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Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; P = .027).

BACKGROUND: Tumor-specific methylation changes in DNA CpG sites commonly occur in cancer and are believed to drive oncogenesis through gene silencing. Detection of methylation changes in circulating cell-free DNA (cfDNA) can offer a novel approach for cancer diagnostics. METHODS: Plasma samples from healthy controls and from patients with advanced colorectal and non-colorectal cancers were included in the study. Bisulfite conversion of cfDNA extracted from plasma was performed using EZ DNA Methylation Lightning Kit (Zymo Research) and was followed by library preparation using Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and target enrichment using xGen Hybridization Capture for NGS Kit (IDT). Targeted methylation sequencing was done using NextSeq500 mid-output flow cell (300 cycles) (Illumina). Detection rates of methylation ratios in colorectal cancer samples were compared to non-colorectal cancers and healthy controls. RESULTS: First, we reviewed methylation changes in nearly 9,000 CpG sites in colorectal cancer (through TCGA database) and healthy controls. Subsequently, 32 CpG sites with greater than 50% methylation ratio in colorectal cancer and less than 1% methylation ratio in healthy controls were selected to develop targeted methylation sequencing based cfDNA assay. The assay was performed in 32 plasma samples from 20 individuals with advanced colorectal cancer who had tumor KRAS mutation, 8 individuals with advanced non-colorectal cancer who had tumor KRAS mutation (ovarian, n=2; endometrial, n=2; pancreatic, n=2; and lung cancer, n=2), and 4 healthy controls. Colorectal cancer specific methylation changes in cfDNA were detected in 85% (17/20) of colorectal cancer patients with a specificity of 92%. In colorectal cancer patients with confirmed KRAS mutation in cfDNA, methylation changes were detected in 92% (11/12) in comparison to 75% (6/8) in colorectal cancer patients without KRAS mutation in cfDNA. Median methylation ratio for target CpG sites was higher in colorectal cancer patients compared to patients with non-colorectal cancers and healthy controls (p CONCLUSIONS: Targeted methylation sequencing of cfDNA demonstrated high sensitivity and specificity for detection of colorectal cancer-specific cfDNA. Colorectal cancer patients with methylated cfDNA had shorter PFS while on cancer therapy. Citation Format: Mohamed A. Gouda, Dzifa Y. Duose, Morten Lapin, Stephanie Zalles, Helen J. Huang, Yuanxin Xi, Xiaofeng Zheng, Amira I. Aldesoky, Alshimaa M. Alhanafy, Mohamed A. Shehata, Jing Wang, Scott Kopetz, Funda Meric-Bernstam, Ignacio I. Wistuba, Rajyalakshmi Luthra, Filip Janku. Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5152.