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Several recent papers have reported the presence of a gammaretrovirus, termed “XMRV” (xenotropic murine leukemia virus-related virus) in prostate cancers (PCa). If confirmed, this could have enormous implications for the detection, prevention, and treatment of PCa. However, other papers report failure to detect XMRV in PCa. We tested nearly 800 PCa samples, using a combination of real-time PCR and immunohistochemistry (IHC). The PCR reactions were simultaneously monitored for amplification of a single-copy human gene, in order to confirm the quality of the sample DNA and its suitability for PCR. Controls demonstrated that the PCR assay could detect the XMRV in a single infected cell, even in the presence of a 10,000-fold excess of uninfected human cells. The IHC used two rabbit polyclonal antisera, each prepared against a purified MLV protein. Both antisera always stained XMRV-infected or – transfected cells, but never stained control cells. No evidence for XMRV in PCa was obtained in these experiments. We discuss possible explanations for the discrepancies in the results from different laboratories. It is possible that XMRV is not actually circulating in the human population; even if it is, the data do not seem to support a causal role for this virus in PCa.
In 2006, a new retrovirus was reported to be associated with prostate cancer (PCa) (1). It was recognized as a murine leukemia virus (a member of the gammaretrovirus genus), and was termed “XMRV”, or “xenotropic murine leukemia virus-related virus”. (Murine leukemia viruses [MLVs] are found in mice; xenotropic MLVs cannot infect mouse cells, but can generally infect human cells.)
Association of a virus with this important cancer could have enormous implications for detection, prevention, and treatment of PCa, just as the discovery of the role of human papillomavirus in cervical cancer has revolutionized our approach to this disease. Accordingly, many laboratories have begun testing for the presence of XMRV in PCa patients. Remarkably, the same virus was also reported in patients with chronic fatigue syndrome (CFS) (2).
Retroviruses are relatively simple RNA-containing viruses. Their unique properties include the copying of their RNA into double-stranded DNA at the time of infection (“reverse transcription”) and the integration of this DNA copy into the chromosomal DNA of the infected cell. Once inserted, this DNA is replicated with the chromosome, and will thus be present in the cell and its descendants into the indefinite future.
The methods that have been used to detect XMRV include nucleic acid hybridization; PCR and reverse transcription-PCR (RT-PCR); fluorescence in situ hybridization (FISH); immunohistochemistry (IHC); screens for anti-viral antibodies in patient sera; and virus cultivation. These diverse methods have only given concordant results in a handful of cases. The field was recently reviewed (3), and the state of the science was also covered in a 1 ½ day meeting held on the NIH campus in September, 2010.
XMRV was initially reported to be more prevalent in prostate cancer tissues from men with homozygous germline RNase L mutations (R462Q) than in men without this QQ genotype (1). In this study, both in situ hybridization and IHC found the virus in approximately 1% of stromal cells, but not in tumor cells. A subsequent study reported that 14 of 233 prostate tumors were positive for XMRV by real-time PCR and 54 of 233 were positive by IHC (4). Surprisingly, a number of cases were positive by IHC, but negative by PCR. The IHC in this study localized XMRV proteins primarily in malignant epithelial cells rather than stromal cells, and positive staining correlated with high tumor grade. No association with RNase L variants was found in this study. Finally, another study reported that 8 of 20 prostate cancer patients with the QQ RNASEL genotype and 3 of 20 with the RQ or RR genotypes were positive in a serum neutralization assay and that 5 of 7 tested tumors were positive by FISH in a subset of stromal cells; FISH and serum positivity correlated with nested PCR results (5). One strong indication that XMRV has infected some human cells in some prostate tumors is the finding of XMRV sequences integrated into human DNA (6, 7).
In contrast, several studies have reported the absence, or extremely low prevalence, of XMRV in PCa. These include a study of 338 samples representing tumor, normal, and BPH tissues from 200 prostate cancer patients using a highly sensitive nested PCR assay; 105 prostate tumors using nested RT-PCR; 589 prostate tumors using nested PCR and nested RT-PCR as well as 146 serum samples using an ELISA assay; and 130 prostate tumors and control tissue samples using RT-PCR(8–11).
