1Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Seoul, Korea
2Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
3Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Copyright © 2024 by the Korean Cancer Association
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ethical Statement
All procedures performed in studies involving human participants were approved by the Ethics Committee at the Asan Medical Center (approval no. 2016-0692) and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. As this study was retrospective, the Institutional Review Board waived the requirement for informed consent based on the study design.
Author Contributions
Conceived and designed the analysis: Sung CO, Jang SJ, Choi CM, Chun SM.
Collected the data: Choi CM, Chun SM.
Contributed data or analysis tools: Lee JY, Jun HR, Chun SM.
Performed the analysis: Lee JY, Jeon S, Chun SM.
Wrote the paper: Lee JY, Chun SM.
Conflicts of Interest
Conflict of interest relevant to this article was not reported.
ULV1 | Assay | Forward primer (5’ → 3’) | Reverse primer (5’ → 3’) | MSP (5’ → 3’)a) | UEP (5’ → 3’) |
---|---|---|---|---|---|
Pool 1 | EGFR E709K | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | TTGATCTTTTTGAATT |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | GCTCTCTTGAGGATCTTGAgGA | CAGTTT | ||
EGFR G719A | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | C AAAAGATCAAAGTGCTGG | |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | CGTGCCGAACGCACCGGgGG | |||
EGFR G719D | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | C AAAAGATCAAAGTGCTGG | |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | CGTGCCGAACGCACCGagGT | |||
EGFR E746_A750delinsK | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | AAAATTCCCGTCGCTATCAA | |
AGCAGAAACTCACATCGAGG | GATCCCAGAAGGTGAGAAAG | CCTTGTTGGCTTTCGGAGATGcTT | |||
EGFR L747_P753>S | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | GATTTCCTTGTTGGCTTTCG | |
AGCAGAAACTCACATCGAGG | GATCCCAGAAGGTGAGAAAG | CCCGTCGCTATCAAGGAtTC | |||
EGFR C797S | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | GGACATAGTCCAGGAGGC | |
TACTGGGAGCCAATATTGTC | CAGCTCATCACGCAGCTCAT | TCACGCAGCTCATGCCCTTCGtCA | |||
KRAS G13D | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | AGGCACTCTTGCCTACG | |
TAGCTGTATCGTCAAGGCAC | TAAGGCCTGCTGAAAATGAC | TGTGGTAGTTGGAGCTGGgGA | |||
KRAS G13V | ACGTTGGATG | ACGTTGGATG | ACGTTGGATGTGTGGTAGTTGGA | AGGCACTCTTGCCTACG | |
TAGCTGTATCGTCAAGGCAC | TAAGGCCTGCTGAAAATGAC | GCTGGgGT | |||
Pool 2 | EGFR E746 _A750del | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | TTGGCTTTCGGAGATGT |
TCGAGGATTTCCTTGTTGGC | GATCCCAGAAGGTGAGAAAG | AATTCCCGTCGCTATCAcGA | |||
EGFR T790M | ACGTTGGATG | ACGTTGGATG | AGCCGAAGGGCATGAGCTGaA | CACCGTGCAGCTCATCA | |
ATCTGCCTCACCTCCACCGT | TGTTCCCGGACATAGTCCAG | ||||
EGFR L858R | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG GT | GCACCCAGCAGTTTGGCC | |
AGCCAGGAACGTACTGGTGA | AAAGCCACCTCCTTACTTTGC | TCAAGATCACAGATTTTGGtCG | |||
BRAF V600E | ACGTTGGATG | ACGTTGGATG | CT | CCCACTCCATCGAGATTTC | |
