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New Paper - Gammaretroviruses - Maureen Hanson, David Bell

Discussion in 'XMRV Research and Replication Studies' started by VillageLife, May 21, 2012.

  1. oceanblue

    oceanblue Senior Member

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    I believe so, if you don't declare it in the paper - that's how science is supposed to work. Being clear and transparent about methodology includes saying how you count mixed results, as Hanson did in her paper.
    Yes, the results are similar, which is why this paper is so interesting. Though I don't know enough about the detail to understand how these results stand up against the other negative studies.

    Agreed - another reason why this paper is so intriguing.
    If JM is unable to separate controls from patients using methods of her choosing, I think it's game over. If, however, the results are more mixed, with more patients than controls detected and sequencing confirming detection of MLVs (rather than a single contaminant eg Silverman) then it would be very interesting indeed.
  2. Bob

    Bob

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    What I meant is that I thought she did declare it in the paper. But I can't remember.
  3. oceanblue

    oceanblue Senior Member

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    Oh, I see! I'm pretty sure it wasn't in the paper (doubt it would have passed review if it had been) but am too lazy to check.
  4. currer

    currer Senior Member

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    This is an interesting analysis, Bob, and I would love to know whether it is correct that so many "positives" can be found.
    Is there any way of asking the authors of this paper whether this interpretation is valid?
    Can we contact them?
  5. VillageLife

    VillageLife Senior Member

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    In case you missed it on the other thread....

    Novel MLV-GAG sequences detected in blood samples of ME/CFS patients

    http://okeefe-lab.blogspot.co.uk/2012/05/novel-mlv-gag-sequences-detected-in.html#comment-form

    ----------------------

    Also Dr O'Keefe has found a new MLV virus in BPH:

    http://www.freshpatents.com/-dt20120503ptan20120107338.php

    ----------------

    Also this from Dr O'Keefe's today:

    Denise S. O'Keefe, PhD.May 24, 2012 7:13 AM
    it is interesting that everyone, us included, seems to have much more trouble amplifying the env and LTR regions - if gag amplification was merely due to mouse contamination, why don't these regions amplify as well? Keeping in mind that tests for mouse DNA are coming up negative...this might suggest that there is more divergence from known MLV-type sequences in these regions - we managed to sequence the variable region of env from one patient -- the sequence was most similar to env from an MLV virus known to cause disease in mice in the wild -- which is pretty interesting if you think about it...
  6. Bob

    Bob

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    Me too! :sleep: (I can't find a 'lazy' smiley, so a sleepy one will have to do.)
  7. Bob

    Bob

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    Hi currer,

    It's usually possible to contact a researcher.

    The paper says that Maureen R. Hanson works for the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.

    So it shouldn't be too difficult to find some contact details.
  8. Bob

    Bob

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    Thanks VillageLife. More very interesting thoughts and info from Dr O'Keefe.

    I wonder why it is so interesting that the env was most similar to an MLV known to cause disease in mice in the wild?
    Because it suggests a wild infection, rather than a lab contaminant?
    Or because it suggests a recombination of VP-62 with a wild virus?
  9. currer

    currer Senior Member

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    O'Keefe says her BPH virus is only related to XMRV

    "The BPH viruses disclosed herein are related to previously identified gammaretroviruses such as murine leukemia virus and xenotropic murine leukemia virus-related virus (XMRV), but are distinct from these viruses based on nucleic acid and amino acid sequences"

    So it doesn't sound like VP62 with another env.
    My guess would be that the env O'Keefe found suggests that the BPH virus has been around for a long time and originally came from wild mice.

    Benign Prostatic Hyperplasia is a chronic condition almost all men get. It is not a new disease.
    Think how closely we have lived with mice over the centuries.
  10. currer

    currer Senior Member

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  11. Cort

    Cort Phoenix Rising Founder

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    ??????

    I guess you're talking prostrate cancer. I think only the Hanson and Lo/Alter studies found more MLV's in CFS patients; Lo/Alter revoked their findings...I havent read the latest Hanson study but at her presentation at the Ottawa she warned about contamination in her study...

