Authenticity of XMRV integration sites

2 March 2011

retroviral integrationIntegration of retroviral DNA into the cellular genome is essential for the production of new infectious particles. A strong argument that the novel human retrovirus XMRV is not a laboratory contaminant is the finding that viral DNA is integrated in chromosomal DNA of prostate tumors. Nucleotide sequence analyses of 14 integration sites in prostate tumor DNAs from 9 different patients previously revealed the expected viral sequences linked to human DNA. But two of these integration sites are identical to those found in a prostate tumor cell line infected with XMRV.

A search of the nucleotide sequence database with the previously identified XMRV integration site sequences revealed that 2 of the 14 sequences (from 2 patients) were identical to two XMRV integration sites in DU145 cells. This cell line was established in 1978 from the brain metastasis of a human prostate tumor. In early 2010 2007 DU145 cells were infected with XMRV, and sequences of two integration sites were determined (the database entries can be found here and here).

Identical retroviral integration sites have never been reported in independently infected cells. Furthermore, XMRV infection of DU145 cells was done in the same laboratory in which the XMRV integration sites were identified in prostate tumor DNA. The conclusion is that two of the 14 XMRV integration sites in prostate tumor DNA are likely to be the result of contamination. These prostate tumor DNA samples were probably contaminated with DNA from XMRV-infected DU145 cells.

These observations do not directly impugn the veracity of the other 12 XMRV integration sites identified in prostate tumor DNA. However, when DNA contamination occurs it is often ubiquitous. Hence the authors write:

Whilst it is conceivable that the other 12 integration sites apparently derived from prostatic tumor tissues are genuine patient-derived sequences, we suspect that some or all of them may also be the result of contamination with DNA from experimentally infected DU145 cells.

This possibility can and must be addressed experimentally.

Update: While writing this post I received an abstract from the 2011 Conference on Retroviruses and Other Opportunistic Infections (CROI) entitled “XMRV probably originated through recombination between two endogenous murine retroviruses during passage of a human prostate tumor in nude mice”. As usual I will await publication of this story in a peer-reviewed journal before discussing it further.

Garson JA, Kellam P, & Towers GJ (2011). Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection. Retrovirology, 8 (1) PMID: 21352548

Stone, K., Mickey, D., Wunderli, H., Mickey, G., & Paulson, D. (1978). Isolation of a human prostate carcinoma cell line (DU 145) International Journal of Cancer, 21 (3), 274-281 DOI: 10.1002/ijc.2910210305

Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, & Silverman RH (2007). An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proceedings of the National Academy of Sciences of the United States of America, 104 (5), 1655-60 PMID: 17234809

  • Anonymous

    Funnily, even those people presenting acknowledged that it was one sided.

  • Guest

    Exact same site to the base-pair – a chance of hundreds of millions to one.

  • Snail

    Not what i said Gob. I said that because they had some mouse DNA in the tumor when they extracted it from the mouse (which of course they would have), they could show that preXMRV sequences were in that very mouse. no speculation – band on a gel which was cloned and sequenced.

    I do however agree with you that CROI should have invited some of the “pro-XMRV/X-MLV” camp. Lo, Alter and Ruscetti are all respectable scientists and their voice should have been heard. Not WPI for obvious conflict of interest reasons (ie: affiliated with the marketing of a deeply suspect unapproved “test”) and because of some of their more way-out pronouncements over the last couple of years for which there is no proof and for which, Gob, i’m sure you will agree, do not belong in a scientific forum.

  • Vaccines

    the retrovirus…contaminates a vaccine ?

  • RRM

    This information is not true. Regarding the Lo et al. study, the samples were not blinded.

    Also, while samples might have been handled in the same manner by the study authors, both studies used banked patient samples, which means that not ‘every sample was handled in the same fashion’ before is was tested, which is kinda like the point.

  • RRM

    Even I would agree, but we don’t know the reason(s) for this.

    As I tend to disbelieve giant conspiracies without specific proof, I would assume that any rejected paper would just not be of the quality required. Just like patients tend to assume that the original authors cannot get any new findings published due to a hostile scientific environment, this seems very, very unlikely to me.

  • RRM

    Again, this is not true. Regarding the Lo et al. study, the samples were not blinded.

    Also, while samples might have been handled in the same manner by the authors of both studies, both used banked patient samples, which means that not ‘every sample was handled in the same fashion’ before is was tested, which is kinda like the point.

  • Crafter Kate

    _____
    Snail says: “Not WPI for obvious conflict of interest reasons (ie: affiliated with the marketing of a deeply suspect unapproved “test”) and because of some of their more way-out pronouncements over the last couple of years for which there is no proof and for which”
    ____

    Do not judge, and you will never be mistaken.’

