Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA
DNA의 특질 분석을 이용한 드롭렛 디지털 PCR 기반의 EGFR 돌연변이 검출 방법 연구
- 약학대학 약학과
- Issue Date
- 서울대학교 대학원
- EGFR; epidermal growth factor receptor; FFPE; Formalin fixed paraffin embedded; DIN; DNA integrity number; ddPCR; Droplet digital PCR; Genetic mutation detection; iQC; internal quality control; Liquid biopsy; ctDNA; Circulating tumor DNA
- 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.