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

Abstract

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.

High MAF of EGFR mutations and high ratio of T790M sensitizing mutations in ctDNA predict better third‐generation TKI outcomes

“…We believe that the different technology used in their research might be the reason for the difference between cutoff ratios. We used capture‐based NGS for cfDNA analysis, which might have a lower efficiency in capture deletions than ddPCR . Since there were 47.83% (22/46) patients who received osimertinib harboring 19del in our study, it is reasonable that T790M sensi‐mutation ratio was higher than the ratio in the study by Del Re et al .…”

Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance

“…However, their clinical performance has not been fully evaluated under a universally accepted standard. The National Institutes for Food and Drug Control (NIFDC) has developed national reference materials for the quality control of IVDs 37 . Therefore, we designed this study for the investigation of a preparation of multiplex cfDNA RMs for the quality control of NGS-based IVDs.…”