  • Anonymous

    This new paper is not on PMRV. You are incorrect about Lombardi et al.

    Lombardi et al. response to comments.

    “All experimental proce- dures were done by the same personnel, in the same physical laboratory space, under identical protocols. Investigators at NCI received 100 samples from individuals without knowing their health status; furthermore, the samples were sent to NCI directly without passing through the WPI laboratory space. Laboratory workers at the NCI and the WPI who performed the polymerase chain reaction (PCR) and immunological studies used coded, blinded samples that did not reveal the CFS status of the individuals. The WPI has examined all 218 control and 101 patient samples by both PCR and serological methods for the presence of XMRV nucleic acid and antibodies. In addition, NCI used plasma from all 100 samples they received in infection experiments with LNCaP cells. It was not feasible to examine all 101 patient and 218 control samples with all four XMRV detection methods described in (1), due to time and resource constraints.”

  • Anonymous

    Yes, I phrased that the wrong, here is the correct information.

    This new paper is not on PMRV.

    Quote on if the samples in Lo et al. were blinded: http://www.cfids.org/cfidslink/2010/090104.pdf

    “We did not specifically blind and mix the two sample groups (CFS and blood donors). However, they were studied in parallel by polymerase chain reaction (PCR). As shown in the figures of the paper, those samples with positive amplicons of the predicted sizes were all sequenced; no sample was considered positive unless sequencing confirmed PCR reactivity. – H.J. Alter, MD, MACP and S‐C Lo, MD, PhD”

    You are incorrect about Lombardi et al.

    Lombardi et al. response to comments. http://www.sciencemag.org/content/328/5980/825.4.full.pdf

    “All experimental proce- dures were done by the same personnel, in the same physical laboratory space, under identical protocols. Investigators at NCI received 100 samples from individuals without knowing their health status; furthermore, the samples were sent to NCI directly without passing through the WPI laboratory space. Laboratory workers at the NCI and the WPI who performed the polymerase chain reaction (PCR) and immunological studies used coded, blinded samples that did not reveal the CFS status of the individuals. The WPI has examined all 218 control and 101 patient samples by both PCR and serological methods for the presence of XMRV nucleic acid and antibodies. In addition, NCI used plasma from all 100 samples they received in infection experiments with LNCaP cells. It was not feasible to examine all 101 patient and 218 control samples with all four XMRV detection methods described in (1), due to time and resource constraints.”

  • Anonymous

    That cannot of course be extended to the other collaborators on Lombardi et al. To remind you, as you have forgotten, it was the NCI and Cleveland clinic. Should they also not invite those who represent Government departments who are meant to keep their eye out for infective agents? Or representatives of Governments who may find this costing billions? Should Stoye, Coffin, Huber, McClure, etc. not be invited because of some of their statement? It would wash both ways.

    How would you know which statements are correct, if they are prevented from speaking? The truth will out, whatever it is. It is unacceptable to hinder scientific research.

    They could not show that preXMRV sequences were in the strains used for the xenograft. They don’t know if they were used.

    From the abstract:
    “Analysis of 15 nude mouse strains indicated that none contained XMRV, but some strains potentially used to passage the xenograft contained both PreXMRV-1 and PreXMRV-2.”

  • Anonymous

    Just as it was with HIV?

  • Anonymous

    It is the only course of action if you believe it is a contaminant. Well that and attempting a replication study. Guess that comes last these days.

  • RRM

    These quotes support my assertion regarding blinding of samples instead of yours.

    I did not assert that both labs handled samples differently, but said that samples were handled differently *before* they entered the authors’ labs. Do you dispute the assertion that both studies used “banked” CFS samples? It is well documented (see the respective studies and the online supporting materials).

  • RRM

    I am sure with such sweeping statements, it would be very simple for you to provide us with some (objective and reliable) sources?

  • Anonymous

    MLV’s can integrate within 1000 bases upstream or down stream of a promoter site. XMRV (not to be confused with an MLV) in particular has a much narrower range than that.

    The DU145 cell line was screened for XMRV 3 years ago and found not to contain any. (Dong et al, 2007)

    Thus, if there is integrated XMRV inside the cell, then they must have been introduced during the experiment and transferred from the patient DNA into the cells and not the other way round.

  • anciendaze

    A simple question, as opposed to various abstruse arguments: if HIV-1 has diverged greatly from SIV in roughly 100 years, and these sequences show markedly less divergence, doesn’t that imply recent insertion? The same applies to suggested pre-XMRV sequences. Shouldn’t someone be looking for a wild-type ancestor with 99% homologous sequences?

