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Open Access Highly Accessed Correspondence

Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection

Jeremy A Garson, Paul Kellam and Greg J Towers*

Retrovirology 2011, 8:13  doi:10.1186/1742-4690-8-13

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XMRV is inserted into 472 sites in Prostate DNA

Gerwyn Morris   (2011-03-06 14:22)  PA institute email

In his reply to my letters of march 02 and 03 AG Miller referenced the study of Kim and others 2008. On close examination the details of the approach reported by Kim and others are revealing and challenge the conlusions of Garson et al and the accuracy of comments made by AG Miller in his most recent letter. Permission to reproduce the following sections has been granted by the authors.


"We sequenced a total of 508 authentic XMRV integration sites from DU145 cells, and 472 of these sites were mapped to unique locations in the human genome. Integration events were found in all 24 human chromosomes (22 autosomes and the sex chromosomes X and Y) (Fig. 1). The frequencies of integration of XMRV were generally proportional to chromosome size, but the overall frequency of XMRV integration into human chromosomes was different from that of uniformly random integration (P < 0.0001). Notably, chromosomes 1, 17, and 19 were significantly overrepresented (P values of 0.0015, 0.0021, and <0.0001, respectively), while chromosomes 5, 13, and X were significantly underrepresented (P = 0.0081, 0.0099, and 0.0002, respectively). Different integration frequencies among the different human chromosomes have also been observed for other retroviruses Additionally, using the criteria previously defined for integration hot spot which is three or more integrations within a 100-kbp region, we identified four integration hot spots for XMRV "(Table 1).

Thus contrary to the assertions made by Miller XMRV is capable of making three insertions within a 100 kilobase region as noted in the follwing table duplicated from the study;


TABLE 1. Integration hotspots of XMRV
Chromosomal region Integration site positions
7q36.1 151018217 151071783 151075248
8q11.21 49590648 49665683 49668524
19p13.2 7363696 7366399 7366635
20q11.22 33358459 33364040 33370664

The next point is that the integration sites within DU145 DNA were compared to those in DNA obtained directly from tumour tissue .While the regions were the same in 472 instances the precise integration points were all different. The reader is invited to consult figure 1 in the study for a more detailed comparison

"FIG. 1. Positions of XMRV integration sites in the human genome. The human chromosomes are shown numbered. Centromere locations are denoted by chromosomal indentations. Sites of XMRV integration in DU145 cells are indicated as red vertical lines along the top, and XMRV integration sites in prostate cancer tissues are indicated as blue "lollipops" on the bottom. Within each chromosome, the top bar shows the relative densities of RefSeq genes, with higher gene-dense regions shown as a more intense cyan. The second bar shows the chromosome cytobands. The third bar shows the cancer breakpoints, and the frequencies of breakpoints in different chromosomal regions are denoted by different colors (see the key at the bottom right-hand corner). The green shading in the bottom bar denotes the locations of common fragile sites."

Thus if two of a possible 470 integrated XMRV sequences were found in DU145, cells which were identical in terms of nucleotide insertion points, then the experimental environment can be the only possible source.In short the DU145 cells were contaminated by human DNA during the experimental procedure.

The difference in the nucleotide integration pattern produced by the infectious clone VP62 compared to xmrv found inserted in the DNA of prostate tissue isolated directly from patients would also seem worthy of further investigation. Differences in genomic sequences could well explain why thus far PCR systems with cycling conditions adjusted to locate the VP62 clone in vitro have failed to find XMRV in vivo.

reference

Kim S, Kim N, Dong B, Boren D, Lee SA, et al. (2008) Integration site preference of xenotropic murine leukemia virus-related virus, a new human retrovirus associated with prostate cancer. J Virol 82: 9964–9977.

Competing interests

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.response rebutting Miller

Gerwyn Morris   (2011-03-05 22:43)  PA email

Dr Miller you are commenting on a different study to the one I raised

I am talking about a study by Dong et al 2007 which first detected xmrv in dna taken straight from prostate tissue where they first screened the DU 145 cells to ensure that they did not harbour XMRV.

If you are going to reply to my posts at least have the courtesy of reading them first

You talk of hypothetical situations as though they were fact

As you know the DU 145 cell line environment is one of high oxidative stress and elevated levels of NF-kappa B.you also know that high NF-kappa B instigates the transcription of XMRV and stimulates its replication. If the DUi25 cell line DNA contained integrated XMRV provirus then the cell line wouldexpress XMRV.

