Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Optimization of the ddPCR Assays
2.2. Determination of the Threshold Number of Minimum Mutated Droplets to Call a Mutation
2.3. Determination of the Limit of Detection of the ddPCR Assays
2.4. Comparison of the ddPCR and UroMuTERT Assays for the Detection of TERT Promoter Mutations in Urinary and Tumor DNA
2.5. Assessment of the Interrater Reliability of ddPCR and UroMuTERT Assays in Detecting Urinary TERT Promoter Mutations
2.6. Correlation of MAFs between ddPCR and UroMuTERT Assays
3. Discussion
4. Materials and Methods
4.1. Study Population, Sample Collection and Processing
4.2. Detection of TERT Promoter Mutations Using ddPCR Assay
4.3. Determination of the False-Positive Measures and Threshold Number of Minimum Mutated Droplets to Call a Mutation
4.4. Determination of the Limit of Detection of the ddPCR Assays
4.5. Comparison of the ddPCR and UroMuTERT Assays for the Detection of TERT Promoter Mutations in the Urine Samples of UC Cases and Controls
4.6. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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C228T or C250T Mutations | ddPCR Assays | UroMuTERT Assay | ||||
---|---|---|---|---|---|---|
DIAGURO Cohort | PORTO Cohort | DIAGURO Cohort a | PORTO Cohort a | DIAGURO Cohort b | PORTO Cohort b | |
US cfDNA or UP DNA | UP DNA | US cfDNA or UP DNA | UP DNA | US cfDNA or UP DNA | UP DNA | |
Total Number analyzed | (n = 181) | (n = 99) | (n = 181) | (n = 99) | (n = 187) | (n = 100) |
True Positive-no | 79 | 33 | 80 | 32 | 81 | 33 |
True Negative-no | 85 | 50 | 88 | 50 | 89 | 50 |
False Positive-no | 7 | 0 | 4 | 0 | 5 | 0 |
False Negative-no | 10 | 16 | 9 | 17 | 12 | 17 |
Sensitivity (95% CI)-% | 86.8 (80.3–94.5) | 67.4 (52.5–80.1) | 90.7 (83.1–95.7) | 65.3 (50.4–78.3) | 87.1 (78.6–93.2) | 66.0 (51.2–78.8) |
Specificity (95% CI)-% | 92.4 (85.0–96.9) | 100.0 (92.9–100.0) | 95.6 (89.2–98.8) | 100.0 (92.9–100.0) | 94.7 (88.0–98.3) | 100.0 (92.9–100.0) |
Accuracy (95% CI)-% | 91.3 (86.2–95.0) | 90.2 (82.6–95.3) | 93.1 (88.5–96.3) | 89.6 (81.8–94.8) | 92.4 (90.6–94.0) | 89.8 (87.8–91.6) |
Sample Type | UroMuTERT-MUT | UroMuTERT-WT | Kappa Coefficient | ||
---|---|---|---|---|---|
ddPCR-MUT | ddPCR-WT | ddPCR-MUT | ddPCR-WT | ||
n | n | n | n | ||
All Follow-up cases (UP cellDNA and US cfDNA) | 173 | 10 | 15 | 196 | 0.87 |
All US cfDNA Follow-up cases | 86 | 3 | 9 | 123 | 0.89 |
All UP cellDNA Follow-up cases | 87 | 7 | 6 | 73 | 0.85 |
Cases US/UP follow-up with MAF < 2% ddPCR | 64 | 10 | 14 | 73 | 0.78 |
Cases US/UP follow-up with MAF > 2% ddPCR | 109 | 0 | 1 | 123 | 0.99 |
Cases US/UP follow-up with MAF < 1% ddPCR | 18 | 7 | 4 | 73 | 0.70 |
Cases US/UP follow-up with MAF > 1% ddPCR | 155 | 3 | 11 | 123 | 0.90 |
Cases US/UP follow-up with MAF < 0.5% ddPCR | 3 | 7 | 3 | 73 | 0.32 |
Cases US/UP follow-up with MAF > 0.5% ddPCR | 170 | 3 | 12 | 123 | 0.90 |
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Hosen, M.I.; Forey, N.; Durand, G.; Voegele, C.; Bilici, S.; Avogbe, P.H.; Delhomme, T.M.; Foll, M.; Manel, A.; Vian, E.; et al. Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer. Cancers 2020, 12, 3541. https://doi.org/10.3390/cancers12123541
Hosen MI, Forey N, Durand G, Voegele C, Bilici S, Avogbe PH, Delhomme TM, Foll M, Manel A, Vian E, et al. Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer. Cancers. 2020; 12(12):3541. https://doi.org/10.3390/cancers12123541
Chicago/Turabian StyleHosen, Md Ismail, Nathalie Forey, Geoffroy Durand, Catherine Voegele, Selin Bilici, Patrice Hodonou Avogbe, Tiffany Myriam Delhomme, Matthieu Foll, Arnaud Manel, Emmanuel Vian, and et al. 2020. "Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer" Cancers 12, no. 12: 3541. https://doi.org/10.3390/cancers12123541