No evidence of XMRV in prostate cancer cohorts in the Midwestern United States
© Sakuma et al; licensee BioMed Central Ltd. 2011
Received: 2 February 2011
Accepted: 29 March 2011
Published: 29 March 2011
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies.
Real-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution.
The lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.
Prostate cancer (PCa) is the most frequently diagnosed noncutaneous malignancy among men in industrialized countries . Although early detection using tests for prostate-specific antigen and improved treatment have emerged as important interventions for decreasing PCa mortality, there is potential for improved prognosis through detection of genetic risk factors. Indeed, a positive family history is among the strongest epidemiological risk factors for PCa, and a number of genetic mutations have been implicated in PCa. For example, an R462Q polymorphism in the RNase L protein, which impairs the catalytic activity of an important effector of the innate antiviral response, has been implicated in up to 13% of unselected PCa cases .
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was first identified in PCa tissues, particularly those with the homozygous RNASEL R462Q mutation . Genetic analysis identified XMRV as a xenotropic gammaretrovirus, closely related to those found in mice [4, 5]. This suggested that XMRV represented a zoonotic transmission from mice to humans. When compared with exogenous and endogenous MLV sequences, XMRV appeared to have a unique, conserved 24 bp deletion in the gag leader region . However, this deletion has recently been found in endogenous MLV proviruses in a variety of mice . Initially, immuno-histochemistry (IHC) and FISH analyses suggested that only prostatic stromal fibroblasts were infected with XMRV . Subsequently, Schlaberg, Singh and colleagues reported the expression of XMRV antigens in 23% of PCa and an association of XMRV infection with higher grade tumors . Contrary to the initial study, Singh's study found viral antigen-positive cells primarily in malignant prostatic epithelium, independently of the RNASEL polymorphism . It is notable that this study found many immuno-histochemistry-positive samples which did not have detectable XMRV DNA . Another study found 11 (27.5%) of 40 PCa patients with XMRV neutralizing antibodies . Importantly, there were correlations between serum positivity and nested PCR results, FISH, or the R462Q RNASEL mutation . In sharp contrast, several recent reports found no or very low prevalence of XMRV (DNA, RNA or antibodies) in PCa samples [9–12].
If the role of XMRV in PCa is confirmed, detection and prevention of XMRV infection could provide a novel intervention strategy for early diagnosis and treatment of PCa. However, the conflicting epidemiological data have made it unclear whether XMRV plays a role in PCa and have questioned whether the virus is truly a human pathogen. In this study we have sought to address the association between XMRV infection and PCa, PCa grades and RNASEL R462Q polymorphism by testing prostate tissues for the presence of XMRV. In addition, to determine the correlation between PCa and seroprevalence of XMRV, serum samples from patients with PCa were compared with age-matched controls for detectable anti-XMRV antibodies. Our study found no XMRV sequences and no XMRV-neutralizing antibodies in 150 prostate tissues (110 PCa and 40 benign/normal) and serum samples (159 PCa and 201 age-matched controls), respectively, indicating no or very low prevalence of XMRV in our cohorts. We did detect MLV sequences in 6 samples, but these samples were also PCR positive for mouse mitochondrial DNA suggesting DNA contamination as a source of the MLV. We were therefore unable to confirm the links between XMRV infection with PCa, PCa grades or RNASEL mutation.
Prevalence of XMRV proviral DNA in PCa
Prevalence of XMRV and tumor grade
Low grade c
To confirm the real-time PCR results, we screened the same DNA samples by nested PCR for XMRV/MLV gag sequences. In order to establish consistency and to minimize the risk of contamination during the procedure, three individuals independently performed the nested PCR experiments using independently aliquoted DNA samples. Four out of 6 real-time PCR positive samples (#15, 51, 52 and 112) were consistently positive by the nested PCR analysis, while the other two positive samples from intermediate grade PCa (#53 and 103) were shown to be nested PCR-positive twice in the first three attempts. Further analysis confirmed that these two samples were nested PCR-positive for viral DNA. The 144 real-time PCR-negative samples were also found to be negative by nested PCR.
No statistical link between the presence of viral DNA and prostate cancer or higher tumor grade
Statistical analysis of XMRV positivity in controls and PCa
Statistical analysis of XMRV prevalence and tumor grade
No correlation between viral DNA detection and RNASEL R462Q mutation
RNASEL genotyping and tumor grade
Statistical analysis of XMRV prevalence and RNASEL genotyping
Phylogenetic analyses of MLV-like sequences in prostate tissue DNA
Comparison of MLV sequences amplified from patient samples with mouse genomic sequences
Sequence (GenBank no.)