Mutations in the cellular and viral DNA would also be expected because of the age of the cell line and the level of oxidative stress involved.None were observed.The most parsimonious hypothesis available which explains all the observations is that human DNA containing integrated XMRV provirus was introduced into the DU125 cells in error.

You speculate about the possibility of contamination without having any empirical evidence whatsoever to base your comments on. It is traditional for scientists to comment on scientific evidence and not to engage in speculation.The abstracts presented at CROI ,for example ,all share the same generic theme.That theme is that XMRV could hypothetically be a contaminant if hypothetical conditions existed. I suggest that many retrovirologists could now benefit from consulting people who are more knowledgeable about study design and adherence to the scientific method Consulting retrovirologists with experience of discovering new human retroviruses would also seem to be adviseable

You saw fit to adopt a condesending attiude in your article and questioned my knowledge.I submit that insulting others has no part to play in anything published in a scientific journal

I suggest that it might benefit you if you spent more time examining the requirements of the scientific method and being knowledgeable enough to at least comment on the correct study.I found.

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Response to the comments by Gerwyn Morris posted 02 March and 03 March 2011

A Dusty Miller   (2011-03-04 15:55)  Fred Hutchinson Cancer Research Center email

You note that I have not replied to your previous comments. This is because of their voluminous nature, your inability to correctly interpret what is basically a sound article, and because I have other things to do!

In your post of 02 March, the main substantive point questions the proposed direction of contaminant transfer, from the XMRV-infected DU145 cells to the prostate cancer tissue. In the Kim et al. 2008 paper describing these studies, they infected DU145 cells with XMRV at a multiplicity of 0.1 and the cells were grown for 3 days. By this time, it is likely that all of the cells carried at least one integrated copy of XMRV, because XMRV is replication-competent and can readily spread in cultured DU145 prostate cancer cells. In contrast, the number of integrated copies of XMRV in fresh prostate cancer tissue is estimated to be low, on the order of 1 copy per 100 cells, if it is there at all. Therefore, contamination of the prostate tissue DNA with heavily-infected DU145 cell DNA is more likely to result in false positive results than the reverse. Furthermore, the real question here is whether XMRV can be found integrated into prostate cancer cells from a human, not whether the DU145 cells are infected. The finding of identical integration sites in these two samples suggests contamination of the prostate cancer samples with XMRV-infected DU145 DNA or PCR products amplified from this DNA, and threatens the conclusion that XMRV is found integrated into the DNA of prostate cancer tissue from humans.

In your post of 03 March, the main substantive point appears to question how DU145 cells could have been involved in the studies of XMRV integration sites in prostate cancer tissue. The answer is that the analysis of XMRV integration sites in XMRV-infected DU145 cells was performed at the same time and in the same lab as was the analysis of XMRV integration into DNA from human prostate cancer tissue, thus the possibility of cross contamination must be considered. Many of your other questions make no sense to me, and I don’t have the time to delve into your underlying meanings. I suggest you seek further clarification and answers to your questions from knowledgeable colleagues.

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Response to the comment by Abigail Smith posted 02 March 2011

A Dusty Miller   (2011-03-04 15:53)  Fred Hutchinson Cancer Research Center email

Thanks for the citation (Mitchell et al., PLoS Biology 2:E234, 2004). While these authors did not mention observing any duplicate integrations, they do state in Materials and Methods that only novel integration site sequences were deposited at the NCBI, implying that some sites were not novel and thus were duplicates. Duplicates would not be unexpected in a single experiment, because PCR amplification results in many copies of what might have initially been a unique site, and if one clones and sequences enough sites, duplicates are bound to be found. For example, if 1,000 cells were infected with 10 of their replication-defective HIV vector particles, resulting in 10 integrated proviruses, cloning of 100 integration sites from DNA isolated from these cells would result in detection of multiple identical (duplicate) integration sites. On the other hand, infection of a million cells with 100,000 vector particles followed by cloning of 100 integration sites would be very unlikely to identify identical sites.

Thus, to get at the question of whether retroviruses integrate at identical sites at much higher rates than would be expected by chance, and thus might explain the identical XMRV integration sites identified by Garson et al., one needs to sequence integration sites from at least two independent experiments, and compare integration sites between experiments for duplicate integrations. The data to make this comparison is obviously available, but I’m not aware of anyone having performed this analysis. If someone has done so, I’d appreciate being informed.

This discussion gets at the heart of the contamination issue raised by Garson et al. In the experiments they discuss, 14 XMRV integration sites in prostate cancer tissue are compared to ~500 XMRV integration sites found in acutely-infected DU145 human prostate cancer cells, and two identical (duplicate) integration sites are observed in these theoretically independent experiments. Given the number of possible integration sites in the human genome (~3 billion), cross contamination almost certainly explains the result. That is, unless there are some unusually hot spots for retrovirus integration, which likely would have been identified by now.