    Huber found more XMRV in the controls than CFS patients; she didn't find any XMRV in the CFS patients - it was controls - that was what sparked her search for a contaminant. In the Blood Safety group Mikovits consistently found more XMRV in the controls and so did her mentor Ruscetti....In fact they found XMRV in different samples from each other and in different samples using different runs.

    I think a more pertinent way to look at this subject is look at the multitude of studies from highly respected labs that found neither XMRV in CFS patients or controls using techniques that were more far more rigorous than employed in the original study. I haven't looked at the XMRV studies in quite a while but the number of negative studies was over 40 as I remember in all sorts of disorders. Not only was XMRV never found in the ill patients it was never found even in background levels. That speaks volumes to me....

    If you remember the Singh study,. XMRV also appeared and disappeared; It took her six months to track the contamination but she finally did.
    Ecoclimber and barbc56 like this.
  12. Mark

    Mark Acting CEO

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    No need to guess: I explicitly stated ME/CFS and prostate cancer in the quote you cited.

    In ME/CFS patients, I think the following found more MLVs in patients than controls:
    - Lombardi et al
    - Lo/Alter
    - Hanson's latest study
    - A previous small-scale study (also by Hanson, I think?)
    - Singh's original results (later found to be contamination, but originally higher levels in patients than controls I think)

    In other studies:
    - A German study into immune-compromised patients
    - Silverman's original prostate cancer study
    - some other prostate cancer studies (not sure how many, I think there were at least 3 PC studies in total)

    I'm glad you've provoked me to list them, because that's more like 9 or 10...and there may be more I've forgotten.

    I haven't seen that, but it doesn't affect the argument. They still found more in patients than in controls. The statistical argument is not affected by anybody's withdrawal of the results.

    That was months ago, the study was only just published. I suggest you read the study, or even just the abstract: the conclusion does not say that the results were contamination and makes it clear that they consider the question still open.

    Esther has cited that, and I believe that's correct though I haven't confirmed it. I don't recall the details, my vague recollection is that she found evidence of contamination right from the start, and found higher levels of contamination than anybody else has observed, and the nature of the contamination was a known problem that Lombardi et al had controlled for...but still, I think you're right that she did initially find more in controls.

    So we have one example where it was the other way round. That brings down the odds of this happening by chance to the same as flipping a coin 10 times and once it's tails. Still unlikely...I won't calculate it right now...

    That particular test is rather a different matter, and yes it was a big fail. But I think it's a misrepresentation to say they found more in controls than in patients. I don't know what the percentages and sample sizes were in that case, but they were very small, and the difference was small, pretty much the same I thought, and not enough to even reach statistical significance I think. So that doesn't count towards the point I'm making, which is that out of those studies that found a statistically significant difference between patients and controls, something like 9 out of 10 found more in patients than in controls.

    I know you find that compelling, but it's certainly not relevant to the point I'm making that lots and lots of studies found nothing at all. Singh did an excellent job in her paper of listing all the problems with the methodology of those negative studies:
    http://forums.phoenixrising.me/index.php?threads/cleaning-up-after-xmrv.17517/page-2#post-267193

    Specifically, she re-iterated comments made by members here: in particular, they generally only searched using PCR assays that rely on conservation of viral sequence, the limits of detection, reproducibility and precision of their assays were unknown, they didn't include enough negative controls, and none of them included positive samples from Lombardi et al. Most of them were looking specifically for the VP62 contaminant XMRV, so most of those results don't speak to the wider question of MLVs at all.

    But regardless of that, no number of negative studies affects my point that of those studies that found a statistically significant difference, nearly all of them found more in patients than controls.

    Hanson and Bell's paper lays this out quite clearly: they found more in patients than in controls on their first run, but then couldn't find any on subsequent runs using different techniques. They couldn't explain their first results as contamination.

    O'Keefe's findings seem particularly relevant: she found that after a successful PCR run, subsequent runs were becoming contaminated in such a way that detection of MLVs was actually being inhibited by artefacts left over from the first positive run. This is a new discovery of previously unknown problems with PCR which can give false negatives.

