The effect of 5-fluorouracil/leucovorin chemotherapy on CpG methylation, or the confounding role of leukocyte heterogeneity: An illustration

• The CpG methylation profiles of leukocyte subpopulations display substantial heterogeneity.
• 5-Fluorouracil/leucovorin chemotherapy reshapes the composition of leukocyte subpopulations.
• 5-Fluorouracil/leucovorin chemotherapy has no effect on blood-based methylation profiles.

Blood-based epigenome-wide association studies that aim at comparing CpG methylation between colorectal cancer (CRC) patients and controls can lead to the discovery of diagnostic or prognostic biomarkers. Numerous confounders can lead to spurious associations. We aimed to see if 5-fluorouracil (5-FU)/leucovorin chemotherapy administered to cases prior to the collection of their blood has an effect on methylation. 304 patients who received treatment and 273 who did not were profiled on the HumanMethylation450 array. Association tests were adjusted for confounders, including proxies for leukocyte cell counts. There were substantial methylation differences between these two groups that vanished once the leukocyte heterogeneity was accounted for. We observed a significant decrease of T cells in the treatment group (CD4 +: p = 10− 6; CD8 +: p = 0.036) and significant increase of NK cells (p = 0.05) and monocytes (p = 0.0006). 5-FU/leucovorin has no effect on global and local blood-based methylation profiles, other than through differences in the leukocyte compositions that the treatment induced.