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the study cannot demonstrate direction of transfer

Gerwyn Morris   (2011-03-03 11:48)  PA institute email


The authors did not follow the scientific method and demonstrate that the DU125 cells did not contain integrated DNA before the start of the experiment. If the cell line was "contaminated" with human XMRV then the results would have been exactly the same

The other problem they have is that none of the studies that identified and isolated XMRV used the the DU145 cell line in any way whatsoever

The experiment that isolated integrated XMRV directly from the prostste tissue of infected patients also did not invole the use of DU145

The question of identical insertion sites is a probabalistic argument and purely determined by sample size and the affinity of a retrovirus for GPG islands

XMRV has been demonstrated a greater affinity for these sites than any known retrovirus.

DU145 cell lines began in 1978 yet Towers asserts that the origin of the XMRV detected in prostae cancer is the "XMRV" in the 22RV1 cell line which began in 1993

Schalberg et al(2009) reported that RT PCR only detected xmrv in samples taken from 6% of prostste cancer suffere while IHC showed 22% of paients to be positive

This is an interesting observation if XMRV is a Mouse contaminant!

I note Miller has not replied to any of the other points I raised


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Citation request by Dr. Miller--

Abigail Smith   (2011-03-02 23:05)  OUHSC

If you do not want a list of every integration site ever (heh), Mitchell et al might be suitable citation: "Retroviral DNA Integration: ASLV, HIV, and MLV Show Distinct Target Site Preferences"

They identified 3127 insertion sites of HIV-1, ASLV, and MLV and they did not mention observing any duplicates. As far as MLV goes, their analysis of Chromosome 11 showed, at max, 3 MLV insertion sites within the same 2 Mb region.

I do not know of any published duplicated insertion sites myself. It certainly strains credulity that a lab only sequenced a handful of integrations, and found not just one duplication, but two. And the duplications just happen to have identical LTRs as well.

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response to Miller

Gerwyn Morris   (2011-03-02 18:20)  PA institute

in response to the letter by Miller

Would he argue with the points below?

The authors state that the sequences were unlikely to have been transfered into the DU125 cells from the patients examined without nothing but their biases as evidence.

Likewise they voice their belief that the other integration sites are probably caused by contamination after failing to provide any evidence despite a rigorous effort to discover said evidence.

I submit that scientists who publish unfounded opinion and not fact is the main issue here

Miller talks of unacceptable phraseology.I am therefore somewhat surprised that he has not highlighted the extract below

"we suspect that some or all
of them may also be the result of contamination with DNA from experimentally infected DU145 cells. It is striking that there have been no independent reports of patient-derived XMRV integration sites nor have there been any descriptions of polytropic or modified polytropic MLV integration sites in human samples despite the apparent detection of these viruses in CFS patients [5]."

The authors are showing bias and are the use of the word "apparent" are denigrating the work of others

I would submit that this is an example of wording which has no place in any journal

On the other hand the authors appear to have no issues with the fact that their findings have not been supported or that they could not demonstate any evidence that the 12 integration sites were not genuine

Considering the evidence as a whole the results can easily be explained by the transfer of proviral DNA from patients into the cell lines.

Would Miller dispute that and why?

How would Miller explain the integration of XMRV into CREB5 and NFATc3?

The essence of the scientific method is hypothesis testing

Hypotheses are tested via challenge or an active attempt to disprove the proposed explanatory model

The authors have clearly not engaged with the scientific method in this study

I am surprised that Miller has not raised this fact

When the authors describe integration sites they appear to be discussing integration into the same genome or region of a genome rather than a single nucleotide as raised by Miller.In which case the statements made by the authors are erroneous as per my original references

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Response to the comment by Gerwyn Morris posted 26 February 2011

A Dusty Miller   (2011-03-02 04:15)  Fred Hutchinson Cancer Research Center, Seattle email

Morris' criticism of the article by Garson et al. is unwarranted, and stems from a misunderstanding of the difference between retrovirus integration near a gene versus integration at exactly the same site in the whole human genome. It is true that murine leukemia viruses (MLV) can often be found within or near oncogenes involved in the progression of tumors in MLV-infected mice. However, integration within 1,000 or 10,000 nucleotides of a gene is quite different from integration at exactly the same nucleotide position in a whole mammalian genome. None of the references provided by Morris show integration of a retrovirus at the same nucleotide position.

