XMRV and prostate cancer-a 'final' perspective

[Firestormm]

10 January 2012: Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/22231291

XMRV and prostate cancer-a 'final' perspective.

Sfanos KS, Aloia AL, De Marzo AM, Rein A.

Source: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Abstract

XMRV was first described in 2006, when it was identified in samples isolated from prostate cancer tissues.

However, studies have since shown that XMRV arose in the laboratory and was formed by genetic recombination between two viral genomes carried in the germline DNA of mice used during serial transplantation of the CWR22 prostate cancer xenograft.

These new findings strongly imply that XMRV does not circulate in humans, but is only present in the laboratory. Thus, there is no reason to believe that it has any role in the etiology of prostate cancer or other diseases.'

This is a paper published in Nature Reviews Urology: http://www.nature.com/nrurol/journal/vaop/ncurrent/full/nrurol.2011.225.html

Just reading through it now. Thanks Jemal for posting the link on another thread: http://forums.phoenixrising.me/showthread.php?15525-New-Paper-by-Dr-Ruscetti&p=232893#post232893

Several have been debating the relevance of prostate cancer and XMRV and I dare say there will be more considerations such as this opinion piece.

Edit:

'Introduction:

Several classes of viruses, including retroviruses, are known to cause cancer in humans and animals. The possibility that additional types of cancer could be caused by as yet unidentified viruses holds great appeal.

However, the attractiveness of this hypothesis has also led to cancer causation being mistakenly attributed to viruses.1,2 It has long been postulated that viruses could have a role in prostate cancer etiology, and reports of viruses in association with prostate cancer date back over 30 years.

Thus far, however, no virus has been causally linked to prostate cancer. The latest entry into this catalog is xenotropic murine leukemia virus-related virus (XMRV).

This Perspectives article will critically evaluate studies that seemed to identify XMRV in prostate cancer samples, address the known sources of false-positive results in XMRV assays, and describe and explain in depth the findings and the implications of a study that showed that XMRV was formed by a rare recombination event in the laboratory.

Thus, we will chart the rise and fall of XMRV from its initial detection in 2006, via claims of its role in prostate cancer, to the 2011 report of its recombinant origin.'

Cool....

Edit:

Link to MECFSForums thread:http://www.mecfsforums.com/index.php/topic,11109.0.html

I should say I suppose that this review includes those studies pertaining to an association of XMRV/MLVs in Chronic Fatigue Syndrome.

Am still reading but so far it is proving quite comprehensive.

 

[Firestormm]

To show the conclusion is not doing justice to the paper, but here it is anyway:

'Conclusions

XMRV arose in the laboratory as the result of a rare recombination event.

We believe that all of the reported PCR-based evidence of XMRV in clinical specimens can be explained by contamination of the assays with mouse DNA, XMRV from cultured cells (CWR22Rv1 cells or cells infected by XMRV from CWR22Rv1 cells), or DNA from infected cells or XMRV plasmids.

Positive IHC data probably reflect the lack of specificity of the antiserum used in these assays. There is no reason to believe that XMRV actively circulates in humans or is present in the environment outside the laboratory.'

 

[Ecoclimber]

I believe the jury is still out until both of Lipkin's research studies are completed but science was advance in regards to researching XMRV such as stated in Dr. O' Keefe's blog located here: http://okeefe-lab.blogspot.com/2011/11/so-lets-get-this-straight-cfs-patients.html?spref=tw. It made scientists look outside the box and perhaps a few will review those negative research papers based on O'Keefe research. It certainly made Dr. O' Keefe aware of new ideas and issues. But Miller did find some interesting aspects regarding the Xpr1 receptor that could be useful done the road. It did open the eyes of a few in the scientific community in its rather contentious debate concerning XMRV as mentioned in Racaniello's article in Discovery Magazine.

Eco

 

[Firestormm]

I think it also and perhaps most importantly raised the concern and awareness that XMRV/MLVs are contaminating - well - pretty much everything in labs.

I am very interested in learning what - if anything - can be done to ensure methods taken previously re: detection are improved as a result of all these research papers and the conclusions they have wrought.

If scientist do look again at their papers - including the 'positive' ones perhaps most especially - I would like to see them locate the source of the contamination.