TTCATGAAGACCTCACAG | AGCCTCAATTCTTACCATCCA | GTGATTTTGGTCTAGCTACgGA | |||
TAAAAA | |||||
KRAS G12D | ACGTTGGATG | ACGTTGGATG | ACTTGTGGTAGTTGGAGCgGA | CACTCTTGCCTACGCCA | |
TTTATTATAAGGCCTGCTG | ATTGTTGGATCATATTCGTCCAC | ||||
AAAATG | |||||
KRAS G12V | ACGTTGGATG | ACGTTGGATG | ACTTGTGGTAGTTGGAGCaGT | CACTCTTGCCTACGCCA | |
TTTATTATAAGGCCTGCTG | ATTGTTGGATCATATTCGTCCAC | ||||
AAAATG |
Patient | Stage | cfDNA amount (ng, for DNA library) | Gene | Mutation | Observed VAF by CT-ULTRA (%, variant allele reads/total reads) | Observed enriched VAF by ULV1 (%) | Semi-quantitative dynamic range (%) | Expected VAF (%) |
---|---|---|---|---|---|---|---|---|
AMC036 | II | 2.4 | TP53 | p.A161Pfs*9 | 3.1 (5/163) | NA | NA | NA |
EGFR | p.L858R | 1.1a) (2/183) | 82 | 29.7-75.4 | > 1 and ≤ 5 | |||
AMC044 | III | 6.5 | MTOR | p.A134V | 1.4 (4/285) | NA | NA | NA |
EGFR | p.L858R | 0.33a) (1/305) | 51 | 29.7-75.4 | 0.08 | |||
AMC070 | IV | 10 | EGFR | p.E746_A750delinsK | 1.2 (5/422) | 64 | 5.8-62.9 | > 1 and ≤ 5 |
EGFR | p.T790M | 0.23a) (1/441) | 31 | 11.2-65.5 | 0.20 |
ULV1 | Assay | Forward primer (5’ → 3’) | Reverse primer (5’ → 3’) | MSP (5’ → 3’) |
UEP (5’ → 3’) |
---|---|---|---|---|---|
Pool 1 | EGFR E709K | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | TTGATCTTTTTGAATT |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | GCTCTCTTGAGGATCTTGAgGA | CAGTTT | ||
EGFR G719A | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | C AAAAGATCAAAGTGCTGG | |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | CGTGCCGAACGCACCGGgGG | |||
EGFR G719D | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | C AAAAGATCAAAGTGCTGG | |
AGGGACCTTACCTTATACACC | AGTGGAGAAGCTCCCAACCA | CGTGCCGAACGCACCGagGT | |||
EGFR E746_A750delinsK | ACGTTGGATG | ACGTTGGATG | AATAAATCATAA | AAAATTCCCGTCGCTATCAA | |
AGCAGAAACTCACATCGAGG | GATCCCAGAAGGTGAGAAAG | CCTTGTTGGCTTTCGGAGATGcTT | |||
EGFR L747_P753>S | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | GATTTCCTTGTTGGCTTTCG | |
AGCAGAAACTCACATCGAGG | GATCCCAGAAGGTGAGAAAG | CCCGTCGCTATCAAGGAtTC | |||
EGFR C797S | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | GGACATAGTCCAGGAGGC | |
TACTGGGAGCCAATATTGTC | CAGCTCATCACGCAGCTCAT | TCACGCAGCTCATGCCCTTCGtCA | |||
KRAS G13D | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | AGGCACTCTTGCCTACG | |
TAGCTGTATCGTCAAGGCAC | TAAGGCCTGCTGAAAATGAC | TGTGGTAGTTGGAGCTGGgGA | |||
KRAS G13V | ACGTTGGATG | ACGTTGGATG | ACGTTGGATGTGTGGTAGTTGGA | AGGCACTCTTGCCTACG | |
TAGCTGTATCGTCAAGGCAC | TAAGGCCTGCTGAAAATGAC | GCTGGgGT | |||
Pool 2 | EGFR E746 _A750del | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG | TTGGCTTTCGGAGATGT |
TCGAGGATTTCCTTGTTGGC | GATCCCAGAAGGTGAGAAAG | AATTCCCGTCGCTATCAcGA | |||
EGFR T790M | ACGTTGGATG | ACGTTGGATG | AGCCGAAGGGCATGAGCTGaA | CACCGTGCAGCTCATCA | |
ATCTGCCTCACCTCCACCGT | TGTTCCCGGACATAGTCCAG | ||||
EGFR L858R | ACGTTGGATG | ACGTTGGATG | ACGTTGGATG GT | GCACCCAGCAGTTTGGCC | |
AGCCAGGAACGTACTGGTGA | AAAGCCACCTCCTTACTTTGC | TCAAGATCACAGATTTTGGtCG | |||
BRAF V600E | ACGTTGGATG | ACGTTGGATG | CT | CCCACTCCATCGAGATTTC | |
