J Cancer 2018; 9(9):1680-1688. doi:10.7150/jca.24126

Research Paper

Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma

Donglai Liu1, Haiwei Zhou1, Dawei Shi1, Shu Shen1, Yabin Tian1, Lin Wang2, Jiatao Lou2, Rong Cong3, Juan Lu3, Henghui Zhang4, Meiru Zhao5, Shida Zhu6, Zhisheng Cao7, Ruilin Jin8, Yin Wang9, Xiaoni Zhang10, Guohua Yang3, Youchun Wang1✉, Chuntao Zhang1✉

1. Division II of In vitro Diagnostics for Infectious Diseases, Institute for In vitro Diagnostics Control, National Institutes for Food and Drug Control, Beijing, China
2. Department of Laboratory Medicine, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China
3. GenoSaber Biotech Co. Ltd., Shanghai, China
4. Genecast Precision Medicine Technology Institute, Beijing, China
5. Geneplus-Beijing, Beijing, China
6. BGI-Shenzhen, Shenzhen, China
7. Novogene Bioinformatics Technology Co., Ltd., Beijing, China
8. Annoroad Gene Technology Co., Ltd., Beijing, China
9. Berry Genomics Co., Ltd., Beijing, China
10. HaploX Biotechnology Co., Ltd., Shenzhen, China

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Liu D, Zhou H, Shi D, Shen S, Tian Y, Wang L, Lou J, Cong R, Lu J, Zhang H, Zhao M, Zhu S, Cao Z, Jin R, Wang Y, Zhang X, Yang G, Wang Y, Zhang C. Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma. J Cancer 2018; 9(9):1680-1688. doi:10.7150/jca.24126. Available from http://www.jcancer.org/v09p1680.htm

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Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations.

Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ).

Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency (CVs ranging from 18% to 106%). It was worth noting that the CV value seemed to correlate with a particular mutation as well. The repeatability of determination of different mutations was L858R>T790M>19del.

Conclusions: The results indicated our reference materials would be pivotal for quality control of NGS-based cancer IVDs and would guide the further development of reference materials covering more onco-relevant mutations.

Keywords: reference materials, next-generation sequencing, circulating tumor DNA, formalin-fixed paraffin-embedded.