Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer–Related Single-Nucleotide Variants and Copy Number Variants Using Simulated Cell-Free DNA

Abstract

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Co‐mutational assessment of circulating tumour DNA (ctDNA) during osimertinib treatment for T790M mutant lung cancer

“…cfDNA extraction and library construction were performed according to previous publication . Peripheral blood lymphocytes (PBLs) from the first centrifugation were used for the extraction of germline genomic DNA.…”

Immune and Circulating Tumor DNA Profiling After Radiation Treatment for Oligometastatic Non-Small Cell Lung Cancer: Translational Correlatives from a Mature Randomized Phase II Trial

“…Single nucleotide variants were called using MuTect2 (3.4-46-gbc02625) and NChot, a software developed in-house to review hotspot variants. 11 Small insertions and deletions were determined with GATK. Candidate variants were verified manually in the Integrative Genomics Viewer.…”

Biospecimen Science of Blood for cfDNA Genetic Analyses

“…Diagnostic non-invasive prenatal testing (NIPT) routinely uses maternal and fetal cfDNA and several FDAapproved and CE-marked kits [2,3]. Recently, sequencing of cfDNA in maternal plasma has even been suggested as a tool for paternity testing [4]. Analysis of cfDNA for prognosis of cancer recurrence or metastasis, based on monitoring and increase of cfDNA concentrations, as well as for prediction of success of cancer therapies, and adaptation of targeted therapies, based on appearance of new variants, is likely to be implemented in clinical diagnostics [5].…”