DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92–0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. Significance: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven-translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human HSPCs. Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of pre-leukemia/clonal hematopoiesis.

148 Background: 5-hydroxymethylcytosine (5hmC) is an epigenetic modification which regulates gene expression and is associated with active transcription. The optimization of 5hmC sequencing in cell-free DNA (cfDNA) could therefore enable assessment of gene activity through a liquid biopsy. We aimed to investigate the 5hmC landscape of metastatic castration-resistant prostate cancer (mCRPC) and to evaluate the potential of 5hmC modifications in cfDNA as biomarkers of outcome in mCRPC patients. Methods: Genome-wide 5hmC modifications were analyzed with a low-input whole-genome 5hmC sequencing method based on selective chemical labeling in DNA from 93 mCRPC tissue biopsies previously profiled with whole-genome sequencing (WGS), RNA-sequencing and whole-genome bisulfite sequencing (WGBS). In addition, we analyzed 64 cell-free DNA (cfDNA) samples, from men with mCRPC before first-line abiraterone or enzalutamide, with both 5hmC sequencing and a conventional targeted ctDNA panel assessing common genomic alterations. Results: In mCRPC tissue samples, 5hmC enrichment was more strongly associated with gene expression than promoter methylation or copy number. Among cancer hallmark pathways, the androgen response genes had the strongest association between 5hmC and gene expression, suggesting a disease specific marking of gene activation. 5hmC patterns in cfDNA could be used to estimate the circulating tumor DNA fraction (ct-fraction), which was prognostic for overall survival (tertiles of ct-fraction, HR = 1.6 95%CI 1.1-2.3, p = 0.007). Further, 5hmC levels were indicative of gain of oncogene activity (such as AR, MYC, and PIK3CA) and loss of tumor suppressor gene activity (such as RB1, TP53 and BRCA2). The number of alterations, by 5hmC levels, of common drivers of mCRPC was prognostic for overall survival, also after adjusting for ct-fraction (adjusted p = 0.00001), and the prognostic value of common alterations detected by 5hmC sequencing versus conventional targeted ctDNA sequencing was similar. Finally, 5hmC levels in cfDNA of genes not significantly altered by copy number gain or loss (and thus not routinely included in targeted ctDNA sequencing assays), such as TOP2A and EZH2, identified a high-risk subgroup of mCRPC, which was highly prognostic for overall survival independent of ct-fraction (adjusted HR = 1.8 95%CI 1.2-2.8, p = 0.007). Conclusions: 5hmC in mCRPC tissue demonstrated an association with gene expression that was highest for prostate cancer driver genes, highlighting the ability to track disease-specific biology. 5hmC in cfDNA from men with mCRPC can be used to estimate the ct-fraction of the sample, infer activity gain and loss of common drivers of mCRPC, and identify high-risk groups of mCRPC based on alterations not commonly detected with conventional ctDNA sequencing, showing its potential as a liquid biomarker. Further studies are aimed at optimizing and validating 5hmC-based biomarkers in larger cohorts.