An ultrasensitive method for detecting somatic mutations from circulating tumour DNA- AVENIO ctDNA Expanded Kit.

Abstract

Background Circulating tumour DNA (ctDNA) is extensively studied as a non-invasive, real-time biomarker that is advantageous in cancer diagnosis, early detection, prognostication, monitoring tumour dynamics and minimal residual disease. However, except in advanced disease conditions only small amount of ctDNA originate from tumour cells and is typically of low abundance. Therefore sensitive methods are required to detect mutations as low as allele frequency (AF) <0.1% from <100ng of ctDNA. Methods We evaluated the AVENIO ctDNA kit; a library preparation method for NGS based Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) and its concordance with an in-house developed Tagged-Amplicon deep sequencing (TAm-Seq) and Capture Sequencing (CS) for detection of somatic mutations. 4 control reference standards and 8 patient samples were tested at PeterMacCallum Cancer centre. Results All the samples produced >150nM libraries, indicating compatibility with diverse range of DNA inputs. The overall percent agreement for the control reference standards was 100%, with strong correlation between expected and observed AF. The AVENIO assay proved to be very sensitive in detecting mutations at low allele frequencies <0.1%, for patient samples with an overall percent agreement of 54.5% and positive percent agreement of 92.31%. Variants detected by AVENIO but missed by in-house methods were below the analytical sensitivity (0.5%) of these assays. Conclusions The AVENIO assay is a powerful tool for the sensitive detection of variants from ctDNA. The assay could be used for molecular profiling and disease monitoring in patients. AVENIO assay is a comprehensive solution for detecting different variant classes like Single nucleotide variations (SNV), Indels, Fusions and Copy Number Variations (CNV) in a single workflow. This assay was able to detect mutations at low allele frequencies <0.1% that would be otherwise missed using conventional ctDNA analysis techniques.