The above paper from Sfanos talks about this although not to a great extent unfortunately. I will add later what she has to say about it - I found it very interesting and as a researcher I would imagine this long tale will prompt greater vigilance and awareness as I said.

I was on a forum or reading some blog the other day and someone was asking why they are still looking at XMRV. I raise it now because as Sfanos infers there is still a lot to learn from it even if it isn't a human pathogen.

It has been identified and the origin explained but there are good reasons to keep looking at it aren't there? From a scientific point of view. Quite fascinating really...

 

[alex3619]

Hi Firestormm, the disturbing thing is not just that MLLVs can easily contaminate labs and cultures, but that methods to detect such contamination routinely fail. In the case of the Lo et. al. and Lombardi et. al. papers only one small part of Lombardi et. al. was shown to be due to contamination. Other attempts to find it in these studies have failed. How is that to be interpreted?

This raises a second point. If these viruses are contaminating results, it may be difficult to find the a virus in a sample even if its there, as the contamination will invalidate the tests. Experimental design is very important in these kinds of studies, and I can see the cost of research in this area spiraling which will dampen and slow the rate of research. This is not good news, but it is important.

Bye, Alex

 

[Firestormm]

Kind of what I was saying Alex but you have said it far better I think. Yes. Exactly that - if detection was employed why did it fail? As I said Sfanos does talk about this - not directly - and of course contamination can possibly occur after detection is employed - indeed possibly I suppose - by the detectant itself...

 

[Firestormm]

One reason I kinda like these critical review papers is because they tend to summarise things in an easier to understand format - and of course one knows the source which is rather comforting when one encounters things said on-line etc. I digress:

'Box 1 | Murine leukemia viruses

Murine leukemia viruses (MLVs) are prototypical gammaretroviruses.65,66 Their overall structure and replication cycle are similar to those of other retroviruses, including HIV 1.

The virus particle is roughly spherical, ~100 nm in diameter, and bounded by a lipid bilayer. It is constructed from the virus-coded Gag proteins and also contains the viral enzymes protease, reverse transcriptase, and integrase (known together as Pol) as well as the Env proteins that mediate the entry of the particle into new host cells and two copies of the viral RNA genome.

Proteins from the virus-producing cell are also present within the particle. MLVs are among the simplest retroviruses, encoding only the three polyproteinsGag, Pol and Envthat will be assembled into progeny virus particles.

After the virus particle is released from the virus-producing cell, each of the polyproteins is cleaved by the viral protease. This maturation step is essential for infectivity.

When an MLV particle infects a new cell, its reverse transcriptase copies the viral RNA into double-stranded DNA. Integrase physically inserts this DNA copy into the chromosomal DNA of the cell, and it is then replicated as part of the cells genetic material and is transcribed and translated by normal cellular machinery. Productive infection is generally harmless for the cell.

MLVs are found in mice in the wild and have been studied extensively in the laboratory. They have been engineered to create vectors for gene therapy.

Both MLV in MLV-infected mice and the vectors administered to children can cause tumors by insertional mutagenesiswhile the chromosomal integration site of the viral DNA is nearly random, it is occasionally near a gene involved in regulation of cell growth.

It can then interrupt the gene or alter its expression. The resulting disruption of the signal-transduction pathways controlling cellular replication is a principal mechanism by which these viruses cause tumors.

Box 2 | Endogenous and xenotropic MLVs

Integration of the viral DNA into the chromosomal DNA of the host cell is an essential step in the retroviral replication cycle. This DNA (the provirus) is then copied along with the rest of the cells DNA and is faithfully transmitted to the daughter cells at cell division.

Infection sometimes occurs in cells of the germline of the infected organism, so that the provirus is also transmitted to the organisms offspring. Such a provirus is referred to as an endogenous virus. The genomes of laboratory mice contain over 100 endogenous MLV genomes67

One of the factors that determines which cells can be infected by a given retrovirus is the interaction of the viral Env protein, which mediates entry into the cell via a receptor on the cell surface.

MLV Env proteins are polymorphic and different MLV isolates use different cellular receptors. Some endogenous MLVs give rise to infectious MLVs with a surprising property: although these particles can infect cells of other species, they cannot infect mouse cells.