TTCATGAAGACCTCACAG | AGCCTCAATTCTTACCATCCA | GTGATTTTGGTCTAGCTACgGA | |||
TAAAAA | |||||
KRAS G12D | ACGTTGGATG | ACGTTGGATG | ACTTGTGGTAGTTGGAGCgGA | CACTCTTGCCTACGCCA | |
TTTATTATAAGGCCTGCTG | ATTGTTGGATCATATTCGTCCAC | ||||
AAAATG | |||||
KRAS G12V | ACGTTGGATG | ACGTTGGATG | ACTTGTGGTAGTTGGAGCaGT | CACTCTTGCCTACGCCA | |
TTTATTATAAGGCCTGCTG | ATTGTTGGATCATATTCGTCCAC | ||||
AAAATG |
ULV1 | Primer | Final conc. (each) (μM) | Ratio (OF:MSP:OR) | MgCl2 (mM) |
---|---|---|---|---|
Pool 1 | EGFR E709K/G719X OF | 0.12 | 1:1:0.35 | 2.75 |
EGFR E709K/G719X OR | 0.04 | |||
EGFR E709K MSP R | 0.12 | |||
EGFR G719A MSP F | 0.12 | |||
EGFR G719D MSP F | 0.12 | |||
EGFR E746_A750delinsK/L747_P753>S OF | 0.04 | 0.1:1:0.1 | ||
EGFR E746_A750delinsK/L747_P753>S OR | 0.04 | |||
EGFR E746_A750deinsK MSP F | 0.36 | |||
EGFR L747_P753>S MSP R | 0.36 | |||
EGFR C797S OF | 0.12 | 1:1:0.35 | ||
EGFR C797S OR | 0.04 | |||
EGFR C797S MSP R | 0.12 | |||
KRAS G13D/V OF | 0.24 | 1:1:0.5 | ||
KRAS G13D/V OR | 0.12 | |||
KRAS G13D MSP R | 0.24 | |||
KRAS G13V MSP R | 0.24 | |||
Pool 2 | EGFR E746_A750del OF | 0.12 | 1:1:0.2 | 2.75 |
EGFR E746_A750del OR | 0.02 | |||
EGFR E746_A750del MSP R | 0.12 | |||
EGFR T790M OF | 0.12 | 1:1:0.2 | ||
EGFR T790M OR | 0.02 | |||
EGFR T790M MSP R | 0.12 | |||
EGFR L858R OF | 0.02 | 0.2:1:1 | ||
EGFR L858R OR | 0.12 | |||
EGFR L858R MSP F | 0.12 | |||
BRAF V600E OF | 0.04 | 0.35:1:1 | ||
BRAF V600E OR | 0.12 | |||
BRAF V600E MSP F | 0.12 | |||
KRAS G12D/V OF | 0.06 | 0.5:1:1 | ||
KRAS G12D/V OR | 0.12 | |||
KRAS G12D MSP F | 0.12 | |||
KRAS G12V MSP F | 0.12 |
Parameter | EGFR |
||||||||
---|---|---|---|---|---|---|---|---|---|
E709K | G719A | G719D | E746_A750delinsK | E746_A750del | L747_P753>S | T790M | C797S | L858R | |
Sensitivity (%) (95% CI) | 100 (94.87-100) | 100 (90-100) | 94.3 (80.84-99.3) | 95.7 (87.98-99.11) | 94.3 (80.84-99.3) | 85.7 (69.74-95.19) | 88.6 (73.26-96.8) | 100 (90-100) | 100 (90-100) |
Specificity (%) (95% CI) | 97.2 (93.64-99.09) | 98.6 (95.98-99.71) | 98.1 (95.31-99.49) | 98.9 (96.04-99.87) | 99.3 (96.22-99.98) | 95.3 (91.61-97.75) | 98.6 (95.11-99.83) | 100 (98.3-100) | 96.6 (92.14-98.87) |
AUC (%) | 99.3 | 99.8 | 98.8 | 98.9 | 96.9 | 91 | 96.9 | 100 | 99.8 |
Cutoff point (%) | 4.9 | 17.2 | 17.2 | 1.8 | 4.8 | 7.4 | 10.6 | 38.0 | 13.5 |
Limit of detection (%) | 0.025 | 0.025 | 0.05 | 0.025 | 0.05 | 0.05 | 0.1 | 0.025 | 0.025 |
PPV (%) (95% CI) | 93.33 (85.51-97.08) | 92.11 (79.13-97.29) | 89.19 (75.69-95.62) | 97.1 (89.4-99.25) | 97.06 (82.37-99.57) | 75 (61.74-84.8) | 93.94 (79.57-98.41) | 100 | 87.5 (74.74-94.31) |
NPV (%) (95% CI) | 100 | 100 | 99.06 (96.49-99.75) | 98.34 (95.15-99.45) | 98.63 (94.94-99.64) | 97.62 (94.79-98.93) | 97.28 (93.43-98.9) | 100 | 100 |
Accuracy (%) (95% CI) | 98 (95.39-99.35) | 98.8 (96.53-99.75) | 97.6 (94.85-99.11) | 98 (95.39-99.35) | 98.33 (95.21-99.65) | 94 (90.3-96.6) | 96.67 (92.89-98.77) | 100 (98.54-100) | 97.22 (93.64-99.09) |
Parameter | BRAF |
KRAS |
||
---|---|---|---|---|
V600E | G12D | G12V G13D | G13V | |
