Abstract 105: Quantification of circulating tumor DNA (ctDNA) in patients using cancer-specific, methylation-based, tissue-free tests for the detection of molecular residual disease (MRD).

Abstract While MRD detection by differential methylation patterns has been shown to predict disease recurrence, clinical decision-making and disease management are increasingly dependent on the exact level of the measured biomarker. We assessed quantitative results by methylation-based MRD assays designed for colorectal (CRC), breast, lung, and muscle-invasive bladder cancer (MIBC). Each cancer-specific, methylation-based, tissue-free ctDNA assay uses next-generation sequencing to query regions of the human genome that are differentially methylated in patients with colorectal, breast, lung, or MIBC cancer compared to cancer-free individuals. Plasma samples from Signatera-tested cancer patients with stage I-IV disease (CRC N=105, breast N=112, lung N=113, MIBC N=60) and from cancer-free individuals (N=223, sex-matched for breast cancer) were analyzed in a cross-validation setup. We compared ctDNA levels measured by the methylation-based assay (estimated by the fraction of differentially methylated alleles for circulating cell-free DNA targets across regions, DMAF) with those from a bespoke, tumor-informed ctDNA assay (SignateraTM, measured as variant allele fraction, VAF), which served as the reference, by calculating the mean squared error (MSE) and Pearson’s Correlation Coefficient (⍴). For each comparison of the cancer-specific, tissue-free, methylation-based assay with the personalized tumor-informed assay, DMAF and VAF were strongly correlated for patients with CRC (VAF median [range]: 1.43% [0.003-8.26%], MSE: 0.064, ⍴: 0.955), breast cancer (VAF median [range]: 1.08% [0.002-7.80%], MSE: 0.161, ⍴: 0.863), lung cancer (VAF median [range]: 0.93% [0.005-12.9%], MSE: 0.179, ⍴: 0.829), and MIBC (VAF median [range]: 2.33% [0.005-9.26%], MSE: 0.079, ⍴: 0.954). Among patients with breast cancer, the correlation between DMAF and VAF was maintained across subtypes, including HR+ (MSE: 0.132, ⍴: 0.888), HER2+ (MSE: 0.272, ⍴: 0.815), and triple-negative (MSE: 0.109, ⍴: 0.897). Similarly, the correlation between DMAF and VAF was strong across lung cancer subtypes, non-small cell lung cancer (MSE: 0.207, ⍴: 0.802) and small cell lung cancer (MSE: 0.069, ⍴: 0.941). For all cancer-specific, tissue-free, methylation-based assays, DMAF levels were independent of patient characteristics such as sex (excluding breast), age, and stage (I-III), as well as cell-free DNA input. These data demonstrate that the abundance of differential methylation in patients with CRC, breast, lung, or MIBC cancer correlates with tumor-informed ctDNA levels, supporting each tissue-free methylation-based assay as a promising tool for informing prognosis and patient management. Future studies will investigate the quantitative abilities of the tissue-free assay in different patients with specific clinicopathological features. Citation Format: Princy Parsana, Tzu-Chun Chen, Nathan Liang, Amanda Kennedy, Veronica Rodriguez, Jie Zhang, Boris Gutman, Ehsan Haghshenas, Garima Kushwaha, Breeana L. Mitchell, Minetta Liu, Ehsan Tabari, Joshua Babiarz, Trupti Kawli, Johannes G. Reiter, Matthew Rabinowitz, Alexey Aleshin. Quantification of circulating tumor DNA (ctDNA) in patients using cancer-specific, methylation-based, tissue-free tests for the detection of molecular residual disease (MRD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 105.