Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA

TLDR

Circulating tumor DNA is emerging as a diagnostic, prognostic, and predictive tool, yet the growing list of actionable gene targets demands reliable, multiplexed detection technologies. This study introduces the InVision liquid‑biopsy platform, employing enhanced TAm‑Seq to detect clinically relevant somatic mutations at low allele fractions across 35 cancer genes. Analytical validation in two laboratories assessed reproducibility and quantitative performance of eTAm‑Seq against digital PCR using DNA standards and full‑process controls. The platform detected mutant alleles as low as 0.02 % AF with 99.9997 % specificity, achieving 94 % detection at 0.25–0.33 % AF and 90 % at lower DNA inputs, demonstrating robust, reproducible clinical applicability.

Abstract

Introduction Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.