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

et al. (2012) In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer. PLoS ONE 7(9): e44954. doi:10.1371/journal.pone.0044954 In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer Deanna Lee Jaydip Das Gupta Christina Gaughan Imke Steffen Ning Tang Ka-Cheung Luk Xiaoxing Qiu Anatoly Urisman Nicole Fischer Ross Molinaro Miranda Broz Gerald Schochetman Eric A. Klein Don Ganem Joseph L. DeRisi Graham Simmons John Hackett Jr. Robert H. Silverman Charles Y. Chiu Gilda Tachedjian, Burnet Institute, Australia 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 laboratoryderived 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 reanalysis. 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. - Funding: The authors gratefully acknowledge support from NIH/NCI CA103943 (R. Silverman), as well as the Charlotte Geyer Foundation, the Maltz Family Foundation, and Abbott Laboratories (to R. Silverman and E. Klein). G. Simmons is supported by grant 1R21HL109761 from the National Heart, Lung, and Blood Institute (NHLBI). J. DeRisi is supported by the Howard Hughes Medical Institute. C. Chiu is supported by NIH grants R56-AI08952 and R01-HL105704, as well as an Abbott Viral Discovery Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. In 2006, sequences corresponding to a novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) were identified in tissue from prostate cancer patients following radical prostatectomy [1]. The discovery of XMRV was accomplished using a broad-spectrum microarray assay (ViroChip) designed to detect all known viruses as well as novel viruses on the basis of sequence homology [1,2,3]. The results from this study also revealed an association between the presence of XMRV and patients known to be homozygous for the R462Q variant of RNAse L, a gene previously linked to the hereditary prostate cancer 1 locus [4]. Mutations in RNAse L that impair the apoptotic response to viral infection were postulated to reflect enhanced susceptibility to infection by XMRV and suggested a potential role for the virus in carcinogenesis [5,6,7,8]. Although the initial study reported a link between RNAse L-variant prostate cancer and XMRV infection, most, but not all, subsequent studies have failed to detect such an association [9,10,11,12]. Since this initial discovery, XMRV and MLV-related virus sequences resembling polytropic MLVs (P-MLVs) were also found in patients with chronic fatigue syndrome (CFS) [13,14]. Subsequent reports have cast doubt on the association of XMRV with prostate cancer or CFS, and indeed on whether XMRV is even found in humans (reviewed in [15]). Moreover, the viral sequences from XMRV-positive patients lacked the level of genetic diversity expected for retroviral infections [1,14], implying that XMRV may have arisen from sample contamination and not true viral infection. Nearly all follow-up studies using specific PCR have largely failed to confirm the presence of XMRV in either CFS or prostate cancer cohorts [12,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32], resulting in retraction of the initial papers linking XMRV and P-MLVs with CFS [33,34]. A 2009 study found, unexpectedly, that a common laboratory cell line called 22Rv1, derived from the CWR22 human prostate cancer xenograft, produced high titers of XMRV [35]. This was followed by a study from Garson, et al. demonstrating that identical XMRV integration sites were shared between putatively infected prostate tumor tissues and an experimentally infected laboratory cell line [36,37], further undermining the prospect that XMRV is a genuine human pathogen. Finally, a 2011 study from Paprotka, et al. provided strong evidence that XMRV is a 22Rv1-derived laboratory contaminant originating from recombination of two mouse endogenous retroviruses during serial passage of CWR22 in nude mice [38]. The recent demonstration that XMRV and related viruses are not present in the primary prostate tumor tissue from the patient CWR22 lends additional support for this hypothesis [39]. Given the clinical and public health implications of potential XMRV infection in humans, we sought to confirm or refute the association between XMRV and prostate cancer. To date, most of the negative studies have been carried out in CFS and not in prostate cancer, and some have speculated that the original discovery of XMRV may in fact reflect bona fide viral infection but that subsequent studies were potentially tainted by mouse genomic contamination and/or widespread circulation of positive control plasmids containing the XMRV infectious molecular clon (...truncated)