Averaged differential expression for the discovery of biomarkers in the blood of patients with prostate cancer

Abstract

Background: The identification of a blood-based diagnostic marker is a goal in many areas of medicine, including the early diagnosis of prostate cancer. We describe the use of averaged differential display as an efficient mechanism for biomarker discovery in whole blood RNA. The process of averaging reduces the problem of clinical heterogeneity while simultaneously minimizing sample handling.

Methodology/principal findings: RNA was isolated from the blood of prostate cancer patients and healthy controls. Samples were pooled and subjected to the averaged differential display process. Transcripts present at different levels between patients and controls were purified and sequenced for identification. Transcript levels in the blood of prostate cancer patients and controls were verified by quantitative RT-PCR. Means were compared using a t-test and a receiver-operating curve was generated. The Ring finger protein 19A (RNF19A) transcript was identified as having higher levels in prostate cancer patients compared to healthy men through the averaged differential display process. Quantitative RT-PCR analysis confirmed a more than 2-fold higher level of RNF19A mRNA levels in the blood of patients with prostate cancer than in healthy controls (p = 0.0066). The accuracy of distinguishing cancer patients from healthy men using RNF19A mRNA levels in blood as determined by the area under the receiving operator curve was 0.727.

Conclusions/significance: Averaged differential display offers a simplified approach for the comprehensive screening of body fluids, such as blood, to identify biomarkers in patients with prostate cancer. Furthermore, this proof-of-concept study warrants further analysis of RNF19A as a clinically relevant biomarker for prostate cancer detection.

Figure 1. ADE analysis of whole blood RNA from prostate cancer patients vs. healthy men.

ADE analysis identified two RNA transcripts with levels significantly higher in whole blood RNA from prostate cancer patients (Ca) than in healthy men (H). Nucleotide sequence analysis revealed that these two transcripts shared a common sequence. This sequence had 100% homology to the nucleotide sequence in the E3-ubiquitin ligase Ring Finger Protein 19a (RNF19A) transcript and is located in the 3′ untranslated region (UTR).

Figure 2. RNF19A transcript levels are higher in prostate cancer patients than in healthy men.

Quantitative RT-PCR was performed on whole blood RNA samples from patients with localized prostate cancer (n = 33) as well as healthy male controls (n = 19). Levels of RNF19A transcript were normalized to 18S RNA (reference gene). Normalized results are presented in box plot format, with boxes representing the 25^th, 50^th, and 75^th percentiles and whiskers representing the 10^th and 90^th percentiles of the data. Outliers are also displayed. The difference between the means was statistically significant (p  =  0.0066).

Figure 3. ROC curve evaluating the accuracy of RNF19A as a diagnostic test.

A receiver-operating curve (ROC) was generated as a preliminary estimate of the accuracy of relative levels of RNF19A in classifying patients with cancer or healthy controls in our cohort of patients. The true positive rate (sensitivity) was plotted against the false positive rate (1-specificity). The area under the curve was calculated as 0.7273.