Abstract B093: Enhanced ctDNA extraction with quaternary ammonium salt integrated in a ctKRAS prototype assay significantly improves sensitivity for detecting low-allelic frequency KRAS mutations in plasma

Abstract Background: Liquid biopsy offers opportunities for the management of colorectal cancer, such as: diagnosis, therapy selection, monitoring and minimal residual disease assessment. Yet, routine adoption has been hindered by the need for high technical expertise, sample batching, and high cost. The Idylla™ platform is a fully automated sample-to-result, real-time PCR system that can overcome these obstacles. Assay cartridges for plasma are available to genotype KRAS, NRAS, BRAF, and EGFR. A growing need for enhanced sensitivity triggered the development of a novel 4 ml circulating tumor DNA (ctDNA) extraction method based on quaternary ammonium salt. Design: A proof-of-concept study was performed to evaluate the sensitivity of the novel ctDNA extraction technology, using KRAS G12C, G12D and G12V as demonstration cases. A 2nd generation IdyllaTM ctKRAS prototype was developed to evaluate the sensitivity to detect KRAS mutants in plasma. KRAS mutants (gBlocks of 170 bp) were used for this evaluation, in a background of normal human plasma matrix and fragmented ctDNA (around 170 bp) (Acrometrix), mimicking the natural background of cell-free DNA in human plasma. The 4-ml samples were prepared at two input levels, a medium input level (10ng ctDNA/ml) and a low input level (2.5ng ctDNA/ml) representative for an average and a minimum concentration of ctDNA observed in patient plasma samples, respectively. For each input level and KRAS mutation six replicates of five concentrations of a 2-fold serial dilution were tested to define the lowest level detected with 100% hit rate. The detectability of KRAS G12C was confirmed in seven human plasma samples spiked with 12.5 cps/ml G12C mutant and tested with the prototype. Results: The Table below lists the lowest concentrations of mutations detectable with a 100% hit rate in plasma using the IdyllaTM ctKRAS prototype with the novel ctDNA extraction technology. The detectability of 12.5 cps/ml of KRAS G12C was successfully confirmed in all human plasma samples tested, collected with K2-EDTA (dipotassium ethylenediaminetetraacetic acid; N=4) or with Streck tubes (N=3). Test results from sample to result were available within 3h, with a hands-on time of less than 5 minutes. Conclusions: The novel ctDNA extraction technology integrated in IdyllaTM allows to develop fully automated sample-to-result assays able to reliably detect mutations in plasma as low as 6.25 cps/ml starting from 4 ml of plasma within 3h rendering the technology suitable for therapy selection and response monitoring. Target Medium input (10 ng ctDNA/ml) Low input (2.5 ng ctDNA/ml) G12C 0.4% Allelic Frequency (AF; 12.5 cps/ml) 1.67% AF (12.5 cps/ml) G12D 0.2% AF (6.25 cps/ml) 0.8% AF (6.75 cps/ml) G12V 0.4% AF (12.5 cps/ml) 3.3% AF (25 cps/ml) Citation Format: Bieke Van Dorst, Ludovic Barault, Eef Vankerckhoven, Kristof Gilliaert, Tania Ivens, Lei Du, Anthony Green, Joost C.A. Janssens. Enhanced ctDNA extraction with quaternary ammonium salt integrated in a ctKRAS prototype assay significantly improves sensitivity for detecting low-allelic frequency KRAS mutations in plasma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B093.