2. UroToday Home
  3. Recent Abstracts
  4. Urologic Oncology
  5. Prostate Cancer

Beyond the Abstract - XMRV: A new virus in prostate cancer? by Alan Rein, PhD, et al.

BERKELEY, CA (UroToday.com) - Xenotropic Murine leukemia virus-Related Virus (XMRV) was first described in 2006 as a newly identified murine gammaretrovirus associated with human prostate cancer.

(1) Subsequent studies have failed to reach a consensus regarding the prevalence of the virus in prostate cancer, with reported numbers ranging from 0% to approximately 27% (reviewed in (2)). Intriguingly, studies aiming to detect XMRV in prostate cancer samples are polarized in that they either find the virus at a fairly high frequency (1, 3-5) or report the complete absence or extremely low presence of the virus.(6-10) Likewise, studies which reportedly find the virus do not agree on the cellular localization (rare benign stromal cells versus malignant epithelium) of the virus or the potential association with homozygous germline RNase L mutations (R462Q). We recently reported on our efforts to detect XMRV in close to 800 human prostate cancer samples using two independent techniques, real-time PCR and immunohistochemistry (IHC).(11)We did not detect XMRV in any of these samples and we discuss possible explanations for the discrepancies in the results from different laboratories.

 

The assays used in our study included multiple positive and negative controls and were shown to result in extremely sensitive detection of XMRV, even in the setting of a vast excess of uninfected cells. Our first approach for XMRV detection utilized a duplex real-time PCR assay in which the same PCR wells were simultaneously tested for XMRV and for CCR5, a single-copy nuclear gene. Therefore, the quality of the PCR reaction and amplifiability of the DNA was tested during each PCR reaction that was run to detect XMRV. In positive control experiments, we determined that this real-time assay could detect the XMRV sequences in a single infected cell, even in the presence of a 10,000-fold excess of uninfected human cells. We screened DNA extracted from 161 fresh or frozen prostate tumor tissues, and although CCR5 was successfully amplified from each PCR reaction well, there was no amplification from the XMRV primers in any of the cases. These results do not indicate a “failure” to detect XMRV. In fact, if XMRV proviral DNA was present in the tested tissues, we would indeed have detected it. Thus, our data represents solid evidence against the presence of XMRV DNA in prostate cancer samples from men who live in North America.

Our second approach for the detection of XMRV in prostate tissues utilized an IHC assay and two distinct rabbit polyclonal antisera specific for murine leukemia virus (MLV) p30CA and gp70SU. In positive control experiments we demonstrated that both antisera cross-react with XMRV and can detect the virus in both XMRV-transfected cells and in cells that were previously shown to be infected with XMRV. We tested close to 500 prostate cancer cases on tissue microarrays (TMAs) with both antisera and did not observe any positive staining. We were concerned about the possibility that XMRV was not detected by IHC in our tissue samples because TMA spots represent a small fraction of the total tissue section, and it has previously been reported that XMRV is present in prostate tissues in a relatively small subset of cells (e.g. from ~1% of stromal cells to ~20% of cancer cells). Furthermore, we were also concerned that the age of the archival tissues contained in the TMA may influence the detection of XMRV. We therefore also tested freshly-obtained full tissue sections from over 110 prostate cancer cases, but still did not detect the virus in any of the cases tested. Finally, it is worth noting that the anti-sera we used are broadly reactive against all known MLVs. Therefore, even though our PCR assays were designed specifically to detect XMRV, and not other MLVs, the IHC studies would clearly be expected to detect any MLV present in the prostate cancer specimens. The fact that these IHC studies were also negative, in the face of specific positive and negative controls that were embedded in all experiments, using antibodies raised against two distinct viral proteins, also provides strong evidence against the presence of viral proteins derived from XMRV or other MLVs in human prostate cancer and benign tissues.

In our manuscript, we propose several possible reasons for the highly variable results reported for the presence of XMRV in prostate tissues. These include geographic and genetic variations among infected individuals, sequence variation of XMRV, extremely low viral levels in positive samples, and cross-reactivity of previously published XMRV antibodies with human proteins.(3, 12) Regarding the latter, we pointed out a potential pitfall with a prior study conducted using IHC in human prostate cancer. In the study by Schlaberg et al., in which XMRV proteins were purportedly detected in 23% of prostate cancer samples, the antibodies used for detection were raised against viral particles that were produced by infected human cells. This protocol carries the possibility that antibodies against human cellular proteins, as well as antibodies against viral proteins, may be present in the antiserum used for IHC. In our study, we obtained several “positive” and “negative” cases from Dr. Singh (the senior author on the study) and all of these cases were negative in our laboratory using the two different antisera used in our study.(3) Importantly, there is recent concern regarding the contamination of clinical specimens and molecular biology reagents with MLV sequences present in mouse DNA and RNA; these can be detected in PCR assays for XMRV. In fact, five papers were recently published in the journal Retrovirology regarding this topic.(13-17) These studies demonstrate the following:

