Wednesday, February 22, 2012
Evidence for an XMRV-like sequence in prostate cancer?
Just saw this one today, published in the British Journal of Cancer, a respectable journal-- despite the title of the paper, "No evidence for the involvement of XMRV or MCV in the pathogenesis of breast cancer.", the paper goes on to describe single-round PCR amplification of an XMRV type sequence in two cases of prostate cancer (out of 12). They had optimized the reaction so as to detect 700 copies of purified plasmid DNA -- one sample did not elicit sufficient product to clone, but the other did, and upon sequencing it was the same as the VP62 sequence with two exceptions - indicating it is not likely to be contamination from the plasmid the investigators used as a control. One caveat, the group does not appear to have tested the tissues for mouse DNA -- and they acknowledge this by admitting that the sequence could be from exogenous DNA. If you have access to the journal, you can read the paper here.
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So they found their samples were contaminated with VP62 (plasmid, I suppose), with only two exceptions? Were they didn't test for mouse DNA? How reassuring. Hold the press, we might be onto something here!
ReplyDeleteBut, please, by all accounts, go ahead and suit yourself on a wild goose chases anytime!
awww, Mr. Mach, I'd be disappointed if you didn't post on my blog -- to clarify, they only found two single-round PCR positive samples out of all the samples they examined, 12 of which were prostate tumors. They were only able to amplify enough DNA to purify and sequence from one of those patients, and the sequence had two mismatches when compared to VP62 - suggesting that it is not contamination with the control plasmid...but in the interests of being fair, yes, I was surprised that this got in to BJC, without including mouse testing, which I would have thought was mandatory by now - by the way, I never did see you comment on the next-gen sequencing post -- http://okeefe-lab.blogspot.com/2011/12/next-gen-sequencing-provides-evidence.html -- I'd be interested to get your take on that --
ReplyDeleteXMRV has never been detected with single round PCR, so they are lucky to have found anything as their analytical sensitivity was very poor. A rt PCR with an analytical sesitivity of one copy per reaction tube missed 75% of infected patients. What do people have against repeating experiments?
ReplyDeleteThey should have used a validated mouse assay and if they found contamination they should have repeated the study with the same XMRV assay and in addition those from other positive studies.
Denise I am interested to hear your thoughts on this from Kearney et al. (2012)
“The Taqman probe used for detection of amplified products was designed to span the signature 9–24 nt deletion in the XMRV gag leader absent from all endogenous MLV sequences (with the exception of PreXMRV-2)”
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0030889#pone.0030889-Oakes1
They say that no mouse ERV has a 9 nt deletion in the glycogag leader. But the patients in the study have a 9nt deletion in the 9 nt deletion in the glycogag. You can see this in Figure 1 C.
I apologise, that should have been.
ReplyDeleteThey say that no mouse ERV has a 9 nt deletion in the glycogag leader. But the patients in the study have a 9nt deletion in the glycogag. You can see this in Figure 1 C.
The IAP and COX2 assays are unvalidated, as they are different from those used in other papers.
ReplyDeleteBy the time the COX2 and IAP assays were used the investigators knew which samples were positive in Kearney et al.
ReplyDelete"Because the X-SCA results suggested high levels of mouse DNA contamination in the CFS patient samples, we used three experimental approaches to detect mouse DNA. We tested for mouse mitochondrial DNA using an assay that detects the COX2 gene (Table 1), for mouse genomic DNA using an assay that detects intracisternal A-particle (IAP) sequences [14] (Table 1), and we performed X-SGS of a 1.4 kb fragment of the XMRV/MLV gag gene to determine the source of the amplified nucleic acid in plasma samples""
Does this mean that MLV research should be carried out on survivors of the ME epidemics first?
ReplyDelete