Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine

Abstract

Background: The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs.

Methods: Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing.

Results: RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG.

Conclusions: Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection.

Keywords: ERG; PCA3; biomarkers; prostate cancer.

FIGURE 1

RNA yield from the pellet and extracellular vesicle (EV) fraction of post-DRE urine. (A) Urine was collected following a DRE and processed to isolate the cell pellet and EV fraction. For each sample, the amount of RNA extracted is represented as a stacked bar, with the amount isolated from the EVs shown in dark gray while the amount of RNA isolated from the cell pellet is shown in light gray (n = 105). The y-axis has been split to improve visualization of the low yield samples. (B) Summary data of the data in A is represented with boxplots (outliers are not shown). The RNA yield was significantly higher in the EVs (P < 0.001). (C) The frequency of RNA quality is shown for EVs (upper plot) and cell pellets (lower plot). The RIN scale goes from 1 to 10, with samples of particularly low quality or concentration unable to have RINs determined (shown as N/A). Representative Bioanalyzer plots are shown in Supplementary Fig. S2. (D) The yield of EV RNA was not significantly different amongst patients with no evidence of disease (N.E.D.) or various levels of prostate cancer risk (P = 0.275; n = 34, 28, 36, and 11; one outlier in the GS7 group at 506 ng not shown)

FIGURE 2

Analysis of urinary extracellular vesicles (EVs) by TEM. The EV fractions recovered from two different patients were negatively stained and imaged by TEM. The observed particles include larger microvesicles (arrows) as well as smaller vesicles likely to be exosomes (arrowheads; scale bar on both images represents 200 nm)

FIGURE 3

Prostate-specific genes are enriched in post-DRE urine EVs. The expression of various prostate-, kidney-, or bladder-specific transcripts were measured by TaqMan qPCR and are shown relative to the expression of RAB7A (n = 60). The boxplots for AQP2, CUBN, NPHS2, and SLC34A1 include data for samples where no amplification was observed and the cycle threshold was set to 40 (8, 1, 20, and 2 samples, respectively)

FIGURE 4

Prostate cancer-associated RNAs are detectable and informative in post-DRE urine EVs. The expression levels of (A) KLK3, (B) PCA3, and (C) ERG were measured by TaqMan qPCR assay and normalized to RAB7A. The data are shown for patients with no evidence of disease (N.E.D., n = 12), Gleason score 6 prostate cancer (GS6, n = 14), or Gleason score ≥7 prostate cancer (GS7+, n = 26). The ERG plot includes data for nine samples where no amplification was observed and the cycle threshold was set to 40 (2, 2, and 5 samples for the N.E.D., GS6, and GS7+ groups, respectively). In comparison to the N.E.D. group, the GS6 and GS7+ groups both had significantly higher expression of PCA3 (P = 0.002 and P < 0.001, respectively), while only the GS7+ group both had significantly higher expression of ERG (P = 0.027). There was no significant difference in KLK3 expression between the any of the groups (P = 0.672)

FIGURE 5

Precise Assay measurement of transcripts also shows enrichment of prostate-specific and prostate cancer-associated transcripts. (A) The same prostate-, kidney-, and bladder-specific transcripts shown in Fig. 3 were assessed by Precise Assay and normalized to RAB7A (n = 60). KLK3 is shown on a different scale because it was present at much higher levels than the other transcripts. The boxplots for AQP2, CUBN, NPHS2, PTH1R, SLC12A1, SLC12A3, SLC34A1, UPK1B, and UPK2 include data for samples where no transcripts were detected and the normalized expression was set to 0 (17, 7, 60, 60, 15, 54, 60, 17, and 22 samples, respectively). Transcripts for (B) KLK3 and (C) PCA3 were measured by Precise Assay and normalized to RAB7A (n = 12, 14, and 26 for the N.E.D., GS6, and GS7+ groups, respectively). The PCA3 plot includes data for 14 samples where no transcripts were detected and normalized expression was set to 0 (6, 4, and 6 samples for the N.E.D., GS6, and GS7+ groups, respectively). GS7+ patients had significantly higher expression of PCA3 as compared to the N.E.D. group (P = 0.003). (D) The ratio of sequence reads to molecular index count is shown for all of the prostate-associated genes and the normalizer RAB7A (means and 95% confidence intervals shown)