PLoS One. 2012;7(9):e45656.

A multiplex assay to measure RNA transcripts of prostate cancer in urine.

Quek SI, Ho ME, Loprieno MA, Ellis WJ, Elliott N, Liu AY.

Department of Urology, University of Washington, Seattle, WA, USA. sueq@u.washington.edu

 

Abstract

The serum prostate-specific antigen (PSA) test has a high false positive rate. As a single marker, PSA provides limited diagnostic information. A multi-marker test capable of detecting not only tumors but also the potentially lethal ones provides an unmet clinical need. Using the nanoString nCounter gene expression system, a 20-gene multiplex test was developed based on digital gene counting of RNA transcripts in urine as a means to detect prostate cancer. In this test, voided urine is centrifuged to pellet cells and the purified RNA is amplified for hybridization to preselected probesets. Amplification of test cell line RNA appeared not to introduce significant bias, and the counts matched well with gene abundance levels as measured by DNA microarrays. For data analysis, the individual counts were compared to that of β2 microglobulin, a housekeeping gene. Urine samples of 5 pre-operative cases and 2 non-cancer were analyzed. Pathology information was then retrieved. Signals for a majority of the genes were low for non-cancer and low Gleason scores, and 6/6 known prostate cancer markers were positive in the cases. One case of Gleason 4+5 showed, in contrast, strong signals for all cancer-associated markers, including CD24. One non-cancer also showed signals for all 6 cancer markers, and this man might harbor an undiagnosed cancer. This multiplex test assaying a natural waste product can potentially be used for screening, early cancer detection and patient stratification. Diagnostic information is gained from the RNA signatures that are associated with cell types of prostate tumors.

PMID: 23029164

 

SUPPLEMENT:
The serum prostate-specific antigen (PSA/KLK3) test has been widely used to screen and monitor men for prostate cancer. However, almost 75% of men with abnormal PSA turn out to be negative for cancer upon biopsy and men with normal PSA could harbor cancer. While PSA is produced abundantly by the prostate, it is not a perfect marker because it is not cancer-specific. This test has a poor specificity (around 20% at a sensitivity of 80%) with the result that many men would have to undergo an unnecessary biopsy (Thompson IM et al., 2008). This led recently to the downgrading of the PSA test by the US Preventive Services Task Force. Better informative markers are needed for more accurate cancer diagnosis. More importantly, markers are needed to distinguish prostate cancers that can be lethal from those that are not. Most, if not all, clinical cancer tests are based on single markers with limited diagnostic and prognostic capability. A multi-marker test, in contrast, would not only produce a much higher level of confidence in specificity and sensitivity but also information about the tumors.

Urine collection represents the least invasive route of obtaining test material and it is known that prostate cancer cells can be released into urine. We showed that prostate cells can be spun down from urine and high-quality RNA can be isolated and amplified . This detected RNA signature can be used for diagnosis and prognosis of prostate cancer. The nanoString nCounter technology (Geiss GK et al., 2008) is ideally suited to generate such RNA signatures through digital quantification. The digital data output can be easily archived and tracked, and is easily understandable to doctors and patients. Gene markers are selected based on association with Gleason grades, and with tumor stromal cells.

In this study, a panel of 20 RNA markers was selected for the nCounter codeset. These included known prostate cancer markers: AGR2, AMACR, CRISP3, ERG, HPN, PCA3; markers discovered by our cell-type analysis: CD90v, BRE, CCL3, CD24, IL24; markers for prostate cells: ACPP, ANPEP, AZGP1, CD9, DPP4, KLK2, MME; a marker for bladder cells: UPK3A; a housekeeping marker for all cells: B2M. The first cohort of urine samples tested produced distinct signatures for non-cancer, low-Gleason score disease and high-Gleason score disease. For non-cancer, all signal counts for the six prostate cancer genes were at background. In four low Gleason score cases, these gene counts for the six cancer markers were 2- to 3-fold above those of non-cancer. In one high Gleason score case, the gene counts for the six cancer markers were >70-fold above background. It would appear that more cancer cells and stromal cells associated with cancer were released into the urine. This could be attributed to the loss of tissue integrity, more pronounced in advanced diseases, as tumors produce enzymes that destroy the extracellular matrix. In subsequent experiments, more genes will be included in the nCounter codeset to validate this finding with a large cohort of cancer patients.
Biomed-frontier-figure1Fig 1. A simplified workflow of the multiplex urine test for prostate cancer from urine processing to nanoString nCounter expression analysis.

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