Cell-free circulating DNA (cfDNA) in plasma has gained global interest as a diagnostic material for noninvasive prenatal testing and cancer diagnosis, or the so-called "liquid biopsy". Recent studies ...have discovered a great number of valuable genetic and epigenetic biomarkers for cfDNA-based liquid biopsy. Considering that the genetic biomarkers, e.g., somatic mutations, usually vary from case to case in most cancer patients, epigenetic biomarkers that are generalizable across various samples thus possess certain advantages. In this study, we reviewed the most recent studies and advances on utilizing epigenetic biomarkers for liquid biopsies. We first reviewed more traditional methods of using tissue/cancer-specific DNA methylation biomarkers and digital PCR or sequencing technologies for cancer diagnosis, as well as tumor origin determination. In the second part, we discussed the emerging novel approaches for exploring the biological basis and clinical applications of cfDNA fragmentation patterns. We further provided our comments and points of view on the future directions on epigenetic biomarker development for cfDNA-based liquid biopsies.
Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic ...individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.
We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their ...methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.
There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important ...contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma.
Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus.
Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings.
Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.
Abstract The tissues of origin of plasma DNA can be revealed by methylation patterns. However, the relative DNA contributions from megakaryocytes and erythroblasts into plasma appeared inconsistent ...among studies. To shed light into this phenomenon, we developed droplet digital PCR (ddPCR) assays for the differential detection of contributions from these cell types in plasma based on megakaryocyte-specific and erythroblast-specific methylation markers. Megakaryocytic DNA and erythroid DNA contributed a median of 44.2% and 6.2% in healthy individuals, respectively. Patients with idiopathic thrombocytopenic purpura had a significantly higher proportion of megakaryocytic DNA in plasma compared to healthy controls (median: 59.9% versus 44.2%; P = 0.03). Similarly, patients with β-thalassemia were shown to have higher proportions of plasma erythroid DNA compared to healthy controls (median: 50.9% versus 6.2%) ( P < 0.0001). Hence, the concurrent analysis of megakaryocytic and erythroid lineage-specific markers could facilitate the dissection of their relative contributions and provide information on patients with hematological disorders.
Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict ...cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide nt window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5′ ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson’s absolute
r
> 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.
Abstract Background The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust ...methods for targeted haplotyping. Methods We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). Results For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. Conclusions The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.
Experience in clinical practice and research in systems pharmacology suggested the limitations of the current one-drug-one-target paradigm in new drug discovery. Single-target drugs may not always ...produce desired physiological effects on the entire biological system, even if they have successfully regulated the activities of their designated targets. On the other hand, multicomponent therapy, in which two or more agents simultaneously interact with multiple targets, has attracted growing attention. Many drug combinations consisting of multiple agents have already entered clinical practice, especially in treating complex and refractory diseases. Drug combination database (DCDB), launched in 2010, is the first available database that collects and organizes information on drug combinations, with an aim to facilitate systems-oriented new drug discovery. Here, we report the second major release of DCDB (Version 2.0), which includes 866 new drug combinations (1363 in total), consisting of 904 distinctive components. These drug combinations are curated from ∼140,000 clinical studies and the food and drug administration (FDA) electronic orange book. In this update, DCDB collects 237 unsuccessful drug combinations, which may provide a contrast for systematic discovery of the patterns in successful drug combinations. Database URL: http://www.cls.zju.edu.cn/dcdb/
Measurement of DNA derived from different tissues in the circulating DNA pool can provide important information regarding the presence of many pathological conditions. However, existing methods ...involving genome-wide bisulfite sequencing are relatively expensive and may present challenges for large-scale analysis.
Through identifying differentially methylated regions in the liver and colon compared with other tissues, we identified 2 markers and developed corresponding droplet digital PCR assays. Plasma concentrations of liver-derived and colon-derived DNA were measured for 13 liver transplant recipients, 40 liver cancer patients, and 62 colorectal cancer (CRC) patients (27 with and 35 without liver metastases).
In liver transplant recipients, the fractional concentration of liver-derived DNA measured using the liver-specific methylation marker and donor-specific alleles showed good correlation (Pearson
= 0.99). In liver cancer patients, the concentration of liver-derived DNA correlated positively with the maximal dimension of the tumor (Spearman
= 0.74). In CRC patients with and without liver metastasis, the plasma concentrations of colon-derived DNA (median, 138 copies/mL and 4 copies/mL, respectively) were increased compared with the 30 healthy controls (26 had undetectable concentrations). The absolute concentration of liver-derived DNA provided a better differentiation between CRC patients with and without liver metastasis compared with the fractional concentration (area under ROC curve, 0.85 vs 0.75).
Quantitative analysis of plasma DNA with tissue-specific methylation patterns using droplet digital PCR is applicable for the investigation of cancers and assessing organ transplantation. This approach is useful for differentiating patients with and without metastases to other organs.
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