These endogenous MLVs are termed xenotropic MLVs. Evidently, these viruses were able to utilize the cellular receptor when they infected mouse germline cells many generations ago. However, in the intervening years, natural selection has altered the receptor, rendering contemporary mice resistant to these viruses.

The majority of endogenous MLV genomes are classified into groups called polytropic and modified polytropic. To our knowledge, polytropic and modified polytropic endogenous MLV genomes do not give rise to infectious MLV.'

 

[Firestormm]

Quite fascinating how the story of XMRV and prostate cancer is so similar to XMRV and CFS but I figured we should stick to the part that seeks to explain the contradictions seen in both PS and CFS studies i.e. why perhaps some studies were positive and others not:

By 2010, the XMRV literature was filled with stark, inescapable contradictions and concerns were being raised regarding false-positive results in assays for XMRV caused by laboratory-based contamination.

Detection of XMRV and related viruses in clinical samples often relied on PCR for detection of viral nucleic acids; however, mice contain endogenous viral sequences that can be amplified with XMRV-specific primers in PCR-based assays.40

Thus, PCR assays for XMRV are particularly susceptible to false-positive results owing to the presence of the MLV genomes in mouse DNA and the extraordinary sensitivity of the assays. Every mouse cell contains at least 100 MLV genomes.

Therefore, a millionth of a microliter of mouse blood contains sequences that can potentially be amplified in XMRV-specific PCR. As mice are ubiquitous in biomedical research, it can be very difficult to obtain clinical samples without trace amounts of murine DNA.

An important step that helped to clarify the role of contaminating mouse DNA in XMRV assays was the development of PCR assays for murine sequences, including mitochondrial cytochrome oxidase and intracisternal A particle (IAP) sequences.41

As there are approximately 1,000 copies of the IAP retrotransposon in the mouse genome, testing for IAP sequences is an extremely sensitive way of detecting the presence of mouse DNA. When these assays were applied to a series of clinical samples, every sample that had been scored as XMRV-positive was also found to contain IAP sequences.41,42

A number of commercial reagents used in assays for XMRV (such as Taq polymerases, PCR master mixes, RT PCR kits and extraction columns) have also been found to contain trace amounts of murine nucleic acids and might, therefore, explain positive results.4345

In some studies, XMRV is detected more frequently in samples from patients with a disease than control subjects. This apparent association of the virus with disease is, at first glance, difficult to reconcile with the idea that the positive results represent contamination.

However, it must be considered that the disease samples might be collected or handled differently, or at different times, compared with the controls.

As suggested by Weiss,2 it is also possible that disease samples might simply be handled more often than job-lot control samples, increasing the opportunities for potential contamination to occur.

Given the extraordinary sensitivity of PCR, which can detect single molecules of template, it seems likely that it could even detect airborne nucleic acid molecules in the laboratory.

A very important development in reconciling the contradictions between different laboratories has emerged from a joint study organized as the Blood XMRV Scientific Research Working Group (SRWG).46

This group sent blinded samples (positive control samples spiked with XMRV, negative control samples with no XMRV, and clinical samples from patients previously reported to be positive for XMRV) to nine different laboratories for testing by nested PCR, virus culture and serology.

Two laboratories that had collaborated in the original report on XMRV in CFS samples36 were the only laboratories to report detection of XMRV in clinical samples.

However, these two laboratories also detected XMRV in negative control samples that did not contain the virus, providing direct, unequivocal evidence that the assays used in these laboratories suffer from some artifacts producing positive results in the absence of XMRV.46

Of particular note, in the SRWG study, the Lo et al.39 laboratory found no XMRV in any of five of their previously reported positive samples or in 10 samples previously reported as positive by Lombardi et al.,36 despite their ability to detect XMRV in all five of the spiked positive controls.46

IHC has also been used to detect XMRV in clinical samples. Surprisingly, this method seemed more sensitive than PCR in the work of Schlaberg et al.8

One concern regarding this work is the way in which the anti-XMRV antiserum was generated whole viral particles produced in human cells were used as the immunogen.

However, retrovirus particles contain cellular proteins in addition to viral proteins.47,48 Thus, this immunization is likely to have generated antibodies against human proteins, as well as against the viral proteins.

