DNA의 특질 분석을 이용한 드롭렛 디지털 PCR 기반의 EGFR 돌연변이 검출 방법 연구

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 2. 신영기.Formalin-fixed paraffin-embedded tissue (FFPET) samples are invaluable sources for both translational clinical research and molecular in vitro diagnostics. However, accurate detection of genetic mutations in FFPET is a major challenge due to artifactual results, due to sample age and quality. In a pre-clinical study, we used 315 non-small cell lung cancer (NSCLC) FFPET-derived DNA (FFPET-DNA) samples to establish sample criteria reflecting the minimum DNA quality suitable for PCR by comparing the results of droplet digital PCR-based mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas® EGFR test). Using this criteria, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests of 171 NSCLC FFPET-DNA samples. Based on the pre-clinical study, the FFPET-DNA sample criterion was established as internal quality control (iQC) index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng [1000 genome equivalents] of FFPET-DNA), indicating that more than half of the input DNA was amplifiable. Based on this iQC index, an independent clinical study revealed that both tests were significantly concordant (overall percent agreement (OPA) = 92.98%). Discordants not detected by the cobas EGFR test were detected using the ddEGFR test, indicating that the higher sensitivity of the ddEGFR test is due to its lower limit of detection. iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and at least equivalent clinical performance. ddEGFR test is expected to serve as an appropriate treatment guidance for NSCLC patients.I. Introduction 1 1. EGFR mutations in NSCLC 2 2. Companion diagnostics (CDx) 9 3. DNA damage in FFPETs 13 3.1 DNA fragmentation 15 3.2 Hydrolytic deamination 15 3.3 Formaldehyde-induced abasic site 17 3.4 Formaldehyde-induced cross-link 17 3.5 DNA damage during preparation of FFPE blocks (physical stress) 17 3.6 Strategies for minimizing sequence artifact 19 4. Liquid biopsy 22 5. Third generation PCR: Droplet digital PCR (ddPCR) 24 6. Limit of blank, limit of detection and limit of quantitation 27 6.1 Limit of blank (LoB) 27 6.2 Limit of detection (LoD) 27 6.3 Limit of quantitation (LoQ) 28 II. Purpose of the study 30 III. Materials and Methods 33 IV. Results 43 V. Discussion 81 VI. Conclusion and perspectives 87 VII. Reference 94 국문 초록 114Docto