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, 7 (9), e44954

In-depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer

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In-depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer

Deanna Lee et al. PLoS One.

Abstract

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

Conflict of interest statement

Competing Interests: JDG, EK, JD, RS, KL, XQ and JH are included as inventors on one or more of the following published XMRV-related patent applications which include either the Cleveland Clinic, Abbott Laboratories, or both the Cleveland Clinic and Abbott Laboratories as assignees: International Publication Numbers WO2011/002932; WO2011/002936; WO2010/075414; WO2006/110589; WO2012/024518 and WO2012/024513. Abbott is the sole assignee of issued United States Patent No. 8,183,349 relating to XMRV. NT, KL, XQ, GS and JH are employees of Abbott Laboratories. DG is an employee of Novartis Institutes for Biomedical Research. These potential competing interests do not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Study Workflow.
Colored boxes refer to the research laboratory in which the described analysis was performed. To minimize the risk of PCR amplicon contamination, blinded XMRV-specific PCR testing was performed separately in 3 independent laboratories.
Figure 2
Figure 2. Detection of XMRV in Prostate Cancer Tissues and Archival RNA Extracts by Microarray.
Samples were analyzed using the ViroChip, a pan-viral DNA detection microarray (x-axis). The heat map shows a selected cluster consisting of 96 gammaretrovirus probes (y-axis) and corresponding to the same cluster observed in the 2006 study by Urisman, et al . The red color saturation indicates the normalized magnitude of hybridization intensity. Microarrays corresponding to key samples are highlighted (arrows). Only prostate cancer samples VP35 and VP42 were found to be consistently positive for XMRV from both total and polyA RNA .
Figure 3
Figure 3. Detection of XMRV in Prostate Cancer Cell Lines and Tissues by FISH.
A probe hybridization mix containing XMRV-SO probe (full-length XMRV VP62) and CEP8-SA internal control probe (complementary to a centromeric region of human chromosome 8) was applied to each slide. (A) A representative image of XMRV-SO orange staining from a cell mixture of DU145 (uninfected; negative XMRV staining) and 22Rv1 (XMRV-infected; strong positive XMRV staining), showing two positively stained cells. (B) DAPI nuclear staining. (C) CEP8-SA aqua staining illustrating two and three CEP8 aqua signals per 22Rv1 and DU145 cell, respectively; (D–F) Representative images of FFPE prostate cancer tissue sections from patient VP62 (XMRV-SO, DAPI, and CEP8-SA, respectively). No XMRV-SO orange staining is observed. The white rectangle outlines the region magnified in panels G-I (G–I) A magnified image of FFPE prostate cancer tissue sections from patient VP62 (XMRV-SO, DAPI, and CEP8-SA, respectively). At this magnification, CEP8-SA aqua staining is clearly visible (panel I; white arrows highlight two representative CEP8 aqua signals). (J–L) Representative images showing no XMRV-SO orange staining in FFPE prostate cancer tissue sections from 3 representative patients (among the prospectively collected cohort of 39 patients).
Figure 4
Figure 4. Serological Detection of Antibodies to XMRV in Prostate Cancer Patients.
Evaluation of 39 plasma samples from prostate cancer patients for the presence of antibodies to XMRV/MLV using recombinant-based XMRV p15E and gp70 chemiluminsescent microparticle immunoassays (CMIAs) . The x-axis represents the CMIA signal expressed in units of natural log–transformed signal ratio of sample to the cutoff (log N of S/CO); values greater than 0 are considered positive. Signals of the positive controls (PC1 and PC2) corresponding to XMRV-infected macaque plasma and negative control (NC) corresponding to a normal blood donor are highlighted in dark green.
Figure 5
Figure 5. Genomic Coverage of Cell Line and Prostate Cancer-Associated XMRV Strains by Deep Sequencing.
RNA extracts of 22Rv1 and LNCaP cells, prostate cancer tissues from the 2006 Urisman, et al. study [VP35, VP42, and VP62(2006)], and re-extracted tissue from the VP62 sample, VP62(2012), were analyzed by unbiased deep sequencing. Reads are mapped to the previously sequenced XMRV genome corresponding to each of the samples, with the exception of reads from LNCaP, which are mapped to the 22Rv1-associated genome (Genbank accession number FN692043). The coverage (y-axis) achieved at each position along the ∼8.2 kB XMRV genome (x-axis) is plotted on a logarithmic scale. Abbreviations: nt, nucleotide.
Figure 6
Figure 6. Lack of Diversity Among XMRV Strains Detected in Laboratory Cell Lines and Prostate Cancer Tissues.
By SNP analysis, single nucleotide differences between the sequences of 22Rv1-associated XMRV and XMRV genomes detected in prostate cancer tissues [VP35, VP42, and VP62(2006)] (red lollipops) are corrected by the deep sequencing coverage data (black lollipops). The depth of read coverage achieved at the nucleotide position corresponding to each SNP is displayed below the x-axis. All reads covering a given position yielded the same (corrected) nucleotide, indicating that previous nucleotide differences in published genomes (red lollipops) are due to sequencing error. A natural A→G polymorphism in the XMRV genome , is present at position 790 (cyan lollipop). Note that XMRV consensus genomes associated with 22Rv1, LNCaP, and the 3 XMRV-positive prostate cancer tissues are identical.
Figure 7
Figure 7. Evidence for Contamination of Prostate Cancer Tissues by XMRV-Infected LNCaP Cells.
SNP analysis of deep sequencing reads corresponding to the XMRV genomes of 22Rv1 (A) and LNCaP (B), as well as the mitochondrial genomes of these two cell lines (C) was performed. (A and B) SNP variants within the XMRV genome for 22Rv1 and LNCaP (x-axis) are shown in order of decreasing frequency (y-axis). Shared SNPs in XMRV genomes corresponding to 3 XMRV-positive prostate cancer samples (VP35, VP42, VP62) and LNCaP (z-axis) at a frequency cutoff of 0.5% are plotted on the graph, with key SNPs highlighted in red. SNPs with variant frequencies <0.5% are plotted as zero; missing values (blank squares) refer to SNP positions for which the coverage is <10X. (C) SNP variants within the mitochondrial genome for 22Rv1 and LNCaP are shown (x-axis), with the frequency of each 22Rv-1−/LNCaP-associated SNP in the general human population, as determined by a population-level human mitochondrial database , given in parentheses. For each prostate cancer-associated mitochondrial genome (z-axis), the minority SNP frequency (y-axis) is plotted against the cell line-associated SNP variant (x-axis), using a frequency cutoff of 3%. For each minority SNP identified, the variant frequency and coverage at the corresponding nucleotide position is shown. Minority SNPs with variant frequencies <3% are plotted as zero; missing values (blank squares) refer to SNP positions for which the coverage is <30X. Note that the VP62 sample shares a 146C mitochondrial SNP with LNCaP (asterisks).
Figure 8
Figure 8. Proposed Model for Laboratory Contamination by XMRV.
Early contamination of VP35/VP42 prostate cancer tissues and/or extracted RNA by XMRV-infected LNCaP cells resulted in mistaken identification of XMRV in association with prostate cancer.

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