At this point, it would be hard to overstate the discrepancies between different laboratories on the basic question of whether XMRV is actually present in the human population, and there is no understanding of its possible role in disease. In an effort to resolve the discrepancies concerning the prevalence of XMRV in PCa, we have tested for the virus using both real-time PCR and IHC, with two antisera specific for different viral proteins; our results are described below. Methods are detailed in Supporting Information.
We developed a real-time PCR assay for detection of XMRV sequences in PCa tissues. The quality and concentration of the sample DNAs were confirmed by a duplex PCR procedure, in which the same PCR wells were simultaneously tested for XMRV and for CCR5, a single-copy nuclear gene. For a positive control, we tested the genomic DNA (gDNA) of 22Rv1 cells, an XMRV-infected PCa cell line (12). 22Rv1 gDNA was diluted into 293T or HeLa cell gDNA (typical results shown in Fig. 1). We could routinely detect XMRV sequences in 10 pg of 22Rv1 gDNA (Fig. 1A, blue line), even in the presence of 100 ng or more of background human gDNA. Tests of 1 pg (orange and pink lines) were occasionally positive, but viral sequences were never detected in 0.1 pg of 22Rv1 gDNA (data not shown). Tests with the XMRV plasmid VP62 as standard indicate that there are ~ 15 copies of XMRV per diploid genome in 22Rv1 gDNA, a number similar to that reported by Knouf et al. (12) (data not shown). Thus our assay can always detect ~20 copies of XMRV DNA, and can occasionally detect ~ 2 copies. The CCR5 assays in the same PCR wells were uniformly positive (Fig. 1A). All assays included parallel tests of 293T or HeLa gDNA alone (XMRV negative control, Fig. 1B, red line) and E. coli gDNA (CCR5 negative control, Fig. 1B, green line). Using this duplex assay, we screened DNA from 161 prostatic adenocarcinomas, including 12 that had been micro-dissected and 10 that were metastases (Table 1). In all cases CCR5 was successfully amplified, confirming the quality of the DNA preparation, but there was no amplification from the XMRV primers in any of the cases (Fig. 1C).
Similar real-time PCR assays were also performed on 54 of these samples using a primer-probe set directed at a highly conserved region within the Gag gene. In addition, nested RT-PCR was performed on 41 additional cases using the primer set described in Urisman et al. (1) and nested PCR was used on 24 cases using the primers of Hohn et al. (9). In all of these tests, XMRV sequences could be efficiently detected in the RNA or DNA from infected cells, but no positive results were obtained with any of the PCa samples (data not shown).
The MLV proteins p30CA and gp70SU are cleavage products of the viral Gag and Env polyproteins, respectively. We tested the ability of the MLV30 and MLV70 antisera to react with XMRV proteins. As shown in Fig. S1 in Supplementary Information, both antisera reacted with the expected viral proteins in virus particles (the MLV30 blot shows that some uncleaved Gag polyprotein, Pr65Gag, as well as the normal cleavage product p30CA, is present in the virus particles). Thus, these antisera cross-react with the corresponding polypeptides from XMRV. Western blots with MLV30 and MLV70 using lysates from 22Rv1 cells were similarly positive (data not shown), but not using lysates of the human PCa cell lines DU145 and PC3, which were negative for XMRV by PCR (data not shown).
By IHC both sera showed clear and reproducible staining of 293T cells transfected with the VP62 clone of XMRV (Fig. 2B) but never stained 293T cells that had been transfected with an empty plasmid (Fig. 2A). Additionally, both antisera stained 22Rv1 cells (Fig. 2C), indicating that the staining did not require the overexpression typically associated with transient transfection. A total of 596 prostatic adenocarcinomas and 452 benign prostate tissue specimens, prepared either as full tissue sections or as tissue microarrays (TMAs), were analyzed with MLV30 and/or MLV70 (Table 1). Many of the prostatic tissues evaluated from these cancer patients included areas of acute and chronic inflammation, atrophy, benign prostatic hyperplasia and high grade prostatic intraepithelial neoplasia (Table 1). Each experiment included positive and negative controls, which always gave results as in Figs. 2A and 2B. However, no staining of prostate tissue samples was ever observed with either antiserum (Fig. 2D).
