A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor-specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53-regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53-(wild-type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank ¼ 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino-embryonic antigen (P ¼ 0.029) and with poorly differentiated tumors (P ¼ 0.039) and tended to have shorter overall survival than patients with low levels (P ¼ 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival.

MicroRNAs (miRNAs) are noncoding RNAs that regulate expression of target mRNAs and are controlled by tumor suppressors and oncogenes. Altered expression of specific miRNAs in several tumor types and its association with poor prognosis parameters have been reported. Fewer data are available on its impact on patients' survival. We studied the impact of the expression of miR-17-5p, miR-106a, and miR-126 on survival and its correlation with the levels of their target mRNAs and host gene and TP53 alterations. We assessed in 110 colon cancer patients the levels of miR-17-5p, miR-106a, miR-126, E2F1, and EGFL7 by quantitative real-time RT-PCR and loss of heterozygosity (LOH) in the TP53 region. Tumor characteristics, disease-free survival (DFS), and overall survival (OS) were examined in each patient. Altered expression of miR-17-5p, miR-106a, and EGFL7 was associated with pathological tumor features of poor prognosis. Downregulation of miR-106a predicted shortened DFS (P = 0.03) and OS (P = 0.04). miR-17-5p correlated with DFS only at early stages (P = 0.07). Inverse correlations were found between miR-17-5p and miR-106a levels and their target expression, E2F1 (P = 0.04 and P = 0.03, respectively). No correlation was found between miR-126 expression and its host gene levels, EGFL7. miR-106a deregulation was revealed as a marker of DFS and OS independent of tumor stage. The lack of association between expression of miR-126 and its host gene EGFL7 suggests their regulation by independent stimuli. Inverse correlation between miR-17-5p and miR-106a and E2F1 levels supports E2F1 as a target mRNA for the two miRNAs.

The identification of tumour biomarkers that detect the presence of disease using noninvasive diagnostic procedures is a key part of cancer research. We determined in plasma the vesicle-related microRNA (miRNA) expression profile of nonsmall cell lung cancer (NSCLC) and evaluate whether plasma miRNAs can be both discriminating (between patients and healthy controls) and prognostic markers.365 human miRNAs were analysed by Taqman1 low-density arrays (Applied Biosystems, Foster City, CA, USA) in the plasma from 28 NSCLC patients and 20 controls. Five selected miRNAs (let7f, miR-20b, miR-30e-3p, miR-223 and miR-301) were validated independently by real-time PCR in plasma from 78 NSCLC and 48 controls and correlated with pathologic parameters and survival.Levels of let-7f, miR-20b and miR-30e-3p were decreased in plasma vesicles of NSCLC patients. Moreover, levels of let-7f and miR-30e-3p distinguished between two groups of patients for stage of disease and therefore possibility of surgery. Plasma levels of miR-30e-3p and let-7f were associated with short disease-free survival and overall survival, respectively. NSCLC patients and healthy controls differ in vesicle-related miRNAs in plasma. Levels of let-7f and miR-30e-3p in NSCLC patients are associated with poor outcome. Thus, plasma vesiclerelated miRNAs obtained by noninvasive methods could serve as circulating tumour biomarkers of discriminating and prognostic value.

Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1-to 2-mm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin.

Introduction Deregulation of Polycomb member Bmi-1 is involved in cell proliferation and human oncogenesis. Modulation of Bmi-1 is found in several tumor tissues, including primary breast carcinomas; however, analysis of Bmi-1 in plasma of cancer patients has not been reported. This is the first study that evaluates Bmi-1 in plasma by using a large series of primary breast carcinomas to investigate the presence at diagnosis of detectable Bmi-1 mRNA in plasma and possible correlations between this event and a series of clinicalpathological parameters of the tumors.