All murine retroviral primer sequences amplified specific products of the appropriate size when tested on pXMRV isolate VP62, an infectious molecular clone of XMRV, kindly provided by R. Silverman. The IAP primers did not amplify sequences from human DNA extracted from LNCaP cells (prostate cancer cell line) or from six PBMC samples from human prostate cancer patients (data not shown).
FFPE prostate tumour slices (two 10 micron slices from each lobe of the prostate) were provided to us blinded by the Histopathology Department at St Mary's Hospital in batches and randomised with benign prostate hyperplasia specimens. For samples received from Thailand and Korea, those carrying out the PCR were blinded to sample provenance. In all cases, care was taken to use a fresh blade for slicing each patient sample, and the top slice was discarded. In total, of 292 UK prostate cancers analysed, 14 were XMRV-positive by PCR using the gag leader primers, as were five out of 139 Korean samples and two out of six from Thailand. A representative result is shown in Figure .
Figure 1 Nested PCR on DNA extracted from FFPE tissue of prostate cancer patients. Figure (a) shows samples that produced a PCR product of the expected size using primers specific for XMRV (lanes 1-5). UK patient 308 and UK 244 (Lane 1 and 2); Thailand 1 and Thailand (more ...)
All FFPE prostate cancer specimens from the Pathology Department at St Mary's hospital London were provided to us in batches. The tissue slices were coded and assayed blind. Initially, the PCR amplification and sequence analysis of the amplicons encouraged us to deduce that we had detected a genuine XMRV infection in some of the samples. When on unblinding we found a concordant result from the same patient whose duplicate specimens had been provided in different batches, this appeared to reaffirm a genuine XMRV infection. Moreover, in two patients in whom the tumour was unilateral, XMRV was detected only in the cancerous lobe. Taken together with the consistently negative PCR water controls, the probability of contamination appeared to be low.
Upon sequencing, however, we noticed differences in the obtained PCR products. Some sequences displayed the deletion characteristic of XMRV upstream of the gag
ATG (Korean samples 12, 35, 15), and UK (sample 244), while others did not (Thai patients 1 and 2 and Korean 16) (Figure ). Downstream of this deletion all sequences are identical, apart from an A > G mutation at position 647, but there is no correlation of the A647G mutation with the presence of the deletion. Sequences that did not contain the deletion were amplified, indicating that the primers were not as specific to XMRV as expected. A BLAST search showed the best match for sequences without the deletion but containing a G at position 647 to be mouse endogenous polytropic provirus clone 15 [Genbank:FJ544577.1
], which suggests the presence of mouse DNA in these samples. It has been shown recently that the 24 bp deletion specific to XMRV [1
], is also present in the sequence of a polytropic endogenous MLV sequence [Genbank:AAHY01591888.1
] in the laboratory mouse strain 129X1/SvJ, commonly used in gene knock-out experiments [21
]. Our sequences (Korean samples 12, 35, 15; and UK sample 244) are identical to this PMLV sequence present in strain 129X1/SvJ (Figure ). We therefore, sought to investigate this potential contamination further.
Figure 2 Sequence alignment of XMRV LTR from 7 prostate cancer patients. The gag leader primer set XMRV-R-I/XMRV-F-O bind either side of the XMRV specific deletion. Sequences were aligned against VP62 [Genbank:EF185282], MLV-releated virus CFS isolate CSF-type1 (more ...)
The IAP and mtDNA PCR assays were applied to 10-fold dilution series of McCoy cell and RAW 264.7 cell DNA to compare the sensitivity of the methods for detection of genomic mouse DNA. We found the mtDNA PCR (14) 100-fold less sensitive than that for IAP in both cell lines (see Additional File 2
; Figure S1). The IAP PCR, thus, provided a far more reliable indicator of contaminating murine sequences. The IAP and mtDNA PCR assays were applied to the same sample to test whether the apparent XMRV positivity might have been due to mouse DNA contamination. Amplification of XMRV-specific sequences was completely concordant with amplification of IAP sequences from the same DNA (Table ). Samples from 292 UK patients, of which 212 (73%) were cancerous, 68 (23%) were benign and 12 (4%) were lost to follow up, along with 139 Korean samples (all cancerous) and 6 Thai samples (50% cancerous, 50% benign) were tested by XMRV, IAP and mtDNA by PCR. Twenty-one samples were positive for XMRV using the gag
leader primers, and of these, 17 were from cancerous tissue and 4 from benign tissue. Overall, 115/437 (26.3%) of the samples, including all 21 of the XMRV-positives were positive for IAP sequences and 21/115 (18.2%), of the IAP positives contained mouse mtDNA. To confirm that the sequences amplified by the IAP primers were indeed murine in origin, we cloned and sequenced one of the amplicons. Several IAP sequences were obtained (see Additional File 3
; Figure S2). The fact that not all IAP positives were XMRV positive may be explained by the low level of contamination of murine DNA that would contain only a few copies of endogenous XMRV like sequences compared to the many IAP copies per genome.
Frequency of positive PCR reactions using XMRV LTR primers, mtDNA primers and IAP primers.