Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study
Abstract
:1. Introduction
2. Results
2.1. Derivation of a Vn96-Isolated EV Reference-Free mRNA Panel for Prostate Cancer Diagnosis
2.2. Derivation of a Vn96-Isolated EV Combined mRNA and miRNA Model for Prostate Cancer Diagnosis
2.3. Additive Value of Combining Vn96-Isolated EV mRNA and miRNA Panels with PCA3 and Clinical Characteristics
3. Discussion
4. Materials and Methods
4.1. Study Participants
4.2. Urine Collection and Processing
4.3. Isolation of Extracellular Vesicles Using the Vn96 Peptide
4.4. RNA Extraction from Urinary EVs
4.5. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
geomean(REχ1,…,REχη), where χ = individual mRNA of interest.
4.6. miRNA Detection and Quantification
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ACTB | Actin Beta |
ANXA3 | Annexin A3 |
AUC | Area Under the Curve |
BC | Benign Control |
BPH | Benign Prostate Hyperplasia |
CFD | Complement Factor D |
DNA | Deoxyribonucleic acid |
dNTP | Deoxynucleoside triphosphate |
DRE | Digital Rectal Examination |
DTT | Dithiothreitol |
ERG | Erythroblast Transformation-Specific (ETS) Transcription Factor ERG |
EV | Extracellular Vesicle |
FOLH1 | Folate Hydrolase 1 |
GOLM1 | Golgi Membrane Protein 1 |
GOLPH2 | Golgi phosphoprotein 2 |
GSTM4 | Glutathione S-Transferase Mu 4 |
HPN | Hepsin |
Hsp/c70 | Heat shock protein/cognate 70 |
ITSN1 | Intersectin 1 |
PIN | Prostatic Intraepithelial Neoplasia |
KLK3 | Kallikrein Related Peptidase 3 |
LTBP4 | Latent Transforming Growth Factor Beta Binding Protein 4 |
mRNA | Messenger ribonucleic acid |
NELL2 | Neural EGFL Like 2 |
PBS | Phosphate-buffered saline |
PCA3 | Prostate Cancer Associated 3 |
PDCD6IP | Programmed Cell Death 6 Interacting Protein |
PrCa | Prostate Cancer |
PSCA | Prostate Stem Cell Antigen |
qPCR | Quantitative Polymerase Chain Reaction |
ROC | Receiver Operator Characteristic |
SDS-PAGE | Sodium dodecyl sulphate-polyacrylamide gel electrophoresis |
SLC45A3 | Solute Carrier Family 45 Member 3 |
SNORD44 | Small Nucleolar RNA, C/D Box 44 |
SPINK1 | Serine Peptidase Inhibitor Kazal Type 1 |
sPSA | Serum Prostate Specific Antigen |
TMN | Tumour, nodes, metastasis |
TMPRSS2 | Transmembrane Serine Protease 2 |
TRUS | Transrectal Ultrasound |
XBP1 | X-Box Binding Protein 1 |
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Patient Group | |||
---|---|---|---|
Total | Benign Controls (BC) | Patients with Prostate Cancer (PrCa) | |
Number (n) | 56 | 28 | 28 |
Age (years) | |||
Mean (±SD) | 67.6 (±6.8) | 67.3 (±7.4) | 68.1 (±6.3) |
Range | 48–79 | 50–79 | 48–79 |
Serum PSA (ng/mL) | |||
Mean (±SD) | 4.6 (±2.6) | 4.5 (±3.0) | 4.8 (±2.1) |
Range | 0.6–14.7 | 0.6–14.7 | 0.9–9.1 |
0–4 ng/mL | 22 (39%) | 13 (46%) | 9 (32%) |
4–10 ng/mL | 32 (57%) | 13 (46%) | 19 (68%) |
>10 ng/mL | 1(1.8%) | 1 (3.6%) | 0 |
Unknown | 1 | 1 | 0 |
Other prostate conditions | |||
BPH * | 16 | 7 | 9 |
PIN † | 13 | 5 | 8 |
Both BPH and PIN | 7 | 1 | 6 |
Nodule(s) | 11 | 5 | 6 |
LUTS †† | 6 | 1 | 5 |
Firm to touch | 13 | 7 | 6 |
Increased volume | 22 | 13 | 9 |
Lobe Asymmetry | 11 | 3 | 8 |
