Thoracic high-dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4–6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of >35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.

Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we characterize the methylomes of healthy human and mouse tissues to establish sequencing-based, cell-type specific reference DNA methylation atlases. Identified cell-type specific DNA blocks were mostly hypomethylated and located within genes intrinsic to cellular identity. Cell-free DNA fragments released from dying cells into the circulation were captured from serum samples by hybridization to CpG-rich DNA panels. The origins of the circulating DNA fragments were inferred from mapping to the established DNA methylation atlases. Thoracic radiation-induced tissue damages in a mouse model were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type specific effects of radiation and provide a quantitative measure of the biologically effective radiation dose received by healthy tissues. Graphical