMRV infect species other than the original host.) Of the 34 men with prostate cancer, five or 14.5 percent were also positive. One reason for the hefty numbers in controls may be that Chinese keep mice as pets. Chow said that older men were more likely to have an MLV than younger men. In addition, Chow reported that XMRV has entered the blood supply in China: 3.4 percent of blood donors were positive for XMRV. There was no association of the RNASE-L genetic defect in the Chinese study.
In contrast, Dr. Robert Silverman of the Cleveland Clinic and co-author of the first study linking prostate cancer to XMRV, talked about the RNASE-L mutation in his cohort. In one family of five sons, for instance, four had prostate cancer, along with the RNASE-L mutation. He also discussed the study he coauthored on macaques infected with XMRV. In these animals, he explained, the prostate epithelium is infected early on, followed by the seminal vesicles and epididymis, and finally the stromal fibroblasts. Prostate cancer is often preceded by inflammation and XMRV stimulates proinflammatory genes, so it’s conceivable, he said, that XMRV infection could cause inflammation and eventually prostate cancer. The enzyme APOBEC3G, a potent inhibitor of XMRV, mutated XMRV DNA in blood cells of the macaques. Silverman also said that androgen stimulates XMRV, and anti-androgens inhibit the retrovirus.
--Dr. Eric Klein, chair of the Glickman Urological and Kidney Institute at the Cleveland Clinic and co-author of the first study linking prostate cancer to XMRV, discussed his findings on prostate cancer and XMRV. Klein said that XMRV is in urine. He also gave possible explanations for why some groups haven’t found the retrovirus, including geographical differences, which is what is seen with another human retrovirus, HTLV. Klein stressed that PCR “details” are important in doing studies correctly.
He also discussed how most prostate cancer is relatively benign: though 17 percent of men get the disease, only 3 percent will die from it. But scientists can’t always predict which prostate cancers need to be treated aggressively, and as it stands now, 90 percent of men with the disease opt for treatment. Treatment, however, carries it own constellation of possible long-term complications, including incontinence and erectile dysfunction. Perhaps, Klein said, the XMRV positive patients will prove to be among those who should be treated. Klein said given that many viruses have been shown to cause cancer, “It’s not unreasonable to assume that the retrovirus XMRV could cause cancer.” Mutations, he said, in RNASE-L and MSR1 genes are associated with an increased risk of prostate cancer. In addition, eunuchs may have a decreased risk of prostate cancer not because of their diminished production of testosterone, but due to the fact that they’re not exposed to pathogens through sex.
--Dr. Ila Singh of the University of Utah and co-author of a study linking XMRV to prostate cancer voiced that XMRV may become a marker for prostate cancer. It’s conceivable, she said, that XMRV status may eventually prove to be a better indicator than the current PSA test, and perhaps antiretroviral therapy will be effective in treating the disease. She explained that the XMRV gag (the basic physical infrastructure of the retrovirus) cross-reacts with Moloney mouse MLV. However, the envelop (the retroviral protein that makes up the outside of the retrovirus) reacts only to XMRV. Of 233 men with prostate cancer in her study, 27 percent were XMRV positive. Interestingly, the higher the Gleason score—which is used to determine the prostate-cancer prognosis and ranges from two to 10—the more likely the men were to be infected with XMRV.
Singh’s CFS study involves 200 healthy controls and 105 patients, all from the Utah area. Singh said she won’t use nested PCR, because in her view contamination can result. Other ways to reduce contamination: Handle only one specimen at a time, and have a negative control. Her tests, she said, are very sensitive at a 97.5 percent detection rate, with a specificity of 100 percent (i.e., no cross reaction with other retroviruses).
--Dr. Maureen Hanson of Cornell University is studying 30 subjects, with a mean age of 39. Ten of those patients supplied by longtime CFS physician Dr. David Bell were severely ill with CFS, with less than three hours of daily upright activity. Another ten had recovered from CFS, with 13.5 hours of upright activity daily. And, finally, 10 healthy controls weighed in with more than 15.5 hours of daily upright activity. Hanson’s findings are consistent, she said, with the Lombardi Science study (which found XMRV in 67 percent of CFS patients) and the recent FDA/NIH/Harvard MLV study (which found XMRV-related MLVs in 86.5 percent of patients). She also said that she’s never worked with mice in her lab, making contamination with a mouse retrovirus less likely.
--Dr. Judy Mikovits of the Whittemore Peterson Institute and principal investigator of the first study linking XMRV to CFS talked about a London CFS cohort (and then, I believe, it was British researcher Dr. Jonathan Stoye who corrected her and said that the cohort was nearer to Kent) who met the Canadian consensus and suffered from severe cognitive dysfunction, joint pain, vertigo and tender lymph nodes; more than 50 percent were homebound. The predominant MLV was XMRV, and the rate of infection among CFS patients was 70 percent. Fifty blood donors served as controls. Dr. Jonathan Kerr (who, like Jonathan Stoye, was an author on the second negative British XMRV CFS study) drew their blood. Four percent of the controls were positive for XMRV.
