Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Samples and DNA Extraction
2.2. Next-Generation Sequencing
2.3. Analysis of the Sensitivity and Specificity of the Detection of the p.Thr790Met Alteration
2.4. PNA-Mediated PCR Clamping on the Wild-Type EGFR Sequence
2.5. Pyrosequencing
2.6. Statistics
3. Results
3.1. Determination of the Detection Limit of Our PNA-Mediated PCR Clamping
3.2. Detection of the p.Thr790Met Alteration of EGFR in Patients’ Somatic DNA
3.3. Patient with Discordant Results
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
TKI | tyrosine kinase inhibitor |
NSCLC | non-small-cell lung cancer |
PNA | peptide nucleic acid |
SCLC | small-cell lung cancer |
ctDNA | circulating tumor DNA |
NGS | next-generation sequencing |
PBNC | peripheral blood nuclear cells |
FFPE | formalin-fixed paraffin-embedded |
ICO | Institut de Cancérologie de l’Ouest |
Appendix A
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Names | Sequences |
---|---|
PCR forward primer | 5′-GCA TCT GCC TCA CCT CCA A-3′ |
PCR reverse primer | 5′-biotin-CGA TCT GCA CAC ACC AGT TG-3′ |
PNA sequence | NH2-CTCATCACGCAGCTCA-COOH [32] |
Sequencing primer (pyrosequencing) | CCG TGC AGC TCA TCA |
Dispensation order (pyrosequencing) | ACTAGCAGC |
Samples | Without Preamplification | With Preamplification | ||
---|---|---|---|---|
PNA− (%) | PNA+ (%) | PNA− (%) | PNA+ (%) | |
Sample 28 | 0 | NA | 2 | 28 |
Sample 30 | 5 | NA | 5 | 59 |
Sample 31 | 12 | 100 | 10 | 68 |
Sample 32 | 8 | NA | 7 | 72 |
Sample 34 | 7 | NA | 9 | 84 |
Sample 35 | 8 | 100 | 9 | 84 |
Sample 38 | 16 | NA | 22 | 88 |
Sample 39 | 22 | 100 | 20 | 90 |
Sample 41 | 18 | 100 | 20 | 91 |
Sample 42 | 21 | 100 | 23 | 91 |
Sample 43 | 13 | 92 | 11 | 92 |
Sample 45 | 26 | 100 | 28 | 93 |
Samples | Ages | Sexes | [DNA] (ng/µL) | Cellularity | NGS Results | PNA-Mediated PCR | Concordance Results | |
---|---|---|---|---|---|---|---|---|
PNA− | PNA+ | |||||||
Sample 1 | 50.8 | M | 10.7 | 30 | WT | 0 | 0 | OK-WT |
Sample 2 | 53.7 | W | 14.2 | 40 | WT | 0 | 0 | OK-WT |
Sample 3 | 62.1 | M | 25.9 | 20 | WT | 0 | 0 | OK-WT |
Sample 4 | 55.5 | W | 10.2 | 20 | WT | 0 | 0 | OK-WT |
Sample 5 | 62.7 | M | 13.3 | 30 | WT | 0 | 0 | OK-WT |
Sample 6 | 73.4 | M | 39 | 10 | WT | 0 | 0 | OK-WT |
Sample 7 | 64.2 | M | 6.4 | 20 | WT | 0 | 0 | OK-WT |
Sample 8 | 67.2 | M | 6.6 | 40 | WT | 0 | 0 | OK-WT |
Sample 9 | 51.4 | W | 11.6 | 40 | WT | 0 | 0 | OK-WT |
Sample 10 | 81.6 | M | 11.4 | 20 | WT | 0 | 0 | OK-WT |
Sample 11 | 56.6 | M | 52.8 | 70 | WT | 0 | 0 | OK-WT |
Sample 12 | 83.6 | W | 1.4 | 10 | WT | 0 | 0 | OK-WT |
Sample 13 | 56 | M | 15.6 | 30 | WT | 0 | 0 | OK-WT |
Sample 14 | 52.7 | M | 7.2 | 30 | WT | 0 | 0 | OK-WT |
