Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

Deconvolution of immune cell composition and biological age of hepatocellular carcinoma using DNA methylation.

Hepatocellular carcinoma (HCC) has been an approved indication for the administration of immunotherapy since 2017, but biomarkers that predict therapeutic response have remained limited. Understanding and characterizing the tumor immune microenvironment enables better classification of these tumors and may reveal biomarkers that predict immunotherapeutic efficacy. In this paper, we applied a cell-type deconvolution algorithm using DNA methylation array data to investigate the composition of the tumor microenvironment in HCC. Using publicly available and in-house datasets with a total cohort size of 57 patients, each with tumor and matched normal tissue samples, we identified key differences in immune cell composition. We found that NK cell abundance was significantly decreased in HCC tumors compared to adjacent normal tissue. We also applied DNA methylation "clocks" which estimate phenotypic aging and compared these findings to expression-based determinations of cellular senescence. Senescence and epigenetic aging were significantly increased in HCC tumors, and the degree of age acceleration and senescence was strongly associated with decreased NK cell abundance. In summary, we found that NK cell infiltration in the tumor microenvironment is significantly diminished, and that this loss of NK abundance is strongly associated with increased senescence and age-related phenotype. These findings point to key interactions between NK cells and the senescent tumor microenvironment and offer insights into the pathogenesis of HCC as well as potential biomarkers of therapeutic efficacy.

Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment.

Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and 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 readout of the biologically effective radiation dose received by healthy tissues.