Noerholm et al. BMC Cancer 2012, 12:22 http://www.biomedcentral.com/1471-2407/12/22 sources and it is likely that tumour microvesicles in many cases constitute a relatively small fraction of the total population. There is a large contribution of microvesicles from normal cells and platelets in blood. In control plasma, for example, platelet-derived microvesicles have been reported to constitute the majority of all microvesicles [27] and although tumour-derived microvesicles increase in serum as a function of degree of malignancy in ovar- ian cancer [30] and that the total number of microvesi- cles in blood increase with disease progression [29], it is not clear what proportion of the increased number of vesicles is coming from the tumour vs. from normal cells as a response to the tumour. Thus, down-regulated genes in GBM exoRNA could either be caused by altered biological processes in normal cells as a response to or consequence of the disease, or by large numbers of tumour microvesicles with low levels of particular RNAs. A major fraction of the genes we observed to be sig- nificantly down-regulated in serum microvesicles from GBM patients in this study code for various ribosomal proteins (e.g. RPL11, RPS29, RPLP1, RPS27A, etc.). However, these genes were not found to be among the most abundant transcripts in platelets in a study by [31], which we also confirmed in our analysis of the public dataset from GEO (GSE11524). We speculate that these RNAs would therefore also not be abundant in the pla- telet-derived microvesicles, which constitute a substan- tial fraction of the plasma microvesicles [27]. However, these genes have been shown to be very highly expressed in lymphocytes relative to other blood cells [23,24] and were also found to be up-regulated in our analysis of the public dataset for PBMC (GSE22224) relative to GBM. This led us to speculate that the lower abundance of these mRNAs in circulating microvesicles from GBM patients may be a consequence of a reduced level of lymphocyte-derived microvesicles in patients as compared to normal controls, since cancer patients are known to often be immune-compromised [26]. Evalua- tion of the GBM patients in this study confirmed that their lymphocyte counts were lower than the normal reference level interval, but because we were unable to measure lymphocyte counts in the normal controls, it is impossible to completely correlate transcript down-regu- lation with lymphocyte depletion in the GBM patients. None of the GBM patients had received chemotherapy prior to diagnosis, but several of them were on steroids and seizure medications at the time of blood draw, which are two potential sources of lymphocytopenia. Evaluation of gene expression differences between lym- phocytopenia caused by cancer and by medication would require a much larger study of more carefully chosen subjects. Page 8 of 11 The observation that many ribosomal protein genes are down-regulated in GBM serum microvesicles may prove to be a valuable contributory marker for GBM and other pathological states. Sharma et al. [5] also observed a reduction in many of the same genes in PBMCs including lymphocytes, from breast cancer patients compared to normal controls. The down-regu- lation of ribosomal protein genes is therefore unlikely to provide a specific GBM component for a diagnostic classification, but may serve as a more generic patholo- gical indicator and can provide confirmatory support when combined with other exoRNA profiles. Interest- ingly, many ribosomal proteins have extra-ribosomal functions that go beyond the function of protein bio- synthesis. Ribosomal protein genes have been shown to have important regulatory functions also in cancer cells. Some ribosomal proteins are tumour suppressors [32], oncogenes or have regulatory functions in tumour pro- gression, invasion and metastasis [33]. The “ribosome” GO-clusters from down-regulated genes had very high enrichment scores from DAVID (> 50) compared to any of the clusters obtained with up-regu- lated genes (< 2.5), confirming that the patterns of down- regulated genes were much more significant. For gene ontology analysis it is important to analyze a relatively large number of genes (e.g. 100 [22]) in order to avoid sto- chastic errors, e.g. if a list of 10 genes is analyzed, the pre- sence of a single gene (10%) of a certain class or belonging to a certain pathway will appear overrepresented com- pared to the prevalence in the genome, when in fact this is just a sampling artifact. The lack of strong GO associa- tions between the up-regulated genes in our study does not mean that individual genes might not be strongly dys- regulated, significant and predictive in GBM exoRNA, but simply that the up-regulated genes do not appear to be associated by already known relationships. Up-regulation of specific genes in exoRNA from GBM patients could be a biological response from normal cells to the presence of the tumour, but they could also be specifically derived from the tumour cells and as such be useful as markers of the tumour. However, identifying uniquely elevated levels of tumour-derived exoRNAs in a high background of nor- mal exoRNA from other serum microvesicle sources is more challenging than identifying systemic changes, such as the depressed lymphocyte count and ribosomal protein RNA levels. The observation that the up-regulated genes chosen for qRT-PCR validation was not verified may be attribu- ted to the fact that we used two different sets of serum samples for the microarray analysis and the qRT-PCR validation, making borderline significant increases diffi- cult to confirm. Similarly, the positive validation of the down-regulated genes in a separate set of samples makes this observation stronger.