 

  1. that purportedly “XMRV-specific” PCR primer sequences can amplify common murine endogenous viral sequences, highlighting the importance of screening for mouse DNA contamination in human samples and molecular biology reagents,(14)
  2. that a commercial reverse transcriptase PCR (RT-PCR) kit used in XMRV research is at times contaminated with an endogenous murine leukemia viral RNA,(15)
  3. that human tissues and DNA samples used in both prostate cancer and chronic fatigue syndrome research can be widely contaminated with mouse DNA, (16, 17) and
  4. that XMRV sequences detected in some prostate cancer samples are very similar to those in the XMRV-infected cell line 22Rv1, raising the possibility that cross-contamination from this cell line could be the source of these sequences.(14, 18)

 

Extremely small amounts of mouse DNA are sufficient to produce positive signals in a PCR assay for XMRV. In all, these new studies prompt serious reservations concerning the PCR evidence that supports the existence of XMRV in prostate cancer.

In summary, with close to 800 cases and using two different well-controlled techniques we found no evidence of XMRV in prostate cancer. Given the recent evidence that describes plausible mechanisms by which positive results for XMRV may represent laboratory contamination or other artifacts, and the lack of variability in the sequences of isolated XMRV genomes, we feel that the question of whether XMRV is actually infecting humans is still unresolved. Even if it is infecting humans, it appears to be present at levels so low that it is unlikely to be a causative agent for prostate cancer.


References:

  1. Urisman A, Molinaro RJ, Fischer N, et al. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2006;2(3):e25.
  2. Silverman RH, Nguyen C, Weight CJ, Klein EA. The human retrovirus XMRV in prostate cancer and chronic fatigue syndrome. Nat Rev Urol 2010;7(7):392-402.
  3. Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proceedings of the National Academy of Sciences 2009;106(38):16351-6.
  4. Arnold RS, Makarova NV, Osunkoya AO, et al. XMRV infection in patients with prostate cancer: novel serologic assay and correlation with PCR and FISH. Urology 2010;75(4):755-61.
  5. Danielson BP, Ayala GE, Kimata JT. Detection of xenotropic murine leukemia virus-related virus in normal and tumor tissue of patients from the southern united states with prostate cancer is dependent on specific polymerase chain reaction conditions. Journal of Infectious Diseases 2010;202(10):1470-7.
  6. Verhaegh GW, de Jong AS, Smit FP, Jannink SA, Melchers WJ, Schalken JA. Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in dutch prostate cancer patients. Prostate 2010.
  7. Sfanos KS, Sauvageot J, Fedor HL, Dick JD, De Marzo AM, Isaacs WB. A molecular analysis of prokaryotic and viral DNA sequences in prostate tissue from patients with prostate cancer indicates the presence of multiple and diverse microorganisms. Prostate 2008;68(3):306-20.
  8. Fischer N, Hellwinkel O, Schulz C, et al. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol 2008;43(3):277-83.
  9. Hohn O, Krause H, Barbarotto P, et al. Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients. Retrovirology 2009;6:92.
  10. Martinez-Fierro ML, Leach RJ, Gomez-Guerra LS, et al. Identification of viral infections in the prostate and evaluation of their association with cancer. BMC Cancer 2010;10:326.
  11. Aloia AL, Sfanos KS, Isaacs WB, et al. XMRV: A new virus in prostate cancer? Cancer Research 2010;70:10028-33.
  12. Switzer WM, Jia H, Hohn O, et al. Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology 2010;7:57.
  13. Smith R. Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses. Retrovirology 2010;7(1):112.
  14. Hue S, Gray E, Gall A, et al. Disease-associated XMRV sequences are consistent with laboratory contamination. Retrovirology 2010;7(1):111.
  15. Sato E, Furuta R, Miyazawa T. An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV. Retrovirology 2010;7(1):110.
  16. Oakes B, Tai A, Cingoz O, et al. Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology 2010;7(1):109.
  17. Robinson M, Erlwein O, Kaye S, et al. Mouse DNA contamination in human tissue tested for XMRV. Retrovirology 2010;7(1):108.
  18. Knouf EC, Metzger MJ, Mitchell PS, et al. Multiple integrated copies and high-level production of the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) from 22Rv1 prostate carcinoma cells. J Virol 2009;83(14):7353-6.

 


Written by:
Amanda L. Aloia, Karen S. Sfanos, Angelo M. De Marzo, and Alan Rein as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

XMRV: A new virus in prostate cancer? - Abstract

UroToday.com Prostate Cancer Section

Read other Beyond The Abstract submissions

More Information about Beyond the Abstract