Sample 15 | 67.6 | M | 10.7 | 30 | WT | 0 | 0 | OK-WT |
Sample 16 | 59 | W | 18.2 | 80 | WT | 0 | 0 | OK-WT |
Sample 17 | 69.7 | M | 29.7 | 60 | WT | 0 | 0 | OK-WT |
Sample 18 | 56.2 | M | 5.7 | 60 | WT | 0 | 0 | OK-WT |
Sample 19 | 62.4 | W | 2.9 | 5 | WT | 0 | 0 | OK-WT |
Sample 20 | 63.2 | W | 9.5 | 70 | WT | 0 | 5 | L-WT |
Sample 21 | 71.4 | M | 24.5 | 40 | WT | 0 | 5 | L-WT |
Sample 22 | 82 | M | 9.5 | 10 | WT | 0 | 5 | L-WT |
Sample 23 | 61.1 | M | 16.1 | 60 | WT | 0 | 6 | L-WT |
Sample 24 | 83 | W | 5.5 | 20 | WT | 0 | 6 | L-WT |
Sample 25 | NA | W | NA | NA | WT | 0 | 6 | L-WT |
Sample 26 | 62.3 | M | 24.8 | 30 | WT | 0 | 8 | L-WT |
Sample 27 | 51.4 | W | 44.2 | 40 | WT | 0 | 18 | Discordant |
Sample 28 | 86.7 | W | 27.3 | 5 | T790M | 2 | 28 | OK-Mutated |
Sample 29 | NA | M | 7.4 | NA | T790M (1.3%) | 2 | 35 | OK-Mutated |
Sample 30 | 76.4 | W | 10 | 40 | T790M (3.5%) | 5 | 59 | OK-Mutated |
Sample 31 | 74.6 | M | 12.4 | 4 | T790M | 10 | 68 | OK-Mutated |
Sample 32 | 78.2 | W | 2.2 | 10 | T790M | 7 | 72 | OK-Mutated |
Sample 33 | 73.7 | W | 2.1 | 50 | T790M (9%) | 11 | 75 | OK-Mutated |
Sample 34 | 67.2 | M | 4.1 | 10 | T790M | 9 | 84 | OK-Mutated |
Sample 35 | 75.6 | W | 23.7 | 40 | T790M | 9 | 84 | OK-Mutated |
Sample 36 | 87.8 | W | 12.4 | 20 | T790M (12%) | 24 | 87 | OK-Mutated |
Sample 37 | 66.4 | W | 6 | 80 | T790M | 26 | 87 | OK-Mutated |
Sample 38 | 64.3 | W | 2.5 | 60 | T790M | 22 | 88 | OK-Mutated |
Sample 39 | 64.8 | W | 9.4 | 40 | T790M | 20 | 90 | OK-Mutated |
Sample 40 | 87.6 | W | 4.4 | 50 | T790M (15.4%) | 17 | 90 | OK-Mutated |
Sample 41 | 65.3 | M | 30.2 | 10 | T790M | 20 | 91 | OK-Mutated |
Sample 42 | NA | M | 6.4 | NA | T790M (18.1%) | 23 | 91 | OK-Mutated |
Sample 43 | 78.3 | W | 30.9 | 70 | T790M | 11 | 92 | OK-Mutated |
Sample 44 | 77 | W | 0.6 | 10 | T790M (14.7%) | 23 | 92 | OK-Mutated |
Sample 45 | 74 | W | 6.9 | 40 | T790M | 28 | 93 | OK-Mutated |
Sample 46 | 78.5 | M | 3.8 | 60 | T790M (21.3%) | 26 | 95 | OK-Mutated |
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Billaud, A.; Verriele, V.; Dauvé, J.; Chevalier, L.-M.; Morel, A. Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing. Diagnostics 2020, 10, 527. https://doi.org/10.3390/diagnostics10080527
Billaud A, Verriele V, Dauvé J, Chevalier L-M, Morel A. Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing. Diagnostics. 2020; 10(8):527. https://doi.org/10.3390/diagnostics10080527
Chicago/Turabian StyleBillaud, Amandine, Veronique Verriele, Jonathan Dauvé, Louise-Marie Chevalier, and Alain Morel. 2020. "Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing" Diagnostics 10, no. 8: 527. https://doi.org/10.3390/diagnostics10080527