Stieler et al.22 have reported that the antiserum used by Schlaberg can recognize cellular proteins in non-infected human and mouse cell lines. Furthermore, Sakuma et al.16 compared the staining by an anti-MLV p30/gp70 antibody that can detect XMRV precursor Gag, CA, and Env proteins to that by the anti-XMRV antiserum used by Schlaberg and colleagues8 in IHC assays on prostate cancer tissues.

Both antibodies reproducibly stained 293T cells transfected with an XMRV clone in positive control assays. However, only the anti-XMRV antiserum used by Schlaberg et al.8 stained areas of prostate tumor epithelium, whereas the anti-MLV p30/gp70 antibody did not show positive staining in any of the tissues.

Sakuma et al.16 concluded that we cannot detect XMRV in prostate cancer tissues and that the antibody described by Schlaberg, Singh and colleagues recognizes non-viral proteins in addition to XMRV.16

In our own studies, we received prostate cancer tissue sections (kindly provided by Dr Ila Singh, University of Utah) from a number of the same patients tested by Schlaberg et al.8 The samples were predicted to be XMRV-positive based on the previous IHC results with the anti-XMRV antiserum.8

However, the sections did not stain with two different broadly reactive MLV antisera that had previously detected XMRV in positive control assays.18 Taken together, the evidence suggests that it is highly unlikely that the IHC staining observed by Schlaberg et al.8 represents the true presence of XMRV.

Finally, one surprising feature of all XMRV sequences reported in clinical samples is their uniformity. Five sequences purportedly isolated from patient samples were found to be, on average, >99.9% identical to each other at the nucleotide level.49

This near-identity of XMRV sequences is difficult to reconcile with the error-prone nature of retroviral replication. In fact, different HIV 1 genomes isolated from a single infected individual show far more divergence than is seen in the entire set of XMRV sequences published to date.

However, it should be noted that the sequence diversity of different isolates of human T cell leukemia virus type 1 (HTLV) (a member of the deltaretrovirus genus, distinct from both MLVs and lentiretroviruses such as HIV 1) is also far lower than that of HIV 1, although not as low as that in XMRV.50

As sequence diversity is generated during the viral replication cycle, it is likely that there is much less ongoing viral replication in an HTLV-infected individual than in an individual infected with HIV 1.51 The level of replication in an MLV-infected, viremic mouse is not known.'

 

[Firestormm]

Silly pondering:

How did they jump from MLVs known to cause cancer in mice and in children through gene therapy to XMRV (an MLV) as a possible cause of CFS? Or was it simply a case of believing they discovered XMRV in CFS patients and non-patient blood and XMRV as a retrovirus must do something bad?

I mean was it not a stretch to begin with (I know we never really proceeded to causation did we but how did the notion arise in the first place?)

Or is this where the incidence of cancer in CFS patients (and presumably non-patients) came in? Because there is no evidence that XMRV or MLVs cause anything other than cancer is there?

 

[currer]

In mice MLVs cause neurological disease, ( the env protein is neurotoxic,) immunological disorders and cancer (leukaemias.)

Mice are mammals like us and are used as a model for disease in humans.

"Resistance of Mice Deficient in IL-4 to Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)
Osami Kanagawa, Barbara A. Vaupel, Shinyo Gayama, Georges Koehler and Manfred Kopf
Science
New Series, Vol. 262, No. 5131 (Oct. 8, 1993), pp. 240-242"

Abstract
"The murine acquired immunodeficiency syndrome (MAIDS) is induced by a defective murine leukemia virus and has many symptoms similar to those found in patients infected with the human immunodeficiency virus. The presence of both B cells and CD4$^+$ T cells is critical for the development of the disease. Furthermore, a Th2 cytokine response dominates during the progression of the disease. When interleukin-4 (IL-4)-deficient mice that are defective in Th2 cytokine responses were infected, there was no lethality, and the development of the T cell abnormalities associated with MAIDS was delayed. These data suggest that IL-4 or a Th2 response is involved in the development of retrovirus-induced immunodeficiency in mice."

