Abstract 1100: Epigenetic profiling identifies markers of aggressive cancer subtype using a targeted methylation-based multi-cancer early detection (MCED) blood test

Abstract Background: Previous MCED studies have shown differences in detection performance across different aggressiveness cancer subtypes. However, the methylation profiles and ctDNA shedding patterns across different aggressiveness subtypes have not been analyzed. We systematically compared cell-free DNA (cfDNA) tumor fraction and methylation profiles across aggressiveness subtypes in three cancers to identify epigenetic biomarkers that differentiate tumor aggressiveness. Methods: Blood samples from a case-control study (NCT06217900) including 757 cancer cases were analyzed using a targeted methylation-based MCED test. We compared cfDNA tumor fraction and methylation profiles between aggressiveness subtypes, stage-matched small cell lung cancer (SCLC) (n=137) versus (vs) non-small cell lung cancer (NSCLC) (n=137), stage I invasive lung adenocarcinoma (IAC) (n=81) vs minimally invasive adenocarcinoma (MIA) (n=81), triple-negative breast cancer (TNBC) (n=151) vs non-TNBC (n=151), and intermediate/high-grade prostate cancer (Gleason Grade GroupGG 2-5)(n=14) vs low-grade prostate cancer(GG1)(n=6). Tumor fraction was estimated using zero-inflated Negative Binomial model based on the distribution of methylation signals, and methylation profiles were assessed by calculating methylated/unmethylated (M/U) ratios between different aggressiveness subtypes. Marker comparisons were conducted using the Mann-Whitney U test with multiple testing corrections. Results: The sensitivity is higher in more aggressive subtypes (lung cancer 93.73% vs SCLC 99.27%; breast cancer 70.76% vs TNBC 81.46%; prostate cancer 40% vs intermediate and high-grad prostate cancer 42.86%). In lung cancer, SCLC exhibited a significantly higher cfDNA tumor fraction than NSCLC (Wilcoxon p = 2.24×10-24), with 41,136 markers (79.18% hypomethylated) showing elevated M/U ratios. In stage I lung adenocarcinoma, IAC demonstrated a higher cfDNA tumor fraction (Wilcoxon p = 2.55×10-6) and systematic methylation differences compared to MIA. In breast cancer, TNBC had a significantly higher cfDNA tumor fraction than non-TNBC (Wilcoxon p = 2.55×10-6), with 25,717 markers (80.32% hypomethylated) displaying increased M/U ratios. Among prostate cancers of the same stage, GG2-5 cases exhibited higher cfDNA tumor fraction (Wilcoxon p = 0.038) and systematic methylation alterations compared to GG1. Conclusion: Aggressive tumor subtypes consistently display elevated cfDNA tumor fraction and characteristic methylation profiles marked by increased M/U ratios. These findings suggest cfDNA methylation patterns are promising epigenetic biomarkers for distinguishing tumor aggressiveness, potentially improving cancer screening precision and reducing overdiagnosis. Citation Format: Kezhong Chen, Jian Huang, Dahong Zhang, Shu Wang, Hongxu Liu, Wenzhao Zhong, Xiangnan Li, Qiang Zhang, Zhigao Li, Jiaqi Liu, Ziqing Tian, Fei Zhou, Gongsheng Jin, Xudong Xiang, Zhigang Li, Hui Xie, Ya Wei, Guochun Zhang, Guolin Ye, Ming Cai, Junfeng Wang, Yan Zhang, Chao Cheng, Hefei Li, Desong Yang, Jianhong Lian, Sheng Huang, Tao Xu, Zengjun Wang, Xi Guo, Zhuowei Liu, Minfeng Chen, Yang Wang, Yue An, Yanzhan Yang, Min Li, Jing Liu, Baoliang Zhu, Yonghui Li, Xiaohui Wu, Fan Yang, Jun Wang. Epigenetic profiling identifies markers of aggressive cancer subtype using a targeted methylation-based multi-cancer early detection (MCED) blood test 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 1100.