Abstract 3245: Analytical validation of a sensitive, tissue-free blood test for tumor burden monitoring

Abstract Background: Liquid biopsy stands as a valuable tool in the field of precision medicine, with its utility extending across cancer detection, recurrence monitoring, and treatment selection. However, achieving the desired level of sensitivity often requires access to tumor tissue, which can be a limiting factor. Recent studies have shown that analyzing methylation patterns in circulating tumor DNA (ctDNA) enables a sensitive and accurate measurement of tumor burden in a tissue-independent manner, thus opening up new applications in relapse monitoring and therapy response assessment. Here, we report the initial validation of OverC MonitorTM, a multi-cancer blood test that interrogates methylation information from over 2, 000 genes at single-base resolution across various solid tumors. Methods: Analytical performance of OverC MonitorTM was assessed using 672 contrived cell line samples and cell-free DNA (cfDNA) samples with 10-30ng input amounts. Analytical sensitivity was evaluated using a dilution series of cell line and cfDNA samples across different tumor allele fraction (TAF) levels, ranging from 0.001% to 0.5%. The limit of detection (LoD95) was determined as the lowest tumor methylated fraction (TMeF) that achieved ≥95% detection probability through Probit regression for each cancer type. In addition, both ultra-deep sequencing (HS-UMI) and droplet digital PCR (ddPCR) were employed to verify the accuracy of TMeF in these dilution mixtures. The limit of blank (LoB) was evaluated by per-sample positive rate in 72 age-matched cancer-free donors. The combined precision and accuracy study was carried out by testing clinical samples from patients with lung cancer (LC), colorectal cancer (CRC), liver cancer (HCC) and non-cancer donors for a total of 216 replicates across different conditions. Results: For sample-level methylation, the limits of detection (LoDs) were reached as low as 0.004% for cell line dilutions, and 0.02% for circulating-free DNA (cfDNA) titrations. The per-sample false positive rate was 0% (95%CI, 0-5.0%) among age-matched cancer-free donors. The positive predictive agreement (PPA) and negative predictive agreement (NPA) across different instruments, operators, and reagent lots were 100% (95%CI, 97.1-99.8%) and 100% (95%CI, 91.7-100%), respectively. Additional validation is ongoing, and data will be presented as it becomes available. Conclusions: OverC MonitorTM is a patient-care-ready liquid biopsy capable of detecting common solid tumors even at ultra-low tumor burden. It requires no prior knowledge for downstream analysis, offering a new opportunity to improved sensitivity in a cost-effective manner. In addition, it is designed to be compatible with conventional tumor genotyping methods, adding a new dimension in the continuum of cancer management. Citation Format: Qiancheng You, Jing Su, Jiayue Xu, Xingyu Yang, Sa Zhang, Zeliang Deng, Guangyou Li, Xinyue Kang, Xiaoling Li, Si Zhang, Meifang Wu, Bingsi Li. Analytical validation of a sensitive, tissue-free blood test for tumor burden monitoring [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 3245.