Abstract 3255: Analytical validation of cancer gene promoter methylation detection in cfDNA liquid biopsy assay
Authors: Janice A. Patterson, Martina Lefterova, Brooke Overstreet, Colby Jenkins, Katie Quinn, Darya Chudova
Journal: Cancer Research (2025)

Introduction: Promoter methylation (PM) is a critical epigenetic mechanism that is involved in gene silencing and is associated with key pathways in cancer progression. Traditionally, PM detection has relied on single-gene testing of tissue biopsies, which are invasive, less clinically accessible, and impractical for tracking tumor evolution. Here, we evaluate PM within clinical multi-modal assessments using Guardant Infinity smart liquid biopsy platform, providing an innovative approach for monitoring epigenetic alterations. Methods: Data across &amp;gt;17k samples, including &amp;gt;5k cancer-free samples, and cancer-positive samples from &amp;gt;100 cancer types were used to define promoter regions associated with hypermethylation in cancer-related genes. PM regions were refined to ensure (a) the methylation signal is present exclusively in cancer and low in cancer-free samples, and (b) a negative correlation between gene expression and methylation in The Cancer Genome Atlas Program (TCGA) cancer samples. PM regions are quantified relative to constitutively hypermethylated control regions, measuring gene PM relative to total cfDNA. Analytical accuracy was demonstrated using an orthogonal Enzymatic Methyl Sequencing (EM-seq) assay on a cohort of clinical cfDNA and contrived samples. Limit of detection, precision, and limit of blank were established based on cancer cfDNA samples, cell line dilutions, and healthy donor samples. Gene PM prevalence was assessed in a test cohort of &amp;gt;7k samples across multiple cancer types to compare the relative prevalence to published tissue data sets. Results: Guardant Infinity PM limit of detection (&amp;gt;95% variant level) was established at PM mutant allele frequency (MAF) 1.6% across a panel of promoter genes. PM above the limit of detection is highly concordant with orthogonal EM-Seq, with 98.2% positive and 98% negative concordance. MLH1 PM was identified in 54% of patients with colorectal cancer (CRC) and 100% of patients with endometrial cancer (EC) who also had MSI-H detected. MLH1 PM in our cohort of CRC showed significant enrichment for BRAF V600E (36%, p&amp;lt;0.001). High prevalence of BRCA1 PM was observed in breast (BC) and ovarian cancer (OC), consistent with what is observed in literature. Enrichment of BRCA1 PM in BC was observed with loss of heterozygosity (LOH) (6%, p=0.02). The relative PM prevalence for individual genes across tumor types is largely consistent with what has been reported in tissue datasets. Conclusion: PM in liquid biopsy is consistent with tissue-based findings and correlates with underlying tumor characteristics, such as MSI-H and LOH events, thus demonstrating Guardant Infinity’s ability to identify potentially actionable PM patterns in a non-invasive manner. This analysis represents a fraction of the PM landscape observed, highlighting the broader potential of methylation as a novel biomarker for cancer detection and characterization. Citation Format: Janice A. Patterson, Martina Lefterova, Brooke Overstreet, Colby Jenkins, Katie Quinn, Darya Chudova. Analytical validation of cancer gene promoter methylation detection in cfDNA liquid biopsy assay [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 3255.