Title Page
Abstract
Graphical Abstract
Contents
Introduction 13
Material and Methods 15
Preparation of positive controls and plasma samples 15
cfDNA library construction 17
Positive sample dilution 17
Analysis of hotspot mutations using the ULV1 panel 22
Targeted NGS analysis of cfDNA using CT-ULTRA panel 28
LOD comparison with PANAMutyper test for three hotspot mutations 31
Statistical analysis 31
Results 33
Optimization for mutant allele selective amplification using the ULV1 panel 33
Performance of the ULV1 panel with high analytical sensitivity and specificity 41
Application of the ULV1 panel using cfDNA from NSCLC patients 47
Feasibility of semi-quantitative analysis of the ULV1 panel 53
Comparison of the ULV1 and PANAMutyper™ R EGFR performances 57
Discussion 59
Conclusion 69
Bibliography 70
국문 요약 74
Table 1. cfDNA library yield according to the input cfDNA amount from 104 patients 18
Table 2. Primer sequences of the ULV1 panel 24
Table 3. Gene list of CT-ULTRA 30
Table 4. The optimal condition of the ULV1 Panel 39
Table 5. Summary of positive controls for evaluating the analytical performance of the ULV1 panel 42
Table 6. Performance of EGFR assays in the ULV1 panel 43
Table 7. Characteristics of 104 patients for the ULV1 and CT-ULTRA test 48
Table 8. Variants detected in cfDNA by the ULV 1 panel only 56
Table 9. Direct comparison between the ULV1 and PANAMutyper 58
Figure 1. Schematic of the experimental procedure for identifying mutational concordance between the tumor tissue and plasma cfDNA. 16
Figure 2. Schematic of PCR amplification steps for the ULV1 panel. 27
Figure 3. Direct comparison of detection sensitivity between the ULV1 panel and conventional MassARRAY using serially diluted H1975 gDNA. 35
Figure 4. Optimization of PCR conditions depending on the unbalanced primer concentration using diluted H1975 gDNA. 36
Figure 5. Optimization of PCR conditions depending on MgCl₂ concentration using 0.5% Seraseq material. 38
Figure 6. Amplification efficiency and sensitivity of the ULV1 panel. 45
Figure 7. Amplification curve shape of the enriched VAF against the initial VAF. 46
Figure 8. Validation of the ULV1 panel using 104 patient samples. 51
Figure 9. Feasibility of semi-quantitative analysis. 55
Figure 10. Schematic of the difference between the conventional PCR and ULV1 panel. 65
Figure 11. Schematic of the amplification efficiency depending on unbalanced primer concentration. 67
Figure 12. Distribution of plasma storage duration between stages in the cohort. 68