I do agree that it would be nice to have formal support for the statement that "With the exception of a single early publication on avian sarcoma-leukosis virus, which was refuted by later work [10], sequencing studies of thousands of retroviral integration sites have to our knowledge never identified exactly the same site twice." However, given our current understanding that retrovirus integration is semi random and can target most of the 3 billion base-pair-long human genome, the authors' contention seems quite reasonable.

Lastly, derogatory comments like "The authors are clearly not familiar with MLV viruses" have no place in these posts.

Competing interests

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is this the final straw to xmrv...

aidan walsh   (2011-02-28 11:49)  sick since june 1990 with (cfs)??? email

i propose that cfs and prostate cancer is related to 'unintentional chronic dehydration' and there may be no infection involved in the illness...also it could be very possible that these infections are involved... c.pneumonaie dr. charles stratton vanderbilt university...mycoplasmas drs. nancy and garth nicolson laguna beach california...ciguetera (epitope) toxins with now a link to low level radiation and their work has been replicated and they claim is a bio-marker for all 'auto-immune'disorders with links to myelodysplasia syndromes...also not to forget a neuro surgeon found 300 consecutive fibromyalgia patients with chiari malformation and/or stenosis of the spines in cfs patients as well and just last week from new jersey where spinal fluids in chronic lyme and cfs patients showed elevated proteins in their spinal fluids compared to normal controls...last, dr. garth nicolson also found 45% of the hiv virus envelope which was somethibg not naturaly occuring with the mycoplsmas involved...there is way to much research now to indicate that cfs is a physiological illness and not of one's mind that can be cured with get/cbt attitudes...it is also a shame that leading infectious disease scientists are denied further grant fundings when the phycology camps are never refused...it is a disgrace to all serious scientists attemping to unravel this devastating illness...god bless all of you who are trying to find these answers...sincerely aidan walsh southampton, u.k. www.watercure.com www.watercure2.org p.s. i strongly believe that cfs in the end will prove to be an 'undiagnosed' illness that was missed in all patients...god bless all who are sick...

Competing interests

chronic fatigue syndrome is a very serious chronic illness and does cause death in patients...also numerous children continue to suffer and'yes' they die as a result of this illness...the memorial list continues to grow and the neglect continues even at the nih/cdc...see the 'memorial list' at the national cfids foundation and the very serious research they are privately funding with 100% of patients donations where gail kansky is heading these projects...'fatigue' is only one of many symptoms patients suffer with each and every day...

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XMRV class viruses integrate into the same sites repeatedly

Gerwyn Morris   (2011-02-26 00:45)  PA institute


The authors are clearly not familiar with MLV viruses

MLV viruses have a preference for integrating within certain genes involved in the promotion or repression of tumours. A classic example would be PIM-1(1) or P53 (2) n-myc(3)is also a common integration site.
Thus mulv virus' display the propery of integrating into the same sites repeatedly.It is surprising that the authors have not researched into the subject. Hence the following section of the paper is completely incorrect

"With the exception of a single early publication on avian
sarcoma-leukosis virus, which was refuted by later work [10], sequencing studies of
thousands of retroviral integration sites have to our knowledge never identified exactly the
same site twice."

The following table demonstrates just how inaccurate the author's comments are. They illustrate the integration sites within genes of the class of virus to which xmrv belongs

BMC

The reader will note that the activity of these genes are all related to the development of cancer in humans

The authors are stating beliefs in the guise of fact.
They state that the sequences were unlikely to have been transfered into the DU125 cells from the patients examined without nothing but their biases as evidence.

Likewise they voice their belief that the other integration sites are probably caused by contamination despite failing to provide any evidence despite a rigorous effort to discover said evidence.

The integration of a mulv class virus into the PIM-1 gene leads to the overexpression of PIM-1(1).The degree of overexpression of PIM-1 correlates with severity of prostate cancer in humans(1)

I submit that patients with prostate cancer are entitled to anyone ,purporting to be scientists, investigating the link of XMRV to their condition limit themselves to publishing scientific evidence and not speculation in the guise of scientific evidence as is,sadly,the case here




references

(1)Wong KS, Li YJ, Howard J, Ben-David Y. Loss of p53 in F-MuLV 14induced-erythroleukemias accelerates the acquisition of mutational events that confers immortality and growth factor independence. Oncogene. 1999 Sep 30;18(40):5525-34.

2)Cuypers HT, Selten G, Quint W, Zijlstra M, Maandag ER, Boelens W, van Wezenbeek P, Melief C & Berns A. (1984) Cell 37: 141–150.

(3)van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H & Berns A. (1991) Cell 65: 737–752

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