Monitoring radiation-induced heart and lung damage through methylation patterns in cell-free DNA

Radiation therapy is a highly effective treatment for many cancers, although acute and long-term toxicities are common. Radiation-induced damage from off-target exposure is most often confined to tissues near the treatment area, with heart and lung damage being common dose-limiting toxicities among patients who receive thoracic radiation. Cell-free DNA (cfDNA) released from dying cells into the circulation is a potential indicator of tissue damage that could improve adverse event detection. Further, it has been shown that methylation patterns of cfDNA can act as markers to deconvolute tissue-specific cell death in a given sample. In contrast to traditional analyses assessing methylation levels at individual CpG sites, we utilize pattern analysis to assess methylation levels across multiple adjacent CpG sites to increase sensitivity and specificity of signal detection. In the present study, we used mice to model radiation-induced damage by exposure to 3 and 8 Gy radiation doses relative to a sham control. We identified two distinct mouse genomic loci with organ-specific methylation patterns of the heart and the lungs. Amplicon sequencing of bisulfite-treated cfDNA isolated from serum samples was conducted at these organotypic genomic loci. cfDNA amplicons with the specific methylation pattern were quantified relative to non-specific cfDNA amplicons from the respective loci to infer radiation-induced heart and lung damage. We found significant increases of both heart- and lung-specific methylated cfDNA amplicons in mice that received 8 Gy radiation compared to 3 Gy and sham control. Our findings suggest that heart and lung derived methylated cfDNA may be useful in monitoring radiation-induced tissue damage.