Abstract 5900: Tumor-independent cfDNA methylation analysis for cancer monitoring during total neoadjuvant treatment in rectal cancer patients

Abstract Introduction: Total neoadjuvant therapy (TNT) is the standard curative-intent treatment for locally advanced rectal cancer (LARC), where patients receive all of their chemotherapy and radiation therapy before surgery. However, monitoring treatment response during TNT can be challenging since radiographic changes are often delayed. Methylation changes in colorectal cancer (CRC) and other cancers are relatively consistent and can be monitored in real time using cell-free DNA (cfDNA), making them a promising strategy for cancer detection and treatment monitoring. This study aims to determine if methylation changes in cfDNA can be used to monitor treatment response in LARC undergoing TNT. Methods: Plasma of CRC patients stage I-IV and healthy controls were obtained to establish the methylation classifier. Patients with stage II or III rectal cancer undergoing TNT were recruited. Blood samples were collected at baseline and about 6 weeks after the start of treatment. Tumor and normal CRC genomic DNA were analyzed using the Agilent Avida Methyl 3400 DMR panel to generate a methylation index score. Cancer treatment response was assessed by the investigator based on computed tomography and/or magnetic resonance imaging. Results: We confirmed low background methylation signals in cfDNA and peripheral blood mononuclear cells of healthy controls. Subsequently, we analyzed methylation signals from plasma of CRC patients with stage I (n=20), II (n=12), and IV (n=5) disease. The sensitivity of the assay was 60%, 75%, and 100% for stages I, II, and IV, respectively, while the specificity was 87.5%. Among LARC patients undergoing treatment with TNT, we observed a decrease in methylation index scores compared to baseline in all three patients who experienced a treatment response and an increase in all three patients who experienced disease progression. Notably, the methylation index scores did not correlate with carcinoembryonic antigen values. Conclusions: Our preliminary results show the feasibility of monitoring rectal cancer treatment response during TNT using a tumor-independent cfDNA methylation analysis. Methylation analysis is a promising technology that may aid in quantifying treatment response to systemic therapy and/or radiation. Citation Format: Kim Nguyen-Ta, Grace Zhao, Heng Wang, Teressa Celma, Lisa Kim, Jeff Pawelek, Bram Herman, Annie Wu, Yun Bao, Gregory Botta, Aaron Miller, Hitendra Patel, Shengrong Lin, Peter Vu. Tumor-independent cfDNA methylation analysis for cancer monitoring during total neoadjuvant treatment in rectal cancer patients [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 5900.