 

[Firestormm]

Nature flagged this review up again the other day. Turns out (I think) it was because of a 'correspondence' attached to it:

What can academia learn from XMRV studies?: http://www.nature.com/nrurol/journal/vaop/ncurrent/full/nrurol.2011.225-c1.html

Chungen Pan, Xiaochu Ma and Shibo Jiang

In their recent Perspectives article (Nat. Rev. Urol. 9, 111118; 2012), (1) Sfanos et al. have presented an overview on the invalid relationship between human prostate cancer and infection with the retrovirus XMRV.

From the first detection of XMRV in prostate cancer patients in 2006(2) and its proposed role in chronic fatigue syndrome (CFS) in 2009(3) to the final judgment in 2011 that XMRV originated from laboratory contamination,(4) XMRV studies have ridden a rollercoaster that is now coming back to the ground.

During this period, scientists from all over the world spent a huge amount of resourcesboth money and efforttrying to replicate the experiments and confirm the conclusions published previously.

Prostate cancer and CFS samples used in the studies came from countries including China, Japan, The Netherlands and the UK.

So how could such a newly emerging and rather obscure issue spark worldwide interest and then come back to silence so soon?

The answer is complicated but might reflect some serious problems troubling academia at the moment.

The first and probably the most important reason for the XMRV frenzy is because most researchers are eager to work on seemingly hot topics, such as XMRV most recently and the case of research into severe acute respiratory syndrome (SARS) 9 years ago.

This effect seems more understandable under todays adverse economic conditions, where in order to obtain enough funding to support their research, scientists are willing to take risks working on emerging pathogens, so that they are able to publish results as promptly as possible.

Another problem that becomes apparent from the studies on XMRV is that of scientific competition.
The breakthrough experiments in a specific field should be repeated and validated by other groups.

However, the nature of scientific competition means that journals might be more interested in publishing positive results than negative data, increasing the possibility that papers showing that the published results could not be reproduced might be rejected or put on hold.(5)

In the XMRV studies, the most notable exception to this theory was the stringently peer-reviewed journal Retrovirology, which published the series of papers in 2010 showing that the false-positive detection of XMRV in the clinical specimens was due to mouse DNA contamination in human studies.(68)

Furthermore, publication pressure might force scientists themselves to select their positive results over negative data for publication, resulting in a body of literature reflecting what scientists in the field want to see, rather than the reality.

If these data include false positives, publication of the false-positive results from studies on a new pathogen might cause greater damage to the scientific community, not only as a huge waste of resources, but also a biosafety threat to the patients involved and those hoping for news of a cure for their disease.

Generation of a highly infective mutated live virus through a plasmid system could realistically cause an epidemic or pandemic of an uncontrollable emerging infectious disease. Thus, research scientists and the academic community must learn a serious lesson from the case of XMRV studies.

The Institute of Human Virology & Department of Biochemistry, Key Laboratory of Tropical Disease Control of MOE, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China (C. Pan, X. Ma). MOE/MOH Key Laboratory of Medical Molecular Virology, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China (S. Jiang).

Correspondence to: C. Pan panchung@mail.sysu.edu.cn

doi:10.1038/nrurol.2011.225 c1

Competing interests

The authors declare no competing interests.

1. Sfanos, K. S., Aloia, A. L., De Marzo, A. M. & Rein, A. XMRV and prostate cancera final perspective. Nat. Rev. Urol. 9, 111118 (2012).
2. Urisman, A. et al. Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2, e25 (2006).
3. Lombardi, V. C. et al. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 326, 585589 (2009).
4. Paprotka, T. et al. Recombinant origin of the retrovirus XMRV. Science 333, 97101 (2011).
5. Enserink, M. Chronic fatigue syndrome. Conflicting papers on hold as XMRV frenzy reaches new heights. Science 329, 1819 (2010).
6. Smith, R. A. Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses. Retrovirology 7, 112 (2010).
7. Robinson, M. J. et al. Mouse DNA contamination in human tissue tested for XMRV. Retrovirology 7, 108 (2010).
8. Oakes, B. et al. Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology 7, 109 (2010).

 

[barbc56]

Nature flagged this review up again the other day. Turns out (I think) it was because of a 'correspondence' attached to it:

This might be right in front of me but what correspondence was attached to the study?

Thanks.

Barb C.:>)