No association of xenotropic murine leukemia virus-related viruses with prostate cancer

Abstract

Background: The association of the xenotropic murine leukemia virus-related virus (XMRV) with prostate cancer continues to receive heightened attention as studies report discrepant XMRV prevalences ranging from zero up to 23%. It is unclear if differences in the diagnostic testing, disease severity, geography, or other factors account for the discordant results. We report here the prevalence of XMRV in a population with well-defined prostate cancers and RNase L polymorphism. We used broadly reactive PCR and Western blot (WB) assays to detect infection with XMRV and related murine leukemia viruses (MLV).

Methodology/principal findings: We studied specimens from 162 US patients diagnosed with prostate cancer with a intermediate to advanced stage (Gleason Scores of 5-10; moderate (46%) poorly differentiated tumors (54%)). Prostate tissue DNA was tested by PCR assays that detect XMRV and MLV variants. To exclude contamination with mouse DNA, we also designed and used a mouse-specific DNA PCR test. Detailed phylogenetic analysis was used to infer evolutionary relationships. RNase L typing showed that 9.3% were homozygous (QQ) for the R462Q RNase L mutation, while 45.6% and 45.1% were homozygous or heterozygous, respectively. Serologic testing was performed by a WB test. Three of 162 (1.9%) prostate tissue DNA were PCR-positive for XMRV and had undetectable mouse DNA. None was homozygous for the QQ mutation. Plasma from all three persons was negative for viral RNA by RT-PCR. All 162 patients were WB negative. Phylogenetic analysis inferred a distinct XMRV.

Conclusions and their significance: We found a very low prevalence of XMRV in prostate cancer patients. Infection was confirmed by phylogenetic analysis and absence of contaminating mouse DNA. The finding of undetectable antibodies and viremia in all three patients may reflect latent infection. Our results do not support an association of XMRV or MLV variants with prostate cancer.

Figure 1. Identification of XMRV sequences in prostate cancer patients.

Representative nested pol PCR results using prostate tumor DNA. Lanes 1–24, prostate cancer patients, including patients 5956 (lane 8) and 6203 (lane 19); lane 25, negative human PBMC DNA control; lanes 26 and 27, water only controls for primary and nested PCR tests, respectively; lanes 28 and 29, assay sensitivity controls consisting of 10 and 10³ copies of XMRV VP62 plasmid DNA diluted in a background of 1 ug of human PBMC DNA, respectively.

Figure 2. Identification of variant XMRV in prostate cancer patients using phylogenetic analysis.

A. envelope (env), B. polymerase (pol), and C. gag. Stability of the tree topology was tested using 1000 bootstrap replicates in both neighbor joining (NJ) and maximum likelihood (ML) methods. Bootstrap values >60 are shown at major nodes (NJ/ML). New sequences from the current study are boxed. Accession numbers for prototypical MLV sequences available at GenBank are XMRV VP35 = DQ241301, XMRV VP62 = DQ399707, XMRV VP42 = DQ241302, XMRV WPI-1106 = GQ497344, XMRV WPI-1178 = GC497343, XMRV PCA1–PCA17 = GU812341–GU812357, MLV DG-75 = AF221065, MLV MTCR = NC_001702, MLV AKV = J01998, MLV BM5eco = AY252102.1, Moloney MLV = J02255, Moloney neuropthogenic MLV variant ts1-92b = AF462057, Rauscher MLV = NC_001819, Friend MLV = X02794, mERV Chr 7 = AC167978, mERV Chr 7 = AC127565, mERV Chr 8 = AC127575, mERV Chr 12 = AC153658, mERV Chr 9 = AC121813, mERV Chr 4 = AL627077, mERV Chr 1 = AC083892), XMLV A2780 = FR670594, XMLV BHY = FR670595, XMLV Daudi = FR670596, XMLV EKVX = FR670597, XMLV IMR-5 = FR670598, XMLV MUTZ-1 = FR670599, XMLV S-117 = FR670600, XMLV TYK-nu = FR670601. Sequences denoted RAW are from the polytropic MLV isolated in HeLa cells used to develop the in-house WB test. Sequences coded as XMRV VP and PCA and WPI are from prostate cancer and CFS patients, respectively. Additional prostate cancer patient VP gag and pol sequences were kindly provided by Drs. Robert Silverman and Joe Derisi. Viral tropism, as determined by analysis of env sequences, is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres.

Figure 3. Absence of antibodies to XMRV and MLV in prostate cancer patients.

Molecular weight markers (kD) are provided on the left of the WBs in the upper panels. Expected sizes of viral Gag (p30, capsid (CA)), pr65, and Envelope (Env, gp69/71) proteins are provided in each WB in the upper panels. Representative WB results for eleven prostate cancer patients, including patients 5956 and 6203 (indicated with asterisks). Determination of MLV specific reactivity is determined by comparison of seroreactivity to xenotropic MLV-infected HeLa antigens and uninfected HeLa antigens in upper and lower panels, respectively. Ra, Rauscher MLV whole virus goat polyclonal antisera; Fr, Friend MLV Envelope (gp69/71) goat polyclonal antisera; pre-immune, goat sera prior to immunization.