Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA

Sung Su Kim ,M Sun Kim,Sarah Park,Young Kee Shin,Jihun Kim,Jin Ju Kim,Myung Ryurl Oh,Jae Seok Lee,Soo Youn Cho,Seoung Wan Chae,Lina Jia,Jin-Soo Kim,Joon Seok Choi ,Hyun-Ki Choi ,Byung Ho Nam ,Jae Kyung Won ,Yoon La Choi ,In Seon Lee ,B H Byun ,Byung Soh Min,Youngho Moon

doi:10.1038/s41598-017-18642-x

Abstract

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.

Highlights

In light of recent advancements in personalized medicine, nucleic acid-based diagnostics will play a pivotal role in implementation of targeted therapies
Because the Droplet digital PCR (ddPCR)-based test can lead to false positive results due to its intrinsic high sensitivity and FFPET characteristics, the cut-offs of the ddEGFR test were determined based on false-positive analyses using normal FFPET
Mutation calls were identified based on true-positive mutation values higher than limit of blank (LoB) and limit of detection (LoD) mutation index (MI) (1), which were established from analytical performance studies (Table 1)

Summary

Introduction

In light of recent advancements in personalized medicine, nucleic acid-based diagnostics will play a pivotal role in implementation of targeted therapies. Formalin fixation time significantly influences the quality of FFPET-DNA and the results of PCR analysis[17]. Because of the differences in storage duration and fixation procedures among laboratories, FFPET-DNA quality should be checked before being used in clinical studies. The clinical performance of ddPCR-based tests with FFPET-DNA quality measurement has not been subjected to a detailed comparison with gold standards, such as qPCR. We report the development of sample criteria for the minimum FFPET-DNA quality suitable for PCR, and the application of these criteria to a ddPCR-based EGFR mutation test. We compared the performance of the GenesWell ddEGFR mutation test (ddEGFR test) with that of the cobas EGFR mutation test (cobas EGFR test) in a retrospective clinical study using 171 NSCLC FFPET samples. Our results indicate that the use of sample criteria is critical for validating performance in clinical studies

Methods

Results

Conclusion