Gleason grade at diagnosis | |||
≤6 (3 + 3) | NA | NA | 21 |
7 (3 + 4) | NA | NA | 4 |
7 (4 + 3) or higher | NA | NA | 3 |
Clinical Stage | |||
T1 | NA | NA | 9 |
T2 | NA | NA | 11 |
T3 | NA | NA | 1 |
Not Available | NA | NA | 7 |
mRNA Panel Variable | Logistic Regression Analysis | ROC Curve Analysis | ||
---|---|---|---|---|
OR (95% CI) | p Value | AUC (95% CI) | p Value | |
8 mRNA Panel | 1.0396 (1.0062, 1.0741) | 0.0199 | 0.695 (0.553, 0.837) | 0.0071 |
LTBP4 and NELL2 removed | 1.2386 (1.0360, 1.4809) | 0.0189 | 0.694 (0.555, 0.832) | 0.0061 |
5 mRNA Panels | ||||
FOLH1 removed | 1.0488 (0.9439, 1.1655) | 0.3755 | 0.651 (0.502, 0.799) | 0.0473 |
HPN removed | 1.4259 (1.0146, 2.0040) | 0.041 | 0.667 (0.523, 0.811) | 0.023 |
XBP1 removed | 1.1272 (1.0416, 1.2199) | 0.003 | 0.761 (0.636, 0.887) | <0.0001 |
ITSN1 removed | 1.0154 (0.9616, 1.0722) | 0.5817 | 0.67 (0.525, 0.814) | 0.0212 |
GSTM4 removed | 1.2439 (1.0482, 1.4762) | 0.0124 | 0.751 (0.623, 0.880) | 0.0001 |
CFD removed | 1.1995 (0.9935, 1.4481) | 0.0584 | 0.662 (0.519, 0.805) | 0.026 |
4 mRNA Panel | ||||
(FOLH1, HPN, ITSN1, CFD) | 1.1359 (1.0479, 1.2313) | 0.002 | 0.798 (0.681, 0.916) | <0.0001 |
mRNA Panel Variable | Logistic Regression Analysis | ROC Curve Analysis | ||
---|---|---|---|---|
OR (95% CI) | p Value | AUC (95% CI) | p Value | |
4 mRNA Panel | ||||
(FOLH1, HPN, ITSN1, CFD) | 1.1359 (1.0479, 1.2313) | 0.002 | 0.798 (0.681, 0.916) | 6.67 × 10−7 |
New 6 mRNA Panel | ||||
(GOLM1, ANXA3, CD24, TMPRSS2-ERG, PSCA, SLC45A3) | 4.4649 (1.3686, 14.5668) | 0.0131 | 0.725 (0.585, 0.865) | 1.62 × 10−3 |
Combined 10 mRNA Panel | 1.6359 (1.1808, 2.2663) | 0.0031 | 0.759 (0.630, 0.888) | 8.49 × 10−5 |
GOLM1 removed | 1.5596 (1.1723, 2.0748) | 0.0023 | 0.797 (0.677, 0.917) | 1.19 × 10−6 |
ANXA3 removed | 1.0827 (0.9981, 1.1745) | 0.0557 | 0.709 (0.570, 0.849) | 3.33 × 10−3 |
CD24 removed | 1.7905 (1.2128, 2.6433) | 0.0034 | 0.769 (0.643, 0.896) | 3.08 × 10−5 |
TMPRSS2-ERG removed | 1.5597 (1.0128, 2.4017) | 0.0436 | 0.704 (0.563, 0.845) | 4.52 × 10−3 |
PSCA removed | 1.5355 (1.1665, 2.0213) | 0.0022 | 0.781 (0.656, 0.905) | 1.03 × 10−5 |
SLC45A3 removed | 1.1223 (0.9776, 1.2885) | 0.1013 | 0.723 (0.584, 0.862) | 1.66 × 10−3 |
FOLH1 removed | 1.0836 (0.9653, 1.2164) | 0.1733 | 0.699 (0.558, 0.840) | 5.54 × 10−3 |
HPN removed | 1.5006 (1.1264, 1.9991) | 0.0055 | 0.741 (0.608, 0.875) | 4.00 × 10−4 |
ITSN1 removed | 2.1580 (1.2820, 3.6325) | 0.0038 | 0.749 (0.617, 0.880) | 2.05 × 10−4 |
CFD removed | 3.1347 (1.5581, 6.3064) | 0.0014 | 0.787 (0.663, 0.911) | 5.82 × 10−6 |
7 mRNA Panel | ||||
(ANXA3, CD24, TMPRSS2-ERG, SLC45A3, FOLH1, HPN, ITSN1) | 2.2371 (1.4036, 3.5656) | 0.0007 | 0.825 (0.710, 0.941) | 3.18 × 10−8 |
miRNA or Combined Variable | Logistic Regression Analysis | ROC Curve Analysis | ||
---|---|---|---|---|
Univariate | OR (95% CI) | p Value | AUC (95% CI) | p Value |
miR-141-3p | 1.6604 (1.0373, 2.6578) | 0.0346 | 0.645 (0.492, 0.797) | 0.0629 |
miR-375-3p | 1.1049 (1.0130, 1.2051) | 0.0243 | 0.744 (0.603, 0.885) | 0.0007 |
miR-574-3p | 1.7572 (1.0882, 2.8373) | 0.0211 | 0.733 (0.599, 0.866) | 0.0006 |
