Abstract 323: Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput

Abstract Molecular testing that interrogates cell free DNA (cfDNA) from plasma has significant potential value across the cancer care continuum, including early cancer detection, driver mutation identification, and disease recurrence monitoring. To achieve this potential, liquid biopsy assays must be optimized to address multiple challenges. For one, cfDNA quantity in the blood is highly variable, with different amounts present depending on malignancy and stage of progression. This requires optimization to detect low concentrations of circulating tumor DNA (ctDNA) and to accommodate variable tumor fractions. In addition, minimizing assay costs will improve test accessibility and adoption. To address these issues, Genece Health is optimizing their lung cancer liquid biopsy assay to accommodate minimal cfDNA input into library preparation. Furthermore, Genece is testing algorithms that tolerate reduced read depths from low-pass whole genome sequencing (LP-WGS) while maintaining high performance. The Genece lung cancer liquid biopsy assay profiles fragment end-motif and size (FEMS) and fragment coverage signals using LP-WGS (Illumina) and proprietary machine learning algorithms. This study first tested the frequency of end motifs in pooled ctDNA after modifying library preparation (IDT) to reduce reagent volumes from 1X down to 0. 5X, 0. 25X, and 0. 1X of manufacturer specification. A range of cfDNA input from 0. 25 ng to 6 ng was tested with each library preparation volume. In addition, LP-WGS read coverage from 5X to 1X was evaluated to assess impact on end motif frequency and assay sensitivity. Performance characterization was further evaluated with a cohort of 50 clinical lung cancer and 100 healthy donor plasma samples tested at 1X and 0. 25X library reagent volume. We observed an expected increase in library complexity at higher cfDNA input and library reagent volume. Assay performance was minimally impacted by variable cfDNA input and library reagent volume. However, reducing sequencing read depth is tolerated down to 1. 25X depth before increasing the variability of end motif frequencies. These observations are corroborated in the 150 clinical sample cohort. This data shows that by altering standard library preparation conditions and reducing unique read depth, liquid biopsy assays can accommodate a wider range of cfDNA, while concurrently decreasing overall costs. Citation Format: Michael Salmans, Dasom Kim, Andrew Carson, Mengchi Wang, Byung In Lee, Bryan Leatham. Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 323.