  • Revday

    “Not WPI for obvious conflict of interest reasons (ie: affiliated with the marketing of a deeply suspect unapproved “test”) and because of some of their more way-out pronouncements over the last couple of years for which there is no proof and for which, Gob, i’m sure you will agree, do not belong in a scientific forum.”
    Obvious conflict of interest? WPI has told patients to wait for testing until standardized testing was developed and proven.

    You have made an egregious statement that WPI has made “way out pronouncements the last couple of years…”. This is quite a leap isn’t it? Can you prove this statement or are you willing to let stand a subjective argument for the exclusion of published research?

    Where I come from this is character and professional assassination.
    And you wonder why lay people can’t trust you. Don’t you agree?

  • Rob12

    MAN-MADE retroviruses spreading accross the world population, how who knows!!?
    So whats the plan bury it all?
    anyone else freaked out?? You…me…our kids ?? how many of us are carrying these Retroviruses?

  • Norwalk1972

    I was at CROI this week, and I was very encouraged by how quickly, efficiently, and expertly different scientific teams have addressed the XMRV issue. The “other side” of the debate was absent from the conference for good reasons: those people haven’t produced anything reproducible or convincing. Their results looked like contamination at the start, and the case is now all but closed. The true believers can’t even get their own samples to test the same way consistently. CROI should not be obliged to give equal time to people who question the existence of HIV and they should not give equal time to WPI just for the heck of it. I’m glad they didn’t.
    The next year of publications should put this fiasco where it belongs, in the past. Sadly the scam artists who are preying on desperate patients with untested cures and bogus test kits will still be around for a long time.

  • Anonymous

    Dr Racaniello, I am interested to hear your thoughts on the risk MRV’s pose to lab workers and medics.

    Currently we know that places like the CDC still cannot demonstrate diagnostic sensitivity or specificity for their assays. Consequently, when they report negative results, they mean little.

    Anyone handling potentially infected samples is therefore placing their lives in danger. I realise so many do not know of, or are unaware of the devastation that ME/CFS can cause, but at least everyone can understand the risk of cancer.

    Would you not agree that a reliable test, which has been shown to virtually guarantee detection of XMRV and other MRV’s has to be a priority from the HHS?

  • Anonymous

    Did you read my post below?

    Lombardi et al. was blinded. Lo et al. found PMRV.

  • Anonymous

    That humans don’t always do the right thing.

  • Anonymous

    Labs unable to detect MRV’s have not replicated the work of those who have. They are not following the scientific process. It is they who have not produced anything convincing. There is still no evidence of contamination. Singling out one group is misdirection. Lo, Alter, Klein, Silverman, Hanson, Cabrera, De Meirleir, Singh, Ruscetti, have all found MRV’s. Including PMRV and XMRV.

    This fiasco, and that of the CROI conference, will eventually be put to rest. Propaganda and weak science is unacceptable.

  • drosha

    another negative report was published recently: http://www.retrovirology.com/content/8/1/12

  • drosha

    Even HIV is handled in BSL2+ facilities, and retroviral vectors (which have no way of replicating in humans) are in BSL-2. These viruses are not very easily transmitted to begin with. I think they should be fine under conditions similar to retroviral vectors. No need to cause panic among people.

  • Anonymous

    I honestly don’t know how to respond to that. Do you fancy accidentally catching a retrovirus?

  • Anonymous

    New unproven assays (on a positive clinical sample), and old proven not to work assays. On a tired (non ME/CFS) cohort. They also used 22Rv1 – how funny under the circumstances.

  • Anonymous

    Why does it appear to researchers that XMRV is not present at the start?

    Xenografts are made by putting tissue into mice, and then you feed them with a high testesterone pellet. Even if PCR can’t detect XMRV in the cells at the start, the androgen will stimulate XMRV and its titre will increase to such a level that even their PCR assays could detect it. Therefore, they just could not detect it at the start.

  • Snail

    Not sure how i’ve engaged in character assassination. i think you will find Mikovitz in an interview easily found on youtube speculating that XMRV is the cause of everything from CFS to Gulf-war syndrome to “vaccine-induced autism” without pausing for breath – no evidence, just way-out publicity-seeking soundbites. She represents the scientific opinion of WPI.
    And need i remind anyone that Lombardi states his conflict of interest in the Science paper as heading up the development of a diagnostic test for XMRV – a test that is not FDA approved or reproducible by other groups using blood drawn from the same patients that were previously told they were “XMRV+”. Ethical patient care or opportunistic scam preying on sick people? i couldn’t possibly comment.

