Rising cell-free DNA level tracks cancer-associated methylation signal and improves multicancer detection

Cancer patients carry more circulating cell-free DNA (ccfDNA)—small DNA fragments shed into the blood—than healthy people. Most of this excess comes not from the tumor itself but from immune cells, suggesting that it would simply dilute the much smaller amount of tumor-derived DNA in a blood sample. Using a study of 1,480 previously untreated participants across 43 hospitals—including patients with 14 cancer types spanning all stages, patients with benign growths, and healthy volunteers—we show the opposite: as total ccfDNA rises, it becomes enriched, not diluted, in fragments carrying cancer-associated DNA methylation signatures, independent of cancer type, disease stage, and a broad panel of demographic, clinical, and technical covariates. In fact, total ccfDNA explains more variance in cancer-associated methylation signal than the disease stage itself. This basic biological finding suggests a coupling between immune cell activity and tumor methylation. It also has a practical implication: we show that rising ccfDNA concentration is associated with higher cancer detection sensitivity, even within disease stages and particularly in early-stage disease—stage I sensitivity varied from 0.55 in the lowest ccfDNA concentration tertile to 0.85 in the highest, and stage II from 0.59 to 0.86. This suggests that ccfDNA concentration should be considered as a stage-independent stratification variable for benchmarking cancer-detection tests. Interestingly, similar behavior was observed in patients with symptomatic benign lesions, suggesting a broader biological response to abnormal tissue growth and not something unique to malignancy.