We used a real-time PCR assay capable of detecting XMRV sequences in DNA from a very small number of infected cells, even in the presence of a vast excess (more than 10,000-fold) of uninfected cell DNA. We also performed IHC with two antisera, each specific for a different MLV protein, under conditions where the sera reproducibly stained XMRV-containing cells but not identically treated control cells. Taken together, the two assays surveyed nearly 800 prostate tumors, including microdissected tumor specimens; metastatic tumor tissue; and intermediate and high-grade primary tumors. No signs of XMRV infection were found in any of these tests. The results suggest that the prevalence of XMRV in prostate tumors may be far lower than has been reported previously.
How can our negative results be reconciled with the positive reports from other laboratories? It has been suggested that XMRV might be present in North American, but not European, prostate tumors (9). However, our samples, like those of Schlaberg et al. (4), were from North American men. Also, while we did not select RNase L R462Q homozygotes for analysis, the number of cases we examined was high enough to include a substantial number of these individuals. Another possibility is that XMRV was present in our samples, but we failed to detect it because the viral sequences were somewhat different from the published XMRV sequences. While little variation in XMRV sequences has been observed to date (the reported sequences are ~ 97% identical), this could potentially explain our negative PCR results. However, we used several primer sets, some against highly conserved MLV sequences, and still saw no MLV signals. Further, unlike PCR primers, the sera we used in our IHC assays are both broadly reactive, since they were generated using Mo–MLV proteins but reacted with the XMRV proteins in our positive controls (Mo-MLV and XMRV are 82 % identical at the amino-acid level). Thus it seems extremely improbable that sequence polymorphisms can explain our failure to detect XMRV by IHC.
It could also be proposed that infected cells are present at such a low level in virus-positive tumors that the samples we tested were too small to contain infected cells. (Contrary to this, Schlaberg et al. initially reported that positive samples contained 1–10 XMRV copies per 660 cells; 660 diploid cells contain ~ 5 ng of DNA, while we tested amounts ranging from 25 to 1000 ng (4)). This might explain the negative IHC results with tissue microarrays, but seems unlikely in the >100 tumors for which we analyzed standard slides, which generally contain more than 105 cells.
Finally, another conceivable explanation for the staining seen by Schlaberg et al. (4) is that the anti-XMRV serum used in their experiments contains antibodies directed against cellular proteins, in addition to the antibodies against XMRV proteins. The XMRV used as immunogen by Schlaberg et al. was apparently produced in human cells. It is thus difficult to exclude the possibility that human proteins were present in the virus preparation used as immunogen. HIV-1 virus particles are known to incorporate a wide variety of proteins from the virus-producing cells (13), so that these proteins are impossible to remove from the virus; indeed, early vaccine trials with simian immunodeficiency virus were confounded by this phenomenon (14, 15). Incorporation of major histocompatibility complex proteins into MLV particles has also been reported (16). We received PCa tissue sections (kindly provided by Dr Ila Singh, University of Utah) from a number of cases from specimens used by Schlaberg et al. (4). Based on their results with the anti-XMRV antiserum, these samples were predicted to be IHC-positive. However, the sections did not stain with our MLV30 or MLV70 antisera (data not shown). While we cannot fully explain the discrepancies in staining results, Switzer et al. have also demonstrated that under immunoblotting conditions, the anti-XMRV antiserum (4) reacts with proteins in uninfected HeLa cells (17).
