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Schematic illustration of the principle of plasma DNA tissue mapping by genome-wide methylation sequencing and its applications.
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Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA, we obtained a bird’s eye view of the identities and contributions of these tissues to the circulating DNA pool. The tissue contributors and their relative proportions are identified by a bioinformatics deconvolut...
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... was previously performed on the plasma DNA obtained from five pregnant women carrying fetuses with trisomy 21 (24). The gestational ages ranged between 13 and 14 wk. In the present study, we performed methylation deconvolution on the sequencing data, and ΔM values were cal- culated using Eq. 1 for multiple tissue types. As can be seen in Fig. 10, the placenta possessed the highest ΔM values for chromosome 21 among the studied tissue types. When the analysis was per- formed for the other chromosomes, no single tissue consistently showed a raised ΔM value (Fig. ...
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... deconvolution on the sequencing data, and ΔM values were cal- culated using Eq. 1 for multiple tissue types. As can be seen in Fig. 10, the placenta possessed the highest ΔM values for chromosome 21 among the studied tissue types. When the analysis was per- formed for the other chromosomes, no single tissue consistently showed a raised ΔM value (Fig. ...
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... at least a 30-Mb region (i.e., ∼1% of the human genome) were observed in the plasma of seven HCC patients. The proportional contributions of each tissue type into plasma based on the genomic regions showing amplifications and de- letions were separately determined. Then, the ΔM values were determined for each of the tissue types using Eq. 2. Fig. 11 shows that the highest ΔM values are observed for the liver for these HCC cases. As a control, we also performed the same analysis using two sets of randomly chosen genomic regions not exhibit- ing copy number aberrations in plasma. As can be seen in Fig. S2, for this control analysis, there is no systematic relationship between the ΔM ...
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... was observed in the follow-up trephine biopsies obtained in October 2011 and April 2013. She subsequently be- came pregnant. At the 11th week of pregnancy (March 2014), blood samples were collected for noninvasive prenatal testing of fetal chromosomal aneuploidies. However, the maternal plasma DNA sequencing analysis revealed gross abnormalities (Fig. 12A). Recurrence of the follicular lymphoma was confirmed by histo- logical examination of lymph node and trephine biopsies. Fig. 12A shows the genome-wide copy number analysis in the buffy coat, lymph node biopsy, pretreatment plasma, and a plasma sample collected 10 wk after the start of chemotherapy. Copy number aberrations were detected ...
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... 11th week of pregnancy (March 2014), blood samples were collected for noninvasive prenatal testing of fetal chromosomal aneuploidies. However, the maternal plasma DNA sequencing analysis revealed gross abnormalities (Fig. 12A). Recurrence of the follicular lymphoma was confirmed by histo- logical examination of lymph node and trephine biopsies. Fig. 12A shows the genome-wide copy number analysis in the buffy coat, lymph node biopsy, pretreatment plasma, and a plasma sample collected 10 wk after the start of chemotherapy. Copy number aberrations were detected in the lymph node bi- opsy and the pretreatment plasma sample but not in the post- treatment plasma sample and the buffy coat of ...
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... this patient, none of the contiguous regions exhibiting copy number losses in plasma were 30 Mb or above in size. As a result, the number of methylation markers located within the deleted regions was insufficient for tissue mapping analysis. Therefore, regions that did not exhibit any copy number aberrations were used as reference. Fig. 12B shows the ΔM values calculated for each of the tissue types. As can be seen, the B lymphocytes show the highest ΔM value, thus confirming that they are the origin of the copy number aberrations in ...
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... demonstrated the feasibility of using genome-wide bisulfite sequencing of plasma DNA and through a process of deconvo- lution to simultaneously deduce the contributions of different types of tissues into the plasma DNA pool. Before this work, efforts had generally been focused on one tissue type at a time, e.g., placental methylation signature in pregnancy (23,25) and donor-derived genetic markers for detecting transplant graft- derived DNA in plasma (13, 14, 19, 34). Our presently reported approach provides a bird's eye view of the major tissue con- tributors of circulating DNA ( Fig. 1 and Tables S1-S5). ...
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... efforts had generally been focused on one tissue type at a time, e.g., placental methylation signature in pregnancy (23,25) and donor-derived genetic markers for detecting transplant graft- derived DNA in plasma (13, 14, 19, 34). Our presently reported approach provides a bird's eye view of the major tissue con- tributors of circulating DNA ( Fig. 1 and Tables ...
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... types. Hence, in addition to applications to prenatal testing, cancer detection/monitoring and transplantation monitor- ing, the approach might also have applications in many branches of medicine for studying cell death or injury of various bodily tissues, e.g., stroke, myocardial infarction, trauma, autoimmune disorders, and infectious diseases (Fig. ...
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... applications of our approach for cancer detection and noninvasive prenatal testing converge in the case of the pregnant woman who suffered from follicular lymphoma. We observed copy number aberrations in the plasma of this pregnant woman (Fig. 12A). Plasma methylation deconvolution revealed a very high contribution from lymphocytes into plasma. The B lym- phocyte is the cell type involved in the pathology of follicular lymphoma. Thus, it was interesting to observe that our method further identified the B cells (Table S5), rather than the T cells, as the major contributor of ...