Mus musculus BAC clone RP23-457E5
Mus musculus chrom 7, clone RP24-220N8
Mus musculus BAC clone RP23-152O2
Mus musculus BAC clone RP23-152O2
Mouse DNA sequence, clone CH29-187G15
Mus musculus chrom 5, clone RP23-280N22
Mouse DNA sequence, CH29-187G15
Comparison of MLV sequences amplified from patient samples  with mouse genomic sequences
Mouse endogenous retrovirus
Mus musculus BAC clone RP23-115O21
Mouse DNA sequence from clone RP23-131N18
Mouse endogenous retrovirus
Mouse DNA sequence, clone CH29-187G15
Mus musculus BAC clone RP23-115O21
Detection of XMRV antigens in PCa tissues
Absence of XMRV antibodies in patients with PCa and age-matched controls
In this study, we have examined the prevalence of XMRV in patients with or without PCa at Mayo Clinic. We were unable to find XMRV sequences or anti-XMRV antibodies in our patients, most of whom are from the mid-west area of the USA, indicating that there is no or very low prevalence of XMRV in this region. Moreover, we were unable to confirm the correlation between XMRV infection and PCa, higher tumor grade or RNASEL R462Q mutation.
A high prevalence of XMRV has been reported in patients with PCa and chronic fatigue syndrome (CFS) in the USA [3, 7, 8], but similar studies in Europe have failed to detect XMRV [10–12]. It has been suggested that geographical differences might explain this striking variation in XMRV prevalence  but our results, as well as recent US studies that also find no evidence for XMRV [9, 21], appear to rule this explanation out. In this regard, it is notable that previous studies to identify XMRV in patients with PCa or chronic fatigue syndrome have relied on very sensitive PCR detection methods. Because of the high similarity between patient associated XMRV/MLV and endogenous MLV sequences and the striking discordance between studies, it has been suggested that PCR-positive results might be attributed to unintentional detection of contaminating mouse DNA in human specimens [6, 22–24]. It is notable that Lo et al.  detected polytropic and modified polytropic MLV sequences, but not XMRV, in blood samples from chronic fatigue patients (Figure 1). These authors were unable to identify the samples as contaminated using mouse mitochondrial PCR. In our study, real-time PCR and nested PCR identified 6 of 150 samples as positive for MLV. However, the amplified sequences were closely related to known endogenous MLV proviruses, rather than XMRV. In fact one patient sample (#52) contained two independent MLV sequences. This might be interpreted as evidence for evolution of the virus in the patient but closer analysis reveals that one of the sequences is identical to a known endogenous modified polytropic sequence whilst the other is a single nucleotide different from a known mouse endogenous xenotropic MLV. This, therefore, suggests either infection of this patient with two independent MLVs or PCR contamination with mouse DNA as a source. As all of the MLV PCR-positive samples contained detectable levels of mouse mitochondrial DNA, we conclude that the amplified sequences originated from mouse DNA that somehow contaminated the study samples.
In order to confirm that the viral sequences were amplified from endogenous MLV in mouse genomic DNA, but not replicating MLV in human tissue, we attempted to determine viral integration sites. We first used the protocol described by Kim et al.  but failed to amplify DNA sequences containing the partial XMRV LTR. We then designed universal primers to recognize LTRs from XMRV and endogenous and exogenous MLVs , as well as a series of primers specific for the viral sequences identified in our clinical samples. Unfortunately, we were not successful, likely due to low viral copy numbers in the clinical samples. Very recently, Robinson et al.  and Oakes et al.  reported similar observations; all XMRV PCR-positive specimens contained detectable levels of mouse mitochondrial or endogenous retroelements (IAPs). Together with our data, these findings highlight the difficulty of avoiding DNA contamination in clinical samples and the risk of testing contaminated samples as XMRV-positive by sensitive PCR detection assays. As a possible source of contamination, Sato et al.  demonstrated that a commercially available hot-start PCR enzyme contained mouse DNA. We used several enzymes and obtained similar results. Thus, it is unlikely that the contaminating mouse genome originated from a PCR kit. Since we could amplify the viral sequences from multiple aliquoted DNA samples, they appeared to be contaminated before or during the DNA isolation step, most likely during tissue sectioning on a microtome.
XMRV antigen-positive cells have been detected in prostatic stromal fibroblasts  or in malignant prostatic epithelium . Our IHC study using two different antisera showed conflicting results. The goat anti-MLV antibody found no viral antigens in clinical samples, while the rabbit anti-XMRV antibody used in the study by Schlaberg, Singh and colleagues  detected antigen-positive cells in prostatic epithelium. Strikingly, the goat anti-MLV serum did not stain the cells, which were IHC-positive by the anti-XMRV rabbit serum, in serial sections of the same tissue. The rabbit antiserum also found antigen-positive cells in PCR-negative sections, confirming the observations of Schlaberg and colleagues who reported frequent detection of IHC-positive samples in PCR-negative tissues . Importantly, both the rabbit and goat antibodies detected XMRV in experimentally infected cells with high sensitivity (Figure 3). Together, these observations strongly suggest that the rabbit antiserum is detecting a non-viral antigen sporadically expressed by tumor cells in the tissue section. We conclude that our PCa samples do not have XMRV antigen-expressing cells that are detectable by IHC.
