Wednesday, December 14, 2011

Next Gen sequencing provides evidence for MLVs and XMRV in prostate cancer! Eh? What's this all aboot?

In an epub that came up in my regular pubmed search today, we see that our Canadian friends have been doing next-gen sequencing of prostate cancer samples.  The publication is entitled "Next generation sequencing of prostate tumours provides independent evidence of XMRV contamination".  Groan. Seriously, how much can this one topic take.  But then I took a closer look at the data, and despite the title, they have some very interesting findings -- the manuscript is from Colin Collins' lab at the University of British Columbia and Vancouver Prostate Centre.  The paper, conveniently super-short, describes some results from genomic and transcriptomic sequencing of tissue samples from 9 prostate tumors or their metastatic deposits, and from 3 human tumor xenografts that were grown in mouse.  The authors conclude that because they detected XMRV in two of the human tumors that were not grown in mouse, but those tumors also have mouse mitochondrial DNA and were prepared on a different day to the other (XMRV negative) tumors, they detected their own contamination of the tissue.  ummm...OK.

Firstly, this study was kind of neat because as opposed to all the PCR-based methods to detect XMRV or MLVs in human tissues, next-gen sequencing is unbiased -- it will sequence whatever is there -- PCR primers are designed against a particular sequence, and if the conditions are not right or there has been some mutation of the original published sequence (as we'd expect from actively replicating or mutating viruses), the product may not amplify.  What these guys did was avoid that issue.  They used xenografts of human tissue implanted in mice as positive controls for what mouse viruses would look like.

 In  Table 1 of the manuscript they summarize the data- as expected, human tissues grown in and excised from mouse had mouse viruses (MLVs and apparently XMRV - I say apparently because it is not clear if the authors mean that the exact same sequence as VP62).  For MLV transcriptomes, there was between 327 and 443 reads per million sequence reads, and for XMRV there was between 169-220 per million reads.  So MLV and XMRV RNA was there.  DNA sequencing from two of the xenografts showed between 6 and 10 MLV DNA reads per million, and 2-4 XMRV DNA reads per million.  So detecting MLV and XMRV DNA copies using next-gen is obviously alot less sensitive than detecting it in the transcriptome - these tissues were contaminated (according to the paper) with significant mouse tissue, so we know there was genomically integrated viral DNA.  Therefore the sensitivity for MLV DNA was 30-70 fold less than for RNA, and for XMRV DNA detection was 40-80 fold less sensitive.  But that's for the controls.  Analysis of the human tumors resulted in ALL being positive for some level of MLV RNAseq reads.  Six samples were positive for XMRV.  The number of reads per virus type was much less than the xenografts grown in mouse -- the authors took the two tumors with the highest level of XMRV (each around 11 reads per million) and did next-gen DNA sequencing on them, as well as on one XMRV negative tumor.  They did not get any DNA reads positive for either MLVs or XMRVs.  But hang on, what was their sensitivity? By comparing their numbers of RNA positive reads, which ranged from as low as 0.1% of that seen in the tissues from mice -- so accordingly, we expect about 0.009 -0.16 MLV reads at the DNA level, if all things were equal.  To counter the argument that they might not have the sensitivity to exclude these viruses at the DNA level, the authors argue that published data suggests XMRV is expected to integrate on every chromosome within infected genomes. They reference Sam Chow's lab's paper from 2008, "Integration site preference of xenotropic murine leukemia virus-related virus, a new human retrovirus associated with prostate cancer".  But that same lab just published (click here) saying that at least from the cases they could tell, their samples were contaminated with DNA from infected DU145 cells.  And I'm pretty sure they never said that XMRV integrates on every chromosome in every infected cell.

So what to make of all this?  I don't know right looks like there are MLV and even XMRV transcriptomes present in some prostate cancer tissues.  There might not have been the sensitivity to detect the viruses at the DNA level.  And assuming this data will be borne out by other next-gen sequencing studies, what does it all mean?  I guess we have to await some more next-gen sequencing to find out - meanwhile, if you want to read the paper - click here-- but you might need access to the journal of clinical microbiology....

Wednesday, December 7, 2011

Dietary Methyl Donors and Progression of Prostate Carcinogenesis...

Carducci's lab at Hopkins have just published a study looking at the effect of excess dietary methyl donors, for example folic acid, methionine, choline, B12 and betaine - on prostate cancer progression in mice.  As this is pretty much what we do in the lab, I was excited -- we have previously published (Tomaszewski et al.) on the very high levels of serum folate in our prostate cancer patients, and the significant increased cancer cell proliferation in patients with high versus low serum folate -- this study concludes that in mice, and in cell culture (in vitro) high levels of folic acid and / or methyl donors, does not result in an increase in tumor growth rate or the ability of a demethylating agent to cause demethyation of the AR promoter, or the Reprimo promoter.  In fact, they find a protective effect of the supplemented diet on the development of higher grades of prostate cancer in the hi-myc mouse model.  The paper is well-written and has an unbiased appraisal of the literature - unfortunately I don't think they can make the conclusions that they are -- I think some of the experiments would be worth doing, but using relevant diets - you'll see what I mean below -

