Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.
Authors
Sung-Su Kim ; Hyun-Jeung Choi ; Jin Ju Kim ; M. Sun Kim ; In-Seon Lee ; Bohyun Byun ; Lina Jia ; Myung Ryurl Oh ; Youngho Moon ; Sarah Park ; Joon-Seok Choi ; Seoung Wan Chae ; Byung-Ho Nam ; Jin-Soo Kim ; Jihun Kim ; Byung Soh Min ; Jae Seok Lee ; Jae-Kyung Won ; Soo Youn Cho ; Yoon-La Choi ; Young Kee Shin
In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, 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.