학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2018. 2. 묵인희.Introduction: Interrogation of circulating tumor (ct)DNA using next-generation sequencing (NGS)-based methods have been proposed as a way to track the dynamics of tumor in real time. However, there was no standard guideline for ctDNA sequencing that I have evaluated the procedure from end-to-end to propose the optimal analysis methods for ctDNA sequencing. Chapter 1* emphasizes the importance of the recovery of unique DNA molecule from the minimal amount of starting material. After that, the systematic evaluation of each step highlights the error-prone step in the sequencing process. In Chapter 2, the utility of ctDNA sequencing has evaluated through the monitoring of tumor genomic in multi-cancer samples.
Method: To maximize the recovery rate of unique DNA molecule, I approached the ligation step during the library preparation in sequencing protocol by optimizing the temperature, time and adapter concentration. Identification of technical errors was conducted with the comparison of background error distribution from the acoustically sheared germline DNA and naturally fragmented cell-free DNA. The utility of ctDNA sequencing analysis was assessed by comparing the standard protein biomarker and imaging changes during the patients therapeutic intervention.
Results: The modified ligation conditions for the minimal amount of starting material able to increase the recovery rate of unique DNA molecule by 20% compared to the standard conditions. A comparison of the characteristic of acoustically sheared gDNA and naturally fragmented cfDNA revealed that gDNA constituted with 64% of C: G> A: T and 39% of C: G> G: C substitution class changes. Through testing of the series of the mild sheared conditions, the reduction of error rate was observed with an average of 40%. Furthermore, the analysis of the vicinity at the ends of the DNA fragments revealed that A> G and A> T preferentially fragmented. The enhanced analytical performance in NGS method able to establish diagnostic utility with the detection sensitivity of 100% and specificity of 97.1% as applied to cancer plasma samples. The level of ctDNA was not only highly correlated with the therapeutic response but also showed an average of two months earlier reaction than the standard protein biomarker and imaging changes. Finally, the determination of tumor heterogeneity was observed through ctDNA analysis, which was not discovered in the matched tumor biopsies.
Conclusions: Overall, the unique characterization of cfDNA could not only emphasize the underlying cause of technical errors but also demonstrate opportunities for early detection of cancer using NGS-based technology. Ultimately, the combined approach of ctDNA and NGS sequencing analysis is believed to address unmet needs in cancer research.GENERAL INTRODUCTION 1
Cell-free DNA 3
Circulating tumor DNA 4
Current detection methods for ctDNA 4
Digital PCR 4
Next generation sequencing 5
NGS-based ctDNA analysis 7
Potential misdiagnosis from background errors 8
CHAPTER1 Practical guidelines for cell-free DNA analysis using enhanced analytical performance of NGS-based method 15
INTRODUCTION 15
MATERIALS AND METHODS 18
RESULTS 22
Comparison of blood collection tubes 22
Optimization of library preparation 22
Optimizing statistical modeling for cfDNA analysis 24
Performance of optimized TDS on cfDNA and PBL DNA 24
Estimation of errors derived by TDS 25
From sequencing reaction 25
Distribution of background errors 25
Sample preparation caused background errors 26
Breakpoint preferences 27
Multi-statistical adjustment for removing the background errors 29
DISCUSSION 31
CHAPTER 2 Ultrasensitive interrogation of circulating tumor DNA from cancer patients using enhanced analytical performance of the NGS-based method 51
INTRODUCTION 58
MATERIALS AND METHODS 54
RESULTS 61
Evaluation of LOD with single mutation 61
KRAS mutations 61
Evaluation of LOD with multi-mutations 61
With primary mutation 61
Biopsy-free manner 62
Monitoring tumor burden by measuring ctDNA 63
Diagnostic utility 65
DISCUSSION 67
GENERAL DISCUSSION 94
REFERENCES 95Docto