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... as the major contributor of plasma DNA in the patient. The ΔM analysis comparing the methylation deconvolution results obtained using methylation markers originating from the genomic regions showing increased copy number aberrations vs. those showing normal copy numbers further confirmed the B cells as the source of the copy number aberrations (Fig. 12B). These results are thus entirely consistent with the diagnosis of follicular lymphoma. With the increase in the clinical utility of noninvasive prenatal testing and the trend of further advances in maternal age, it is likely that more and more cases of malignancy will be detected during the course of such testing (35, 36). The approach ...
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Citations
... Because whole-genome cell-type atlases of DNA methylation have not been available until recently, prior tools have focused on either array-based analysis [7,[17][18][19], or analysis of specific tissue types [8,20]. CelFiE [21] was developed for whole-genome bisulfite sequencing (WGBS) and improved accuracy by incorporating the absolute number of unmethylated and methylated reads at each CpG to account for sequencing depth. ...
... One of these (CelFiE-ISH) is an extension of the model in CelFiE [21], which is the leading non-read aware algorithm. We did not evaluate other methods that were based on array [7], or not general with respect to cell types [8,20,24,31] or wholegenome markers [11]. ...
Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing.
... Another innovative application of the liquid biopsy concept involves using DNA methylation patterns to identify the cell-type origin of cfDNA. This allows for the monitoring of cell death in specific tissues [61][62][63] . Each cell type in the human body is characterized by a unique DNA methylation profile that remains consistent in every individual and is stably maintained throughout life in both healthy and diseased cells. ...
... Patients diagnosed with BKVN-exhibited heightened levels of kidney-specific cfDNA in their urine compared with a normal control group. Additionally, individuals experiencing BK polyomavirus (BKV) reactivation without nephropathy also displayed increased levels of kidney-specific DNA, though not to the same extent as those with BKVN [62,63,[83][84][85][86] . The researchers utilized wholegenome bisulfite sequencing (WGBS) to analyze methylation marks within urinary cfDNA. ...
Urinary tract infection (UTI) constitutes a pervasive health concern. UTIs are dichotomously classified into upper and lower strata, and further categorized as either complicated or uncomplicated. Upper UTIs predominantly afflict the renal and ureteral domains, typified by conditions exemplified in pyelonephritis, whereas lower UTIs manifest within the confines of the urethra and bladder. The conventional diagnosis of UTIs relies upon clinical manifestations and urine culture. Common symptoms encompass dysuria, frequent micturition, hematuria, and suprapubic discomfort. Urine culture serves to identify the specific pathogens instigating the infection and assess their antibiotic susceptibility. However, this procedural protocol generally necessitates an approximate duration of 24 h, coupled with an additional 24‐h interval for antibiotic susceptibility testing. In contradistinction, the detection of cell‐free DNA (cfDNA) in the circulatory system presents a potential avenue for non‐invasive diagnostic modalities. Notwithstanding, cfDNA emanates from deteriorating cells, affording insights into the extant cellular demise within the organism. Extensive scholarly inquiry has established a positive correlation between cfDNA concentration in the bloodstream and the incidence of cell death in conditions, such as severe traumas, sepsis, aseptic inflammation, myocardial infarction, and stroke. However, limited investigative effort has been devoted to elucidating the diagnostic potential of cfDNA in the context of UTIs. Consequently, the concentration and constitution of plasma cfDNA harbor substantial promise for non‐invasively diagnosing UTIs. In summation, the utilization of cfDNA in UTI diagnosis remains an incipient area of scholarly pursuit, underscoring the imperative for further research to elucidate the prospective role of plasma cfDNA in non‐intrusively discerning UTIs.
... However, in the case of injuries and inflammatory or oncological diseases, the resulting foci of inflammation and necrosis contribute to an increase in the concentration of cfDNA in the bloodstream and changes in its composition [10,11]. Thus, cfDNA carries genetic and epigenetic information from the cell and tissue from which it originated [12]. ...
It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.
... Genomic studies of DNA methylation often use the Illumina BeadChip 450K/EPIC arrays, which measure the average DNA methylation levels (beta values) at a predefined set of CpG sites, including a mere 1.5-3% of the 28 million CpG methylation sites across the entire human genome [9,10]. Recent advances in DNA sequencing technologies have facilitated a fragmentcentered view that captures binary DNA methylation patterns of multiple neighboring CpG sites, at a single-molecule resolution [4][5][6][7][8][11][12][13][14]. These technologies include conversion methods such as bisulfite [15] or enzymatic methyl treatment followed by sequencing (EMseq) [16], alongside direct detection of base modifications, using methylation-aware DNA sequencing technologies such as Oxford Nanopore Technologies (ONT) [17] or PacBio [18]. ...
Next-generation methylation-aware sequencing of DNA sheds light on the fundamental role of methylation in cellular function in health and disease. These data are commonly represented at a single CpG resolution, while single-molecule fragment-level analysis is often overlooked.