miR-21-3p | 1.2562 (1.0154, 1.5540) | 0.0357 | 0.698 (0.553, 0.843) | 0.0073 |
4 miRNA Panel | 1.5012 (1.0761, 2.0942) | 0.0168 | 0.719 (0.574, 0.865) | 0.0031 |
3 miRNA Panel (miR-141-3p, miR-375-3p, miR-574-3p) | 1.4851 (1.0815, 2.0393) | 0.0145 | 0.704 (0.559, 0.849) | 0.0059 |
3 miRNA Panel (miR-375-3p, miR-574-3p, miR-21-3p) | 1.3990 (1.0750, 1.8206) | 0.0125 | 0.737 (0.597, 0.876) | 0.0009 |
2 miRNA Panel (miR-375-3p, miR-574-3p) | 1.3390 (1.0592, 1.6928) | 0.0147 | 0.744 (0.607, 0.880) | 0.0005 |
Multivariable | ||||
7 Gene Score + 2 miRNA Model | NA | NA | 0.843 (0.722, 0.964) | 2.55 × 10−8 |
7 mRNA Panel | 1.8463 (1.1375, 2.9968) | 0.0131 | NA | NA |
2 miRNA Panel | 1.1822 (0.9581, 1.4587) | 0.1822 | NA | NA |
Variable | Logistic Regression Analysis | ROC Curve Analysis | ||
---|---|---|---|---|
Univariate | OR (95% CI) | p Value | AUC (95% CI) | p Value |
Urinary Vn96-EV PCA3 | 1.0960 (1.0298, 1.1665) | 0.0039 | 0.816 (0.708, 0.925) | 1.20 × 10−8 |
Age at collection | 1.0181 (0.9420, 1.1004) | 0.6509 | 0.543 (0.387, 0.700) | 0.5864 |
Serum PSA at collection | 1.0742 (0.8671, 1.3308) | 0.5124 | 0.594 (0.440, 0.749) | 0.2303 |
Prostate Volume at collection | 0.9726 (0.9421, 1.0041) | 0.0874 | 0.656 (0.508, 0.805) | 0.0391 |
DRE Result | 0.8627 (0.2972, 2.5041) | 0.7860 | 0.518 (0.387, 0.649) | 0.7895 |
Multivariable | ||||
Model 1 (SOC † Only) | NA | NA | 0.718 (0.583, 0.853) | 0.0016 |
Age | 1.0746 (0.9785, 1.1802) | 0.1322 | NA | NA |
Serum PSA | 1.2333 (0.9385, 1.6209) | 0.1325 | NA | NA |
Prostate Volume | 0.9427 (0.9001, 0.9873) | 0.0123 | NA | NA |
DRE Result | 0.4799 (0.1327, 1.7351) | 0.2629 | NA | NA |
Model 2 †† | NA | NA | 0.901 (0.811, 0.990) | 1.75 × 10−18 |
7 mRNA Panel | 1.7053 (1.0026, 2.9005) | 0.0489 | NA | NA |
2 miRNA Panel | 1.1670 (0.9429, 1.4445) | 0.1088 | NA | NA |
PCA3 Value | 1.0615 (1.0047, 1.1216) | 0.0335 | NA | NA |
Model 3 ††† | NA | NA | 0.955 (0.909, 1.002) | 2.27 × 10−82 |
7 mRNA Panel | 1.4312 (0.7581, 2.7021) | 0.2688 | NA | NA |
2 miRNA Panel | 1.4024 (0.9767, 2.0138) | 0.0669 | NA | NA |
PCA3 Value | 1.0810 (0.9984, 1.1705) | 0.0548 | NA | NA |
Age | 1.0921 (0.8962, 1.3309) | 0.3823 | NA | NA |
Serum PSA | 1.5220 (0.8605, 2.6921) | 0.1489 | NA | NA |
Prostate Volume | 0.8741 (0.7764, 0.9841) | 0.0261 | NA | NA |
DRE Results | 0.1393 (0.0117, 1.6573) | 0.1187 | NA | NA |
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Davey, M.; Benzina, S.; Savoie, M.; Breault, G.; Ghosh, A.; Ouellette, R.J. Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study. Int. J. Mol. Sci. 2020, 21, 8330. https://doi.org/10.3390/ijms21218330
Davey M, Benzina S, Savoie M, Breault G, Ghosh A, Ouellette RJ. Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study. International Journal of Molecular Sciences. 2020; 21(21):8330. https://doi.org/10.3390/ijms21218330
Chicago/Turabian StyleDavey, Michelle, Sami Benzina, Marc Savoie, Guy Breault, Anirban Ghosh, and Rodney J. Ouellette. 2020. "Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study" International Journal of Molecular Sciences 21, no. 21: 8330. https://doi.org/10.3390/ijms21218330