    In regard to what lay people should trust, i guess they need to realise that all retrovirologists at CROI presenting about the lack of evidence for XMRV as a human pathogen did not set out to trash it. They all wanted to find out if it was true. All of them (without exception) want to research retroviruses, particularly if they underlie human disease. There is no conspiracy on their behalf to rubbish this field – it’s ultimately shooting themselves in the foot because the truth will out. Therefore the fact that more and more of them are independently refuting the XMRV hypothesis is very telling.

  • Anonymous

    Here’s an interesting question that keeps popping up.

    The CDC detected XMRV in the Blood Working Group using samples from WPI pedigreed positive patients. They have now abandoned that assay. Why?

  • drosha

    Because you never worked with any of them, or know how they transmit, or understand the lab safety protocols.

  • Crafter Kate

    You have no idea how sick we feel, if you did you would probably understand why we think a retrovirus is connected to our disease.

  • Anonymous

    I’m think this need repeating.

    There is substatial scientific evidence that HGRVs are human pathogens and none whatsoever that they are not.

    The abstracts posted in CROI present conclusions as fact when they are no such thing. The DU145 cell lines were screened for the presence of XMRV by Dong et al in 2007, and no XMRV was present as provirus or cDNA. Therefore, if integrated sequences were found within the cells, then the direction of transfer was from the human prostate DNA, which contained those sequence, to the cells in question. Similarily, the apparent appearence of XMRV, after the production of xenografts can be explained by the fact that the PCR system used was not sensitive enough to detect XMRV in the original tissue. We certainly have enough examples of that. The testesterone pellets used in the process however would ensure rapid replication of XMRV to the levels which the PCR system used could detect it. The other point is that, in science, a study should never be designed to seek evidence in support of an investigators beliefs, but should be designed to actively disprove them. None of the reports produced at CROI qualify as scientific investigations.

  • Snail

    …or another type of virus. Don’t get me wrong, I believe that CFS/ME is associated with some infectious agent, perhaps even multiple agents. Serious research needs to be funded into this. However like some forms of diabetes and other autoimmune diseases which have been postulated to have an infectious etiology, the disease is likely to be multifactorial, and perhaps the agent itself will be fairly inocuous unless combined with various genetic and environmental factors.

  • Anonymous

    Is that why some lab workers are testing positive. Sorry, but you are burying your head in the sand. I understand exactly what can happen in a lab. To dismiss the very real concern is foolish. There is no evidence that MRV’s are a contaminant. It may well have been created in a lab though.

  • Anonymous

    It would be better to make sure that MRV’s be explored first. No point in missing a more likely cause.

  • Alan Dove

    The DU145 cell line didn’t contain XMRV initially, that’s true, but in the 2007 paper Dong et al. cloned the virus (from the original isolates that were discussed at the CROI meeting), then put it into DU145 cells. After that, they had a DU145 cell line with integrated XMRV. They probably also had a lot of XMRV cDNA floating around in the lab. Subsequently, the same lab performed a rather extreme PCR assay on 9 clinical samples and identified 14 apparent XMRV integration sites. Unfortunately, they only cloned one end of those integration sites, didn’t include any negative controls, and didn’t check the sites’ sequences against the DU145 line they’d previously created. I pointed out some of this in an earlier comment on this blog, but I tried to be diplomatic about it so maybe my point didn’t get across. The blunt version is that the XMRV integration paper had some serious flaws.

    Towers did the analysis the reviewers of the previous paper probably should have asked for, and found that two of the apparent prostate integration sites are identical to a sequenced integration site in the DU145 line the lab previously infected. The DU145 cells had the virus first, the prostate cancer cells second, and at least two of the prostate samples got their XMRV from the DU145 cells. No other interpretation makes any sense. It’s still formally possible that the other 7 patients had real XMRV infections, but when almost a quarter of your samples from the same experiment were unambiguously contaminated, the smart money bets against the whole set.

    The reports at CROI were designed to actively disprove a hypothesis, and while I’ll withhold final judgement until the peer-reviewed papers come out, it looks like they probably did. I’m particularly keen to see the paper from Kearney, who was apparently able to detect XMRV in infected macaque blood (i.e. she can see it when it’s there) but unable to find it in any clinical specimens, including two that Lombardi et al. had reported as positive.

    Finally, Gob987, please stop reflexively dismissing every study that disagrees with your preferred outcome. It’s perfectly appropriate to level substantive critiques at people’s techniques or experimental designs, but saying that their research should be ignored makes you sound like a propagandist, not a truth-seeker.

  • RRM

    When I read the Towers paper I remembered that comment you made. Credit were credit’s due: you called it.

  • RRM

    Do you have any reliable evidence that they “abandoned” that assay? Also, those samples were processed/handled by WPI, suggesting contamination as the most likely explanation for the positive CDC results. When the blood from the same patients was distributed independently (and blindly), the WPI did not find anything, while the WPI’s initial results also failed to hold up.

    The only lab that has seemed to abandoned its successful assays is WPI’s lab, as Mikovits just doesn’t use “simple PCR” anymore to detect XMRV, while it should be pretty succesful, looking at the Lombardi et al. paper

  • RRM

    This is bizarre reasoning.

    Your position on XMRV relies on XMRV not being very easy to find, to put it mildly. And not only for the “negative” labs, but also for the “positive” labs. See Mikovits’ and Ruscetti’s results of Phase IIb of the BWG, for instance. So, even if the virus is really out there, it is very difficult to find/at the level of detection/goes dormant, et cetera.

    But when a lab screens a cell line for XMRV and doesn’t find any, you take this as conclusive evidence that it wasn’t infected with XMRV.

  • RRM

    I take this as a “no, I don’t have any evidence for the assertion that very good HIV papers were not being publiehed due to a hostile scientific environment”. : )

  • RRM

    I am sorry, I meant to type:

    “When the blood from the same patients was distributed independently (and blindly), the CDC did not find anything, while the WPI’s initial results also failed to hold up.”

    …in the post above.

  • Gob

    I’m sure you are not suggesting that people should make no reply if they wish to respond to a study. This is the scientific processes after all, and the reason for the blog comments is to discuss the content. Obviously no where did I state that anything should be ignored.

    The conclusions of the studies are not fact though, they are a hypotheses.

    The titres in the infected monkeys would be massively higher than in human PMBC’s, with a natural infection pattern. That is artificial inoculation versus natural infection, and can be used to determine tissue tropism. Thus the observation is not scientific.

    The question is, did Kearney use the Lombardi assay to see if she could locate XMRV, which would be adhering to the scientific method?

  • Gob

    There are so many variables that can explain this. It’s too early to say, but interesting to note that the CDC did detect MRV’s. As did the WPI and NCI in Phase II. The latest study from the CDC, didn’t use the same assay. They also detected no contamination.

    In the BWG, positive results were obtained by the CDC on plasma with nested gag PCR, qRT-PCR (pro) and qRT-PCR (int). Their latest paper used the same assay as that shown by Danielson et al to be incapable of detecting pol or gag in those known to be infected.

  • drosha

    Do you expect them to put XMRV to the same category as Ebola when there is no evidence that this virus causes any disease? and even in BSL-4 facilities there are accidents. If you’re working with an infectious agent, you know the risks involved. For your cancer “concern” XMRV doesn’t carry an oncogene, so the only way it can cause cancer is by insertional mutagenesis (or by landing next to an oncogene in the genome). Retroviral vectors have the same (and probably more likely than XMRV bacause they have stronger promoters) potential. Some of the retroviral vectors I use can get into every single type of cell in the body. No need to work with them under more than BSL-2 conditions.

  • Gob

    Incorrect. It is imperative that the scientific method be employed. Multiple variables can alter results, including sample preparation and processing.

    Thus the hypothesis presented at this time for contamination is weak. Dong et al used an assay proven to detect XMRV in a known positive.

  • gob

    I didn’t state that, I was referring to your comment on there being a hostile environment. There was with HIV.

    Where is your evidence that any paper has been specifically rejected?

  • Gob

    You introduced Ebola into the discussion just now.

    I think everyone can agree that it is unknown whether this virus causes disease at this time, but again something I wasn’t discussing. Not sure why you believe that I would be unaware that XMRV does not carry an oncogene. Still, cancer and neuro-immune diseases are a real concern. It’s very unhelpful to suggest, and I realise you haven’t here, that this is not the case.

    Now, if you re-read my earlier post, the risk is not only to lab technicians. Medics may very well be also at risk of infecting themselves with this retrovirus.

  • RRM

    What exactly would be “incorrect”?

    I know this “this isn’t the scientific method” thing is a meme online, but the scientific method is being employed here.

    This isn’t a criminal trial. In science, I would define evidence as data that supports or rejects a given hypothesis. As such there are multiple, independent lines of evidence that support the hypothesis that XMRV is not a human pathogen as described by (e.g.) Lombardi et al.