1)  the xenograft model using DU145 and PC3 prostate cancer cells
The cells were grown in mice being fed either the Teklad 18% protein diet, or the same diet supplemented with methyl donors, including 15mg/kg folic acid.  Teklad's base diet has 3mg/kg folic acid from the added vitamin mix, and an estimated further 1mg/kg from natural folates -- found in the brewer's yeast, corn, wheat etc. that the diet is made from.  2mg/kg is the basal level of folic acid for rodent diets, so this diet starts out with twice as much.  The supplemented diet has 15mg/kg folic acid on top of this, so is closer to 19mg/kg.  The cup can only get so full guys!  Figure 1c has the ki67 labelling index for the xenograft tumors (both DU145/PC3 together?) -- on the regular and methyl supplemented diet -- yikes!  is that 55% proliferating at any one time in the saline groups?  Is it possible that the cells literally can not grow any faster than they already do on the "regular" diet?  In humans, the proliferation index for prostate cancer is considered high when it is around 5% -- so how do the circulating folate levels compare to humans for these mice?  Well the ones on the so called "regular" diet have a mean plasma folate of around 200nM, the supplemented group has 340nM (mean values, calculated from the ng/ml value x 2.27).  In our lab studies, patients range from <10nM, to our patient with the highest serum folate we've seen, somewhere around 160nM.  Just because the cells aren't growing faster doesn't mean supplemental folic acid is not driving prostate cancer proliferation in humans --  in addition, the statistics used in this manuscript don't seem to mesh with the data as presented -- t-tests are for normal distributions, but as seen in Fig 4 - the distributions aren't all normal - the plasma folates in the methyl supplemented group have outliers -- the mann-whitney rank sum would be better -- although from guestimating the numbers, it would still be significant (as expected).  The problem is many of the other analyses that are not significant have just 4 data points, and as it seems this manuscript wasn't seen by a statistician, we can not be sure that there was sufficient power to make the non significant determination. 

2)  Cell culture assay
The next issue is the media used for the cell culture assay that showed that myc was expressed similarly in regular media, versus folic acid supplemented media.  We assume it was folic acid supplemented, but it is not mentioned in the methods section and could have been another folate.  Anyhow, DMEM has 9 micromolar folic acid in the standard formulation -- and the authors supplemented that with another 226 micromolar, for a comparison of 9 vs 235 micromolar folic acid containing DMEM. Smiraglia's lab at Roswell has shown that the proliferative capacity of prostate cancer cell lines in response  to folic acid is maxed out somewhere around 200 nanomolar.  Yep, 1000-fold less than the 235 micromolar being tested here, and not relevant to the mouse model above, either.

Why do I care?  The last thing I want to do is criticize someone's freshly minted manuscript -- in fact, I usually won't comment on a paper unless I think it is well done -- however we are talking about figuring out what levels of folic acid intake are safe in cancer patients, and once someone gets an idea out into the scientific mainstream that "supplemental folic acid is safe, does not cause increased proliferation of cancer cells and may even reduce cancer", it is very hard to get the idea back again - and that affects funding and publishing decisions.  So while I think that the idea was great, and I'm glad to see that the methylation inhibitor assay was not affected by huge folate doses (although would it be more effective in a low-folate environment?) - this paper, at least to my mind, does not answer the question that it set out to answer.  But hey, don't believe me -- read it for yourself: 

Cancer Prev Res (Phila). 2011 Dec 2. [Epub ahead of print]

Progression of Prostate Carcinogenesis and Dietary Methyl Donors: Temporal Dependence.

Shabbeer S, Williams SA, Simons BW, Herman JG, Carducci MA.


Insufficient dose of dietary methyl groups are associated with a host of conditions ranging from neural tube defects to cancer. On the other hand, it is not certain what effect excess dietary methyl groups could have on cancer. This is especially true for prostate cancer (PCa), a disease that is characterized by increasing DNA methylation changes with increasing grade of the cancer. In this three-part study in animals, we look at (i) the effect of excess methyl donors on the growth rate of PCa in vivo, (ii) the ability of 5-aza-2'-deoxycytidine, a demethylating agent, to demethylate in the presence of excess dietary methyl donors and (iii) the effect of in utero feeding of excess methyl donors to the later onset of PCa. The results show that when mice are fed a dietary excess of methyl donors, we do not see (i) an increase in the growth rate of DU-145 and PC-3 xenografts in vivo, or (ii) interference in the ability of 5-aza-2'-deoxycytidine to demethylate the promoters of Androgen Receptor or Reprimo of PCa xenografts but (iii) a protective effect on the development of higher grades of PCa in the "Hi-myc" mouse model of PCa which were fed the increased methyl donors in utero. We conclude that the impact of dietary methyl donors on PCa progression depends upon the timing of exposure to the dietary agents. When fed before the onset of cancer, i.e. in utero, excess methyl donors can have a protective effect on the progression of cancer.
[PubMed - as supplied by publisher]