Many laboratories have used PCR to detect XMRV in clinical samples. However, the extraordinary sensitivity of this technique magnifies the risk of finding false positives, as well as the ability to find authentic positives. The risk is compounded by the widespread use of mice in biomedical research. Every mouse cell contains, in its DNA, ~ 100 MLV genomes, termed “endogenous viruses”. These genomes reflect past infections of germ cells and the resulting integration of the viral sequences into the mouse germline. As PCR is capable of amplifying and detecting a single molecule of viral DNA, this means that, for example, (depending, of course, on the specificity of the primers) a millionth of a microliter of mouse blood is a potential source of a positive signal in a PCR assay for MLV. Indeed, there are anecdotal reports of false-positive MLV signals ultimately traced to the use of the same microtome blade for cutting mouse and PCa sections, and to the tiny amounts of mouse DNA contaminating the mouse anti-polymerase monoclonal antibody used in commercial “hot start” PCR kits.
The existence of endogenous MLVs may be pertinent to another recent set of observations. In an attempt to reproduce the detection of XMRV in cases of CFS, Lo et al. (18) performed PCR and reverse transcription-PCR on blood samples from CFS patients and healthy blood donors. They obtained positive signals from a high proportion of the CFS cases (and a much lower proportion of the healthy donors). However, when the PCR products were sequenced, they were found to differ from XMRV; thus these results are completely distinct from the reports of XMRV detection. In fact, the sequences match endogenous MLV sequences almost exactly. It should be emphasized that (unlike the studies reporting isolation of XMRV) this report does not include direct evidence for the presence of an infectious virus: the data consisted exclusively of amplification and detection of MLV-like sequences. Notably, the endogenous MLVs that they resemble most closely are defective MLV genomes which do not give rise to infectious MLV. While the authors provided strong experimental evidence arguing against contamination of their clinical samples with mouse DNA, this remains a possible explanation for their results.
In conclusion, the fundamental question of whether XMRV is really an infectious agent circulating in the human population is still unresolved. This question will not be settled until reproducible assays for the virus are established and validated; in turn this will require exchange of samples and testing of well-characterized standards, followed by cross-comparison of results obtained in different laboratories. Efforts in this direction are now underway at the U.S. National Institutes of Health. However, based on the data presented here, as well as that from other investigators (8–11), we are doubtful that XMRV is commonly found in PCa. Over the years, many claims associating viruses with diseases have turned out to be mistaken (19, 20), and it is still possible that XMRV will fall into this category.
Finally, it is crucial to distinguish the question of the existence and prevalence of XMRV in the human population from the question of its causal role in PCa. In general, gammaretroviruses like XMRV induce malignant transformation by insertional mutagenesis, so that tumors induced by a gammaretrovirus are clones in which all the cells are infected (21). This mechanism of carcinogenesis has been observed not only in laboratory animals, but also in children exposed to gammaretrovirus-derived vectors in gene-therapy trials (22, 23). Although some exceptions to this insertional mutagenesis mechanism have been described (24), the viral genome is present in the transformed cells in all known cases. Thus, infection of an extremely minute fraction of the cells in some prostate tumors, even if confirmed, would seem to be incompatible with the possibility that XMRV plays a causal role in prostate tumorigenesis.
Grant Support: Immunohistochemistry studies and a number of the frozen prostate tissues for PCR assays were supported by NCI-SPORE in prostate cancer P50CA58236. K.S.S is supported by a postdoctoral fellowship award from the Prevent Cancer Foundation. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
We thank Helen Fedor at Johns Hopkins for help with prostate tissues and slides for IHC, Ila Singh for FFPE sections of PCa tissue, and W. Marston Linehan, Kenneth J. Pienta, Peter Pinto, Frank Ruscetti, and Cathy Vocke for prostate tumor DNAs. We are also grateful to Jessica L. Hicks, Jane Mirro, and Demetria Harvin for superb technical assistance and to John M. Coffin, Robert J. Gorelick, Stephen H. Hughes, Michael Piatak, and Saraswati Sukumar for many helpful discussions.
Disclosure of potential conflicts of interest.
No potential conflicts of interest were disclosed.