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A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients

Authors Feng Q, Gai F, Sang Y, Zhang J, Wang P, Wang Y, Liu B, Lin D, Yu Y, Fang J

Received 2 August 2017

Accepted for publication 31 October 2017

Published 18 January 2018 Volume 2018:10 Pages 115—121

DOI https://doi.org/10.2147/CMAR.S148134

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Colin Mak

Peer reviewer comments 2

Editor who approved publication: Dr Antonella D'Anneo


Qin Feng,1,* Fei Gai,2,* Yaxiong Sang,2 Jie Zhang,3 Ping Wang,1 Yue Wang,1 Bing Liu,2 Dongmei Lin,1 Yang Yu,2 Jian Fang3

1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Pathology, Peking University Cancer Hospital & Institute, 2Oncology Business Division, Beijing Novogene Bioinformatics Technology Co., Ltd, 3Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Thoracic Oncology II, Peking University Cancer Hospital & Institute, Beijing, China

*These authors contributed equally to this work

Background:
The AURA3 clinical trial has shown that advanced non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations in circulating tumor DNA (ctDNA) could benefit from osimertinib.
Purpose: The aim of this study was to assess the usefulness of QuantStudio™ 3D Digital PCR System platform for the detection of plasma EGFR T790M mutations in NSCLC patients, and compare the performances of 3D Digital PCR and ARMS-PCR.
Patients and methods: A total of 119 Chinese patients were enrolled in this study. Mutant allele frequency of plasma EGFR T790M was detected by 3D Digital PCR, then 25 selected samples were verified by ARMS-PCR and four of them were verified by next generation sequencing (NGS).
Results: In total, 52.94% (69/119) had EGFR T790M mutations detected by 3D Digital PCR. In 69 positive samples, the median mutant allele frequency (AF) was 1.09% and three cases presented low concentration (AF <0.1%). Limited by the amount of plasma DNA, 17 samples (AF <2.5%) and eight samples (T790M-) were selected for verification by ARMS-PCR. Four of those samples were verified by NGS as a third verification method. Among the selected 17 positive cases, ten samples presented mutant allele frequency <0.5%, and seven samples presented intermediate mutant allele frequency (0.5%<AF<2.5%). However, only three samples (3/17) were identified as positive by ARMS-PCR, namely, P6 (AF =1.09%), P7 (AF =2.09%), and P8 (AF =2.21%). It is worth mentioning that sample P9 (AF =2.05%, analyzed by 3D Digital PCR) was identified as T790M- by ARMS-PCR. Four samples were identified as T790M+ by both NGS and 3D Digital PCR, and typically three samples (3/4) presented at a low ratio (AF <0.5%).
Conclusion: Our study demonstrated that 3D Digital PCR is a novel method with high sensitivity and specificity to detect EGFR T790M mutation in plasma.

Keywords: 3D Digital PCR, allele frequency, EGFR TKIs, resistance, osimertinib, erlotinib, gefitinib, icotinib

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