Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion

BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.