Here, we present wgbstools , an extensive computational suite tailored for methylation sequencing data. wgbstools allows fast access and ultra-compact anonymized representation of high-throughput methylome data, obtained through various library preparation and sequencing methods. Additionally, wgbstools contains state-of-the-art algorithms for genomic segmentation, biomarker identification, genetic and epigenetic data integration, and more. wgbstools offers fragment-level analysis and informative visualizations, across multiple genomic regions and samples.
... Since no reliable tools are available for calculating CNVs in WGBS data, we developed an algorithm for identifying CNVs in WGBS data inspired Article https://doi.org/10.1038/s41467-024-47886-1 by previous literature on WGS or WGBS data analysis 60,61 . Fragment counting involved standardizing sample data to a uniform depth and segmenting the genome into 1000-kb regions, yielding 2701 regions, with fragment counts quantified within each region. ...
Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.
... While previous studies have linked NETs [20][21][22]27] and EETs [22,23] to allergic asthma, cfDNA contained numerous unidentified components. The utilization of DNA methylation signatures in cfDNA has been extensively employed for early diagnosis, prognosis prediction, and dynamic monitoring of cancers [52], as well as prenatal testing [53] and organ transplants [54]. Therefore, cfDNA has the potential to serve as an objective biomarker for AAI and warrants further investigation. ...
Allergic airway inflammation (AAI), including allergic rhinitis (AR) and allergic asthma, is driven by epithelial barrier dysfunction and type 2 inflammation. However, the underlying mechanism remains uncertain and available treatments are constrained. Consequently, we aim to explore the role of cell-free DNA (cfDNA) in AAI and assess the potential alleviating effects of cationic polymers (CPs) through cfDNA elimination. Levels of cfDNA were evaluated in AR patients, allergen-stimulated human bronchial epithelium (BEAS-2B cells) and primary human nasal epithelium from both AR and healthy control (HC), and AAI murine model. Polyamidoamine dendrimers-generation 3 (PAMAM-G3), a classic type of cationic polymers, were applied to investigate whether the clearance of cfDNA could ameliorate airway epithelial dysfunction and inhibit AAI. The levels of cfDNA in the plasma and nasal secretion from AR were higher than those from HC (P < 0.05). Additionally, cfDNA levels in the exhaled breath condensate (EBC) were positively correlated with Interleukin (IL)-5 levels in EBC (R = 0.4191, P = 0.0001). Plasma cfDNA levels negatively correlated with the duration of allergen immunotherapy treatment (R = −0.4297, P = 0.006). Allergen stimulated cfDNA secretion in vitro (P < 0.001) and in vivo (P < 0.0001), which could be effectively scavenged with PAMAM-G3. The application of PAMAM-G3 inhibited epithelial barrier dysfunction in vitro and attenuated the development of AAI in vivo. This study elucidates that cfDNA, a promising biomarker for monitoring disease severity, aggravates AAI and the application of intranasal PAMAM-G3 could potentially be a novel therapeutic intervention for AAI.
Allergen stimulates the secretion of cell-free DNA (cfDNA) in both human and mouse airway. Intranasal polyamidoamine dendrimers-generation 3 (PAMAM-G3) scavenges cfDNA and alleviates allergic airway inflammation.
... In comparison to genomic mutations, alterations in the DNA methylation patterns of tumor cells not only occur earlier but also demonstrate a high degree of cell-type specificity. 11,12 This theoretically endows methylation markers with both tumor discrimination and cell lineage tracing capabilities, earning them the designation of ''molecular fingerprints''. Numerous studies have successively reported a series of potential methylation markers applicable to lung cancer diagnosis. ...
... cfDNA in the blood comes from various tissues and cells [22,23]. Recently, it has been revealed that fragments derived from various cancers are shorter than those derived from normal cells due to different biological mechanisms, and the possibility of diagnosis using this is being discussed [24][25][26][27]. ...
Gastric cancer is the 5th most common disease in the world and the 4th most common cause of death. It is diagnosed through esophagogastroduodenoscopy with biopsy, however there are limitations in finding lesions in the early stages. Recently, research has been actively conducted to use liquid biopsy to diagnose various cancers, including gastric cancer. Various substances derived from cancer are reflected in the blood. By analyzing these substances, it was expected that not only the presence or absence of cancer but also the type of cancer can be diagnosed. However, the amount of these substances is extremely small, and even these have various variables depending on the characteristics of the individual or the characteristics of the cancer. To overcome these, we collected methylated DNA fragments using MeDIP and compared them with normal plasma to characterize gastric cancer tissue or patient’s plasma. And we attempted to diagnose gastric cancer using the characteristics of cancer reflected in the blood through the cancer tissue and patient’s plasma. As a result, we confirmed that the consistency of common methylated fragments between tissue and plasma was approximately 41.2%, and found the possibility of diagnosing and characterizing cancer using the characteristics of the fragments through SFR and 5'end-motif analysis.
... Although the biological cause for such an increase remains unclear, studies suggest that nucleic acids from cancer cell necrