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. 2023 Nov 30:10:1279854.
doi: 10.3389/fmolb.2023.1279854. eCollection 2023.

Validation of potential RNA biomarkers for prostate cancer diagnosis and monitoring in plasma and urinary extracellular vesicles

Agnese Brokāne  1 , Cristina Bajo-Santos  1 , Pawel Zayakin  1 , Alberts Belovs  2 , Juris Jansons  2 , Vilnis Lietuvietis  2 , Elena S Martens-Uzunova  3 , Guido W Jenster  3 , Aija Linē  1
Affiliations

Validation of potential RNA biomarkers for prostate cancer diagnosis and monitoring in plasma and urinary extracellular vesicles

Agnese Brokāne et al. Front Mol Biosci. .

Abstract

Introduction: Prostate cancer (PCa), one of the most prevalent malignancies affecting men worldwide, presents significant challenges in terms of early detection, risk stratification, and active surveillance. In recent years, liquid biopsies have emerged as a promising non-invasive approach to complement or even replace traditional tissue biopsies. Extracellular vesicles (EVs), nanosized membranous structures released by various cells into body fluids, have gained substantial attention as a source of cancer biomarkers due to their ability to encapsulate and transport a wide range of biological molecules, including RNA. In this study, we aimed to validate 15 potential RNA biomarkers, identified in a previous EV RNA sequencing study, using droplet digital PCR. Methods: The candidate biomarkers were tested in plasma and urinary EVs collected before and after radical prostatectomy from 30 PCa patients and their diagnostic potential was evaluated in a test cohort consisting of 20 benign prostate hyperplasia (BPH) and 20 PCa patients' plasma and urinary EVs. Next, the results were validated in an independent cohort of plasma EVs from 31 PCa and 31 BPH patients. Results: We found that the levels of NKX3-1 (p = 0.0008) in plasma EVs, and tRF-Phe-GAA-3b (p < 0.0001) tRF-Lys-CTT-5c (p < 0.0327), piR-28004 (p = 0.0081) and miR-375-3p (p < 0.0001) in urinary EVs significantly decreased after radical prostatectomy suggesting that the main tissue source of these RNAs is prostate and/or PCa. Two mRNA biomarkers-GLO1 and NKX3-1 showed promising diagnostic potential in distinguishing between PCa and BPH with AUC of 0.68 and 0.82, respectively, in the test cohort and AUC of 0.73 and 0.65, respectively, in the validation cohort, when tested in plasma EVs. Combining these markers in a biomarker model yielded AUC of 0.85 and 0.71 in the test and validation cohorts, respectively. Although the PSA levels in the blood could not distinguish PCa from BPH in our cohort, adding PSA to the mRNA biomarker model increased AUC from 0.71 to 0.76. Conclusion: This study identified two novel EV-enclosed RNA biomarkers-NKX3-1 and GLO1-for the detection of PCa, and highlights the complementary nature of GLO1, NKX3-1 and PSA as combined biomarkers in liquid biopsies of PCa.

Keywords: RNA biomarkers; droplet digital PCR; extracellular vesicles; liquid biopsies; prostate cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
EV isolation and quality control. (A,B) Transmission electron microscopy images showing EVs isolated from urine (A) and plasma (B). Western blot analysis of ALIX, Calnexin, CD63 and TSG101 in plasma and urinary EVs from 3 randomly selected patients as well as LNCap prostate cancer cells (C). Paired dot plots showing EV concentration per ml of plasma (D) and urine (E) samples before and after radical prostatectomy. Wilcoxon matched-pairs signed rank test was used to assess the statistical significance of the differences between groups. (F–H) Box plots showing EV concentration per ml of plasma (F–H) and urine (G) in patients with PCa and BPH. Figures 1F, 1G represent patients in the test cohort and Figure 1H represents patients in the validation cohort. Mann-Whitney test was used to assess the statistical significance of the differences between groups. p-value < 0.05 was considered significant.
FIGURE 2
FIGURE 2
Comparison of RNA biomarker levels in Pre-Op vs. Post-Op plasma and urinary EVs. Paired dot plots show copy numbers of RNA biomarkers per ml of urine (A–D) and plasma (E) collected before and after radical prostatectomy in 30 PCa patients. Wilcoxon matched-pairs signed rank test was used to assess the statistical significance of the differences between groups. p-value < 0.05 was considered significant.
FIGURE 3
FIGURE 3
Comparison of RNA biomarker levels in patients with PCa vs BPH in plasma EV samples. Violin plots show copy numbers of RNA biomarkers NKX3-1 (A,D), miR-27a (B,E) and GLO1 (C,F) per ml of plasma in the test cohort (20 PCa, 20 BPH) (A–C) and validation cohort (31 PCa, 31 BPH) (D–F). Mann-Whitney test was used to assess the statistical significance of the differences between groups. p-value < 0.05 was considered significant.
FIGURE 4
FIGURE 4
RNA biomarker models. Generalized linear models combining the top two performing candidates NKX3-1 and GLO1 based on Gaussian identity function show an ability to discriminate between PCa and BPH in a test cohort of 20 PCa vs 20 BPH patient plasma EVs (A) and a validation cohort of 31 PCa vs 31 BPH patient plasma EVs (B). Leave-one-out cross-validation of the model (C). ROC curve for the PSA test (D). A combined model of PSA measurements and two RNA biomarkers (NKX3-1 and GLO1) showing the ability to discriminate between PCa and BPH in a test cohort of 20 PCa vs 20 BPH patient plasma EVs (E) and a validation cohort of 31 PCa vs 31 BPH patient plasma EVs (F).
FIGURE 5
FIGURE 5
Comparison of RNA biomarker levels in patients with high Gleason (HG) and low Gleason (LG) scores. Violin plots show copy numbers of RNA biomarkers per 1 mL of patient plasma (A) and urinary EVs (B–D). Mann-Whitney test was used to assess the statistical significance of the differences between groups. p-value < 0.05 was considered significant.
FIGURE 6
FIGURE 6
Expression levels and mutational analysis of NKX3-1 in PCa tissues. (A) The box plot shows NKX3-1 mRNA levels in prostate adenocarcinoma (PRAD) and normal prostate tissue (NT) gene expression datasets from TCGA database. Mann-Whitney test was used to assess the statistical significance of the differences between groups. p-value < 0.05 was considered significant. (B) Paired dot plots show allele frequencies of 2 SNPs obtained from RNA sequencing data in tumor and normal prostate tissues from 10 PCa patients.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the ERDF project No. 1.1.1.1/20/A/045.