Abstract 786: Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer

Abstract Liquid biopsies are being investigated for the detection of cancer from blood. Epigenetic characterization of cell free DNA (cfDNA), specifically DNA methylation, is an emerging approach for sensitive detection and quantification of tumor burden. For breast cancer detection from blood, existing approaches for testing cfDNA have demonstrated a low performance in identifying affected patients. For this study, we developed a new approach that overcame this issue. Addressing the challenge of detecting breast cancer from cfDNA, this new approach relied on single-molecule methylation profiling of cfDNA. We used nanopore single molecule sequencing to provide quantitative characterization of cfDNA methylomes. This study determined if this approach provided the methylation features required for detecting the presence of breast cancer. The study population consisted of an extended cohort (~200 with untreated breast cancer and ~400 healthy controls). We identified 5mC-specific profiles from the nanopore sequencing signals of cfDNA. Importantly, this approach did not require any biosulfite or biochemical processing steps for determining methylation. Thus, we determined methylation profiles that were directly extrapolated from native cfDNA at single molecule resolution. Our results from this study demonstrated higher sensitivity of detecting breast cancer compared to other cfDNA methylation tests. We developed a new analytical framework for characterizing the methylation profiles of individuals with breast cancer compared to healthy individuals. Using a supervised bootstrapped algorithm, we identified CpG sites that were differentially methylated between the case and control groups. Afterwards, we calculated the similarity of any sample’s methylation profile to a training data. Our method also accommodates sparse and missing data. Based on separate validation datasets, we accurately identified those individuals with breast cancer. We validated our study with a different, independent cohort of healthy controls and individuals with breast cancer. Overall, this study demonstrates that a nanopore-based cfDNA sequencing and methylation profiling detects breast cancer from blood with higher performance then has been previously reported. Citation Format: Billy T. Lau, Xiangqi Bai, Alison Notestine, Brett Parkinson, Hanlee P. Ji, Lincoln Nadauld. Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 786.