We recently reported that Mus pahari mice elicit potent XMRV-specific humoral immune response upon XMRV infection . At a serum dilution of 1:640, antisera from infected animals almost completely blocked XMRV infection . Similarly, an animal study using XMRV-infected rhesus macaques and sensitive ELISA detection assays showed that infected animals rapidly develop antibodies against XMRV proteins, including gp70 (Env), p15E (transmembrane), and p30 (CA) . These results indicate that XMRV is strongly immunogenic in these animals. In contrast, we were unable to detect strong XMRV-specific neutralizing antibodies in our 360 patients, age 50-70, with or without PCa. This observation further suggests a lack of XMRV in our cohorts. It is possible, although less likely, that XMRV is not immunogenic in humans or that XMRV-specific immune response might have disappeared in these relatively elderly patients.
In our study population of patients with or without PCa from the USA, we found no evidence of infection with XMRV using PCR, IHC and serological tests. Our negative results are in accordance with previous studies using sensitive PCR, ELISA and Western blot assays, which failed to detect PCR or seropositive samples in a large number of blood donors, HTLV- and HIV-infected, or patients with or without CFS [9–12, 21, 27–31]. Our results indicate the possible false-positive detection of XMRV/MLV-related sequences or antigen-positive cells through laboratory contamination or non-specific immune reaction respectively, and underscore the need for careful validation of previous and future studies.
Materials and methods
Prostate tissues and plasma samples from patients
Prostate tissues and plasma samples were obtained from Mayo Clinic Biospecimen Core with an approval from the Institutional Review Boards. Frozen sections of prostate cancer tissues (10 μm) were identified as 1 through 150 in duplicates. These samples included 40 normal/low grade Gleason score, 70 intermediate (Gleason score 5-7), and 40 high grade (Gleason score 8-10) with men aged between 50-70 years old. For plasma analysis, total of 360 plasma samples from 50-70 year old male patients including 159 prostate patients (Gleason score 5-7) and 201 patients with no prostate cancer or urological disorders were used in this study.
Total cellular DNA was extracted by PureLink Genomic DNA Mini Kit according to the manufacturer's protocol (Invitrogen). All samples were eluted in 50 μl of elution buffer, and the concentration and quality of the DNA were determined by a NanoDrop Spectrophotometer. For the real-time PCR assay, TaqMan Universal PCR Master Mix (Roche) was used along with 2 μl of each sample. Primers were used at a range of 230 nM to 300 nM final concentration. TaqMan probe #51 from Roche Universal Probe Library was used for XMRV-gag at 100 nM final concentration. A standard curve was created by using serially diluted XMRV plasmid (pcDNA3.1(-)/VP62). The assay was analyzed by the ABI 7300 Real-Time PCR System using the default thermal cycling conditions for the two-step RT-PCR method and FAM reporter .
RNASEL genotype was determined by nested PCR amplification using outer primers 5'-CTGGGGTTCTATGAGAAGCAAG-3' and 5'-TGAGCTTTCAGATCCTCAAATG-3', and inner primers 5'-GAGAGAACAGTCACTTGGTGAC-3' and 5'-CAGCCCACTTGATGCTCTTATC-3' with pfx polymerase (Invitrogen). Final PCR products were purified with QIAquick PCR Purification Kit (Qiagen) before sequence analysis.
The neutralization assay was carried out using GFP-encoding XMRV as described previously [13, 14]. Briefly, 293T cells were transfected with pcDNA3.1(-)/VP62 and a GFP-encoding retroviral vector using FuGene 6 (Roche). Serum samples were heat inactivated at 56°C for 30 min. A mixture of plasma samples and 2.5 × 104 infectious units of GFP-carrying XMRV were incubated at 37°C for 30 min before infecting 293T cells (5 × 104). Three days post-infection, cells were resuspended, fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD FACScan). The percentages of GFP-positive cells were measured using CellQuestPro software .
Western blot analysis
For Western blot analysis of XMRV proteins, cell lysates of prostate cancer (PC-3) cell line (ATCC) and PC-3 infected with XMRV were harvested in 1.0 ml of RIPA lysis buffer. Cell debris was removed by centrifugation, and the supernatant was diluted with Laemmli sample buffer containing β-mercaptoethanol. After heat-denaturation at 95°C for 5 min, 10 μl of proteins were subjected to SDS-PAGE with a 4-15% gradient gel (Bio-Rad), and transferred to a polyvinylidene diflouride membrane at 0.7 mA/cm2 for 40 min. Membranes were blocked in 5% milk/PBS, then stained with patient's plasma samples diluted to 1:250, followed by anti-human IgG (1:1000, Jackson ImmunoResearch Laboratories, Inc.).
Immunohistochemistry was performed on tissue samples from patients with or without prostate cancer. Sections were fixed with 4% paraformaldehyde for 20 min and treated with 0.3% Triton X100 for 15 min at room temperature. They were then blocked with 5% FBS/PBS for 30 min and immunostained with rabbit-anti XMRV (kindly provided by Dr. Ila Singh, University of Utah) or goat-anti p30/gp70 (NCI HD625 CAT No. 04-0109, LOT No. 81S000262, Quality Biotech, kindly provided by Dr. Yasuhiro Takeuchi, UCL) at a dilution of 1:500 for 4 h at room temperature. FITC-conjugated anti-rabbit IgG (1:500; Amersham) or DyLight 488-conjugated anti-goat IgG (1:500; Jackson ImmunoResearch Lab) were applied for 2 h at room temperature. Nuclei were then counter-stained with 4'-6-Diamidino-2-phenylindole (DAPI), and analyzed by confocal microscopy (Zeiss).
Nested PCR and sequence analysis of proviral DNA
Sequence analysis was performed as previously described . Briefly, DNA was extracted by PureLink Genomic DNA Mini Kit (Invitrogen). Nested-PCR was performed for XMRV gag (primers for outer gag: 5'-ACGAGTTCGTATTCCCGGCCGCA-3' and 5'-CCGCCTCTTCTTCATTGTTC-3', primers for inner gag: 5'-GCCCATTCTGTATCAGTTAA-3' and 5'-AGAGGGTAAGGGCAGGGTAA-3') with platinum Taq polymerase (Cat. no. 10966-034, Invitrogen). The resulting PCR products from a total of 4 patient samples (#15, #51, #52 and #112) were cloned into the TOPO vector (Invitrogen). Sequences from the two patient samples #53 and #103 were not analyzed. From patients #15, 51, 52, 112, we sequenced 2, 2, 4, 1 clones, and got 2, 2, 2, 1 different sequences, respectively. They were analyzed by DNADynamo (BlueTractorSoftware).
Seven unique gag gene sequences (255 to 528 nt), amplified from our clinical samples (GenBank no. JF288878, JF288879, JF288880, JF288881, JF288882, JF288883, and JF288884), were manually aligned with previously described murine leukemia virus gag gene sequences (n = 79), 22Rv1 cell line derived gag sequences (1605 nt; n = 15), XMRV gag sequences apparently amplified from prostate cancer and CFS samples (n = 7) , as well as 6 MLV virus gag sequences isolated from chronic fatigue syndrome samples . Bayesian phylogenies were reconstructed as previously described . The Markov chain Monte Carlo search was set to 10,000,000 iterations, with trees sampled every 1000th generation, and with a 20% burn in. The phylogeny of the aforementioned sequences was also reconstructed by maximum likelihood (ML) inference under the general time reversible model of nucleotide substitution, with gamma-distributed rate heterogeneity and proportion of invariable sites, using the program RAxML (data not shown) . The ML topology was assessed by neighbor joining bootstrapping with 1000 replicates using the program PAUP*.
A semi-nested mouse-specific mtDNA PCR
We used a PCR assay for mouse mitochondrial DNA reported to be able to detect 2.5 fg of mouse DNA in the presence of 35 ng human background DNA . Using this assay, we tested whether our samples were contaminated with mouse DNA. DNA from PCR positive samples were PCR amplified with KOD Hot Start DNA Polymerase following the manufactures instruction (Novagen) as described . The resulting PCR fragments were further cloned into the TOPO vector and the sequences were confirmed to be identical to the mouse cytochrome b gene by DNA BLAST.
Rabbit-anti XMRV and goat-anti p30/gp70 were kindly provided by Dr. Ila Singh and Dr. Yasuhiro Takeuchi respectively. This work was supported by the National Institute of Health (AI093186), Mayo Clinic Career Development Project in Prostate SPORE grant CA91956-080013, the Mayo Foundation (YI), Wellcome Trust senior fellowship WT090940 (GJT) European Community's Seventh Framework Programme (FP7/2007-2013) under the project 'Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)', grant agreement no. 223131 (SH) and the National Institute of Health Research UCL/UCLH Comprehensive Biomedical Research Centre (GJT).
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