--Dr. Mary Kearney of the National Cancer Institute discussed the assays she developed to detect XMRV, which she said can discriminate between XMRV and mouse retroviruses. She explained that mouse retroviruses emit a lower level of florescence than XMRV. She claimed she can distinguish between the two with 100 percent accuracy. Perhaps her assay will be used to render a final verdict on the mouse-contamination theory.
--Dr. Ross Molinaro of Emory University, co-author on the first study linking XMRV to prostate cancer, said that XMRV encodes a previous unknown Orf3 protein with punctate patterns. In macaques, he explained, the prostate epithelium is infected early on, followed by the seminal vesicles and epididymis, and finally the stromal fiberblasts.
--Dr. Francois Villinger of Emory University chronicled the infection of macaques with XMRV. He said their CD4, CD8 and natural killer cells were infected and was surprised that monocytes were not. "The viral load in blood was contained very quickly," he said. Chronic infections, Villinger said, were seen in the spleen, epididymis, macrophages in the lung, the seminal vesicles, cervix and vagina. Villinger said he didn't look at the breast or spinal cord. Someone in the audience brought up screening sperm banks for MLVs.
--Dr. Jonas Blomberg of Uppsala University in Sweden looked at CFS and fibromyalgia in Swedish patients. Judy Mikovits supplied five samples, and Blomberg found three to be positive, so he was able to detect the retrovirus, just not in the Swedish cohort. Of the 35 CFS patients and 15 fibromyalgia patients, all were negative.
--Dr. Graham Simmons of the Blood Working Group discussed the different phases of study for the blood people. Phase 1: assay evaluation. Of all the assays, he said, the CDC’s and the FDA’s were the most sensitive. Phase 2: pilot clinical study. Phase 3: clinical sensitivity specificity. Phase 4: blood donor clinical panel. While phase 2 to phase 4 are not complete, “We do think we have begun to figure it out,” Simmons said and indicated that proper collection and processing of blood are vitally important.
--Dr. Nicole Fischer of University Medical Center Hamburg-Eppendorf found XMRV in one out of 300 men with prostate cancer and one out of 70 controls in Germany.
--Dr. Shyh-Ching Lo of the FDA talked about the findings in FDA/NIH/Harvard paper, which he co-authored, and said that different primers yield different results.
--Dr. Brigitte Huber of Tufts University focused on contamination as the cause of XMRV positive samples. Veteran CFS physician Dr. Susan Levine supplied the 111 patients, all of whom met the Fukuda CFS definition; 25 percent were severely disabled. Only one of the 111 patients—less than 1 percent—proved positive.
Last fall, Huber collected new blood: three CFS samples and 36 healthy controls. Using nested gag PCR, two out of the three CFS patients and 17 of the 36 controls were positive. Twelve samples were negative. Mouse DNA probes detected mouse DNA in those 17 positive samples from CFS patients, and Huber called the correlation “shocking.” Her conclusion was that a common lab reagent is contaminated with mouse DNA. Most of her samples contained several MLVs. Her lecture drew criticism from several members of the audience. One outspoken critic shouted to Huber that heparin tubes were a “nightmare. Why are you still using it?” The heparin naysayers used EDTA tubes instead.
--Bill Switzer, lead investigator of the CDC’s negative XMRV CFS study, reported on the agency’s prostate cancer study of 162 patients. Switzer said that the CDC found a gag sequence in one and pol sequences in three. (Pol proteins synthesize viral DNA.) Other scientists are looking at RNA, Switzer said, and the CDC is looking at DNA. Switzer made the point that the Robert Koch Institute in Germany didn’t find XMRV in prostate-cancer patients either.
--Dr. Frank Ruscetti of the National Cancer Institute and co-author of the first study linking XMRV to CFS said that there are 17 million people in the world with Chronic Fatigue Syndrome, and all of them have contacted Judy Mikovits. That line drew a huge laugh from the audience. Single round PCR, he explained, locates only 15 percent of cases, whereas nested PCR finds 69 percent. XMRV reservoirs, he said, have not yet been identified. One patient who was studied had both XMRV and a polytropic MLV. When I asked if that person was more ill, Judy Mikovits replied yes.
Coming up: A look at the salient posters presented at the conference.
This article is copyright CFS Central 2010. All Rights Reserved. You may quote up to 150 words from this article as long as you indicate in the body of your post (as opposed to a footnote or an endnote) that the excerpt is by Mindy Kitei for CFS Central. You may not reprint more than 150 words from this article on blogs, forums, websites or any other online or print venue. Instead, refer readers to this blog to read the article.
.jpg){kind=small}