    Sure, and that was one of the examples I was noting. I noted that of these anomalous results, many of which have now been withdrawn or have been concluded to be caused by contamination, nearly all of them (Huber being the sole known exception so far) found more MLVs in patients than in controls. The point is that if the explanation of this is batch contamination, and nothing systematically different about the patient samples, then one would expect that there would also be lots of cases where more was found in controls than in patients. That may perhaps have happened, but it hasn't been reported as far as I'm aware.
    penny, jace and RustyJ like this.
  13. RRM

    RRM

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    The previous "small-scale study" is in fact the very same study as this published Hanson study. Although the original sample size was originally deemed to be too small for publication (and the study was therefore initially regarded as some sort of pilot study), instead of doing a new, larger study, a couple of samples were added to the existing batch.

    And you thought wrong about Singh. She found "it" at the same levels in controls, as she did in patients. The relevant quote from the Singh paper: "...we found approximately 5% of our samples to be positive for products of the expected size, regardless of whether they were patients or healthy volunteers".

    Singh was originally hopeful because she had found some positives and had not yet unblinded her results, which could explain why you would think this. However, in no way has Singh ever reported that she found the virus at a higher rate in ME/CFS patients.



    Again, it is the very same study. It's how these things work - you present these things in conferences before you publish and you explain what you think happened, in a less formal way as you would phrase it in a paper.

    Although you (or anyone else) is certainly free to give your own interpretation of this paper, my guess is that it really adds nothing to the idea that these viruses are "out there", not in the least because this was already known data and, although previously unpublished, had already been taken into account by most people in the field. Lo et al. even mentions this study/data in their retraction notice.

    See this quote from Oakes et al:

    And while not exactly the same, in this context the (early) Groom et al. study is also worth mentioning. Using a non-PCR (neutralisation assay), they found 0/142 of patients and 22/157 (14%) of healthy blood donors to be positive for XMRV (or, due to cross-reactivity, another (retro)virus).

    Purely statistically, the chances of a coin flipping like that would be 10/1024. Note that, technically, the "right" question to ask is what the chances are the coin flips at least 9 times heads, which would be 11/1024. Regardless, it amounts to around 1%.

    However, there are two reasons why this doesn't really apply:

    1. The "samples getting handled more often" argument. You seem to dismiss it earlier without really arguing why (which is curious as it seems to give a satisfactory explanation to what is apparently your most serious problem with the results thus far).

    To give you the example of the Lo et al. study (which I chose because the timeline of sample collecting is pretty well documented) :

    - Lo collected these samples in the mid-nineties for another study. They were handled for that study (tubes were opened, needles went into the samples, etc.). After the study, the samples went into the freezer for 15 years. Perhaps they were moved in and out of the freezer multiple times, perhaps they were stored next to mouse DNA, perhaps they were used for some other experiments - who knows what exactly happened?

    - After reading the Lombardi et al. study, Lo remembers the ME/CFS samples being in his freezer. He calls up Harvey Alter and says: "Harvey, can you send me some fresh control samples to mix up with the other samples?"

    I think it is entirely conceivable that Lo has(/had) some sort of trace contamination in his lab going on (either through reagents or other means) and that, therefore, the samples that were in his lab longer will be contaminated at a higher rate than samples that were present in his contaminated lab for a shorter period of time.

    2. Confirmation bias.

    The results of the BWG were indeed disastrous. Not only in phase III (the Simmons et all study), but (pilot) phase IIb was also pretty bad (although performed on a rather small sample size of 4 patients and one control).

    Mikovits should have no problem whatsover to discriminate between patients and controls. Not only did she reliably do this in the original study,she also did this for the (unpublished but reported at conferences) UK study.Moreover, for the Lombardi et al 2010 study (the cytokine one) they sampled 118 patient samples for XMRV (and it is important to note that they were screened as ME/CFS patients beforehand, not as XMRV-positive) and they found all 118 of them (100%) to be positive for XMRV. Given the supposed problems of detecting XMRV even if it would be present in blood samples, this result is statistically realistically impossible to accomplish. Although samples were supposedly blinded in the original study, blinding does leave room for confirmation bias (for instance by disregarding some test results), not to mention that adding certain substances (e.g. 5-AZA) to your patient samples but not to control samples before you run a test, will not be "saved" by all the blinding in the world.