Sensitivity (%) (95% CI) | 97.1 (85.08-99.93) | 80 (63.06-91.56) | 94.3 (80.84-99.3) 97.1 (85.08-99.93) | 91.4 (76.94-98.2) |
Specificity (%) (95% CI) | 97.9 (94.07-99.57) | 91.7 (85.99-95.65) | 99.3 (96.22-99.98) 89.8 (84.92-93.48) | 87.9 (82.78-91.95) |
AUC (%) | 99.4 | 92.2 | 98.5 98.3 | 92.8 |
Cutoff point (%) | 3.8 | 3.1 | 13.3 6.0 | 5.4 |
Limit of detection (%) | 0.025 | 0.1 | 0.025 0.025 | 0.05 |
PPV (%) (95% CI) | 91.89 (78.69-97.21) | 70 (56.97-80.44) | 97.06 (82.37-99.57) 60.71 (50.88-69.75) | 55.17 (45.84-64.15) |
NPV (%) (95% CI) | 99.3 (95.36-99.9) | 95 (90.72-97.36) | 98.63 (94.94-99.64) 99.48 (96.55-99.93) | 98.44 (95.52-99.47) |
Accuracy (%) (95% CI) | 97.78 (94.41-99.39) | 89.44 (84.01-93.52) | 98.33 (95.21-99.65) 90.8 (86.52-94.08) | 88.4 (83.77-92.09) |
Characteristic | No. (%) |
---|---|
Age (yr) | |
Median (range) | 65 (39-83) |
Sex | |
Female | 48 (46.2) |
Male | 56 (53.8) |
Pathologic stage | |
I | 23 (22.1) |
II | 13 (12.5) |
III | 14 (13.5) |
IV | 54 (51.9) |
Activating EGFR mutation | |
Exon 18 | 3 (2.9) |
Exon 19 | 36 (34.6) |
Exon 20 | 3 (2.9) |
Exon 21 | 37 (35.6) |
Exon 19+Exon 20 | 1 (1.0) |
Wild | 24 (23.1) |
Patient | Stage | cfDNA amount (ng, for DNA library) | Gene | Mutation | Observed VAF by CT-ULTRA (%, variant allele reads/total reads) | Observed enriched VAF by ULV1 (%) | Semi-quantitative dynamic range (%) | Expected VAF (%) |
---|---|---|---|---|---|---|---|---|
AMC036 | II | 2.4 | TP53 | p.A161Pfs*9 | 3.1 (5/163) | NA | NA | NA |
EGFR | p.L858R | 1.1 |
82 | 29.7-75.4 | > 1 and ≤ 5 | |||
AMC044 | III | 6.5 | MTOR | p.A134V | 1.4 (4/285) | NA | NA | NA |
EGFR | p.L858R | 0.33 |
51 | 29.7-75.4 | 0.08 | |||
AMC070 | IV | 10 | EGFR | p.E746_A750delinsK | 1.2 (5/422) | 64 | 5.8-62.9 | > 1 and ≤ 5 |
EGFR | p.T790M | 0.23 |
31 | 11.2-65.5 | 0.20 |
Hotspot | Diluted sample (%) | ULV1 | PANAMutyper |
---|---|---|---|
EGFR exon 19 del | 1 | 3/3 (100) | 3/3 (100) |
0.5 | 3/3 (100) | 3/3 (100) | |
0.25 | 3/3 (100) | 3/3 (100) | |
0.1 | 3/3 (100) | 3/3 (100) | |
0.05 | 3/3 (100) | 3/3 (100) | |
0.025 | 3/3 (100) | 3/3 (100) | |
0 | 0/3 (0) | 0/3 (0) | |
EGFR T790M | 1 | 3/3 (100) | 3/3 (100) |
0.5 | 3/3 (100) | 3/3 (100) | |
0.25 | 3/3 (100) | 3/3 (100) | |
0.1 | 3/3 (100) | 3/3 (100) | |
0.05 | 3/3 (100) | 2/3 (66.7) | |
0.025 | 2/3 (66.7) | 0/3 (0) | |
0 | 0/3 (0) | 0/3 (0) | |
EGFR L858R | 1 | 3/3 (100) | 3/3 (100) |
0.5 | 3/3 (100) | 3/3 (100) | |
0.25 | 3/3 (100) | 3/3 (100) | |
0.1 | 3/3 (100) | 3/3 (100) | |
0.05 | 3/3 (100) | 3/3 (100) | |
0.025 | 3/3 (100) | 3/3 (100) | |
0 | 0/3 (0) | 0/3 (0) |
Lowercase letters in the MSP sequence indicates 3′ terminal mismatch.
AUC, area under the curve; CI, confidence interval;
AUC, area under the curve; CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value; ULV1, ultra-high-sensitivity lung version 1.
cfDNA, cell-free DNA; NA, not available; ULV1, ultra-high-sensitivity lung version 1; VAF, variant allelic frequenc. VAFs were measured by manual curation using Integrative Genomics Viewer (IGV).
Values are presented as number (%) unless otherwise indicated.