Abstract 5879: An optimized sample-to-result workflow for detection of tumor mutations and immune sequencing in liquid biopsies

Abstract Non-invasive liquid biopsies are helping advance various aspects of disease assessment including early detection, disease monitoring, and therapy selection, while also allowing for improved patient follow-up and economic efficiency over traditional tissue biopsies. Plasma cell-free DNA (cfDNA) applications have recently been adopted in prenatal screening, tumor mutation profiling and post-transplant monitoring. Additionally, combining cfDNA profiling with paired RNA analysis of peripheral blood mononuclear cells (PBMCs) can provide useful insights into biomarkers and immune phenotypes, and present a comprehensive multiomic profile from a single blood draw. Several technical and analytical challenges persist in the liquid biopsy field including immediate preservation of draw time levels of cfDNA, minimizing PBMC lysis during storage, low concentrations of cfDNA with even lower tumor fraction, as well as the need for a higher sensitivity in downstream applications to accurately interpret clinical signals. Here, we present an efficient blood-to-result workflow for tumor-specific mutation detection and immune profiling using contrived samples spiked with known mutation controls. Whole blood was collected in Streck Nucleic Acid BCT enabling preservation of draw time concentration of cell-free nucleic acids and prevention of immune cell lysis. Nucleic acid extraction was performed on the isolated PBMCs and plasma fraction using an optimized protocol for the Monarch Mag Viral DNA/RNA Extraction Kit which features a simplified, magnetic bead-based workflow. Isolated cfDNA was used to prepare sequencing libraries using an optimized library prep workflow to maximize conversion efficiency from cfDNA. Isolated PBMC RNA was utilized to prepare RNA-seq libraries and Immune-seq libraries using the NEBNext UltraExpress RNA Library Prep Kit and NEBNext Immune Sequencing Kit, respectively. Combining these sample storage, extraction, and library prep methods resulted in a high yield of cfDNA libraries with minimal contaminating genomic DNA, enabling improved sequencing data quality, high conversion efficiency, and high sensitivity for clinical research, including detecting spike-in mutations. Transcriptome and immune repertoire profiling from the preserved PBMC in the same blood sample provided complementary biomarker information to enable a comprehensive evaluation of the disease status of these samples. Citation Format: Anagha Kadam, Margaret R. Heider, Chen Song, Jian Sun, Ariel Erijman, Nicholas M. George. An optimized sample-to-result workflow for detection of tumor mutations and immune sequencing in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5879.