    Also, in the Lo et al. study, the samples were defintely not blinded, leaving much more room for confirmation bias. This is painfully obvious from the 8 resampled patients, where 7 of them were found to be positive without the use of any controls (!). However, when blindly testing for the BWG (all of the 5 "Lo" BWG samples collected were from these group of 8 patients), Lo could not designate a single one of them as positive.
    Ecoclimber, oceanblue and barbc56 like this.
  14. barbc56

    barbc56 Senior Member

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    Thanks, RRM for putting that in a logical, easy to understand manner. I took a lot of stastical courses in graduate school and I think there are some days that I can only remember mean, median and mode.!!:rolleyes:

    Barb C.:>)
  15. Bob

    Bob

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    According to my records Dr Singh found XMRV in prostate cancer patients at 23%, and in the normal healthy population at 4% (or 6%?). Why do we think differently about this?

    The Hanson paper is explicit that no contamination was found. If Hanson suspects contamination, then she wasn't able to determine either what the contamination was, or its source. Furthermore, my understanding is that the contamination happened to be P-MLV-like virus sequences, and not mouse DNA.

    Yes, and you are equally free to interpret the findings, as you have done here.

    I don't think that Mark was really interpreting the results of the Hanson study. He was asking the question "why?"
    Some of the sequences that have been found, in various studies including the Hanson study, have not been explained.
    So it's not interpreting any results to ask what they are and where they come from, and why they are still being found in CFS patient samples, more than controls.
    I think some of your explanations are plausible, but I don't find them fully convincing simply because the sequences are not explained by mouse contamination, as far as I understand.
    If you can explain exactly what the sequences are, and what the source is, then I'd be interested in your explanation. You did manage to convince me that Switzers' few sequences could have been mouse contamination.
    RL_sparky and jace like this.
  16. Bob

    Bob

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    People keep saying this.
    Is it in relation to her prostate cancer study?
    If it is then I don't have any memory of her annoucement.
    Has anyone got a link or a quote for this at all?
  17. RRM

    RRM

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    Bob,

    If you read back Mark's original post about Singh's result, you'll see that he was mentioning it under the header of ME/CFS patients. In ME/CFS patients, Singh never found XMRV (or variants) at a higher rate than in control samples.


    And why "do we think differently about this?" Singh's prostate cancer study was earlier than the ME/CFS one, before investigators were fully aware of the dangers of the possibility of trace contamination. Therefore, for her ME/CFS study, Singh handled samples from patients and controls in the exact same manner from start (collection) to finish.

    As Coffin/Cingöz pointed out in their 2011 review paper with a reference (number 33) to the Singh Me/CFS study, the following is really essential:

    The original Singh PC study did not report doing this.

    In fact, none of the studies that reported doing what Coffin/Cingöz suggested have detected any asociation of XMRV (or variants) with any human disease.
    Ecoclimber likes this.
  18. Bob

    Bob

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    OK, thanku. We all know that her CFS/ME study was a zero/zero study, so I assumed that Mark was referring to the prostate cancer study. Maybe he was in his mind. :)

    Ah, OK, we are talking about different studies.

    OK, thanks for the info RRM.
    That is a very important issue about controls being treated in exactly the same way.

    But I would still like to know where all of the unknown sequences originate from.
    i.e. I'd like to know what exactly is the original source of the sequences.
    This question led to the important finding of XMRV 22rv1 in the cell line.
    So, searching for these new sequences might lead to further important discoveries.
  19. Cort

    Cort Phoenix Rising Founder

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    An update - I looked at XMRV studies since Feb....

    Three studies demonstrating how susceptible laboratory cultures are to XMRV contamination. Another finding demonstrating that the XMRV found in the WPI's studies came the mice used to culture the prostate cancer cell line and couple more studies unable to find XMRV in prostate cancer patients and two more studies unable to XMRV in immunocompromised patients (who tend to pick up all sorts of pathogens)..


