The signature of cancer methylation markers in maternal plasma: Factors influencing the development and application of cancer liquid biopsy assay

Background: DNA methylation is highly correlated with cancer and embryo development, and plasma-based methylation markers have been widely used for cancer early detection. However, whether the commonly used cancer methylation markers cause "false positives" in the plasma of pregnant women has not been comprehensively evaluated. Methods: We conducted a case-control study from February 2021 to March 2023, which included 138 pregnant women and 44 control women. Plasma cell-free DNA (cfDNA) was isolated and bisulfite-converted, and then the methylation levels of eight methylated markers related to gastrointestinal cancer (SEPT9, SDC2, C9orf50, KCNQ5, CLIP4, TFPI2, ELMO1 and ZNF582) and three markers related to lung cancer (SHOX2, RASSF1A and PTGER4) were analyzed. Results: When comparing the plasma of pregnant women to that of control women, SEPT9, CLIP4, ZNF582, SHOX2, RASSF1A and PTGER showed significantly higher levels of methylation (p < 0.05). These positive signals originate from the placenta/fetus rather than the mother. We found no discernible difference in DNA methylation levels between fetal cfDNA fractions of < 10 % and >= 10 % in pregnant women (p > 0.05), while CLIP4 and PTGER4 showed high methylation levels in the assisted fertilization group compared to the natural fertilization group (p < 0.05). Conclusion: Our study shows that cancer and fetus/placenta exhibit similar DNA methylation patterns, and some gastrointestinal cancer and lung cancer-related methylation markers also show positives in maternal plasma. This is an important consideration in the design and application of plasma-based cancer liquid biopsy assays.