5746 - Next-generation sequencing panel verification to detect low frequency single nucleotide and copy number variants from mixing cell line studies

Background

The next-generation sequencing (NGS) technology has increased the number of genes and types of genomic alterations detectable by a high-throughput assay and has become an essential part of clinical decision-making. We aimed to verify the analytical specifications of TruSight™ Tumor 170 (TST170, Illumina Inc.) panel for detecting 5% variant allele frequency (VAF) and reliable amplifications (copy number variants, CNVs) for ensuring high quality of sequencing results.

Methods

We use well-characterized human cancer cell lines mixtures with known specific gene mutations to evaluate the specifications given by TST170 and defined thresholds for each type of genomic alterations intended to detect. We performed the following mixing studies by using the lung adenocarcinoma cell lines H1975 and H1299 to acquired proportions: 100%, 33%, 10% and 2% H1975. H1975 harbors EGFR p.Thr790Met (c.2369C>T), p.Leu858Arg (c.2573T>G), TP53 p.Arg273His (c.818G>A), CDKN2A p.Glu69Ter (c.205G>T) and PIK3CA p.Gly118Asp (c.353G>A). H1299 harbors an EGFR, CDKN2A, PIK3CA wild type and a homozygous partial deletion of the TP53 gene. We extracted DNA and performed NGS TST170 analysis with each diluted sample.

Results

When we calculated the correlation between the expected and observed VAFs by linear regression analysis, the coefficient of determination was 0.99 in hotspot NSCLC genes. This result further supports the reliability of this system for variant identification. Mutations with a low VAF (<5%) were identified in half of the mutations and all mutations with >5% VAF were detected without exception. In the CNV analysis, the number of copies seen in the undiluted cell line (100% H1975) were found in all 33% dilution cases, in one third of the 10% dilution cases and in no case in the 2% dilution, independently of undiluted cell line fold changes.

Conclusions

Our analytical verification determines the ability of TST170 pipeline to detect 5% VAF with high confidence and CNVs in samples of tumor purity at 33% or more.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.