    1.
    Susceptibility of Human Lymphoid Tissue Cultured ex vivo to Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Infection.
    Curriu M, Carrillo J, Massanella M, Garcia E, Cunyat F, Peña R, Wienberg P, Carrato C, Areal J, Bofill M, Clotet B, Blanco J, Cabrera C.
    PLoS One. 2012;7(5):e37415. Epub 2012 May 16.

    PMID:

    22616002

    [PubMed - in process]
    Free Article

    Related citations
    2.
    No Biological Evidence of XMRV in Blood or Prostatic Fluid from Prostate Cancer Patients.
    Mendoza R, Silverman RH, Klein EA, Miller AD.
    PLoS One. 2012;7(5):e36073. Epub 2012 May 16.

    PMID:

    22615749

    [PubMed - in process]
    Free Article

    Related citations
    3.
    Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1.
    Das Gupta J, Luk KC, Tang N, Gaughan C, Klein EA, Kandel ES, Hackett J Jr, Silverman RH.
    PLoS One. 2012;7(5):e36072. Epub 2012 May 16.

    PMID:

    22615748

    [PubMed - in process]
    Free Article

    Related citations
    4.
    Lack of evidence for a role of xenotropic murine leukemia virus-related virusin the pathogenesis of prostate cancer and/or chronic fatigue syndrome.
    Hong P, Li J.
    Virus Res. 2012 Apr 15. [Epub ahead of print]

    PMID:

    22531412

    [PubMed - as supplied by publisher]

    Related citations

    6.
    No evidence for infection of UK prostate cancer patients with XMRV, BK virus, Trichomonas vaginalis or human papilloma viruses.
    Groom HC, Warren AY, Neal DE, Bishop KN.
    PLoS One. 2012;7(3):e34221. Epub 2012 Mar 28.

    PMID:

    22470540

    [PubMed - in process]
    Free PMC Article

    Related citations
    7.
    Thirty years of research on infection and prostate cancer: No conclusive evidence for a link. A systematic review.
    Hrbacek J, Urban M, Hamsikova E, Tachezy R, Heracek J.
    Urol Oncol. 2012 Mar 27. [Epub ahead of print]

    PMID:

    22459691

    [PubMed - as supplied by publisher]

    Related citations
    8.
    Identification of XMRV infection-associated microRNAs in four cell types in culture.
    Mohan KV, Devadas K, Sainath Rao S, Hewlett I, Atreya C.
    PLoS One. 2012;7(3):e32853. Epub 2012 Mar 16.

    PMID:

    22438885

    [PubMed - in process]
    Free PMC Article

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    9.
  20. Cort

    Cort Phoenix Rising Founder

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    It wouldn't let me put the whole post in there...

    Multiple sources of contamination in samples from patients reported to have XMRVinfection.
    Kearney MF, Spindler J, Wiegand A, Shao W, Anderson EM, Maldarelli F, Ruscetti FW, Mellors JW, Hughes SH, Le Grice SF, Coffin JM.
    PLoS One. 2012;7(2):e30889. Epub 2012 Feb 20.
    PMID:

    22363509

    [PubMed - in process]
    Free PMC Article

    Related citations

    11.​
    No evidence of XMRV infection in immunocompromised patients and HIV-positive individuals from Germany.
    Korn K, Reil H, Ensser A, Knöll A.
    Infection. 2012 Apr;40(2):181-4. Epub 2012 Feb 21.
    PMID:

    22350961

    [PubMed - in process]

    Related citations
    12.​
    Absence of XMRV in peripheral blood mononuclear cells of ARV-treatment naïve HIV-1 infected and HIV-1/HCV coinfected individuals and blood donors.
    Gingaras C, Danielson BP, Vigil KJ, Vey E, Arduino RC, Kimata JT.
    PLoS One. 2012;7(2):e31398. Epub 2012 Feb 13.
    PMID:

    22348082

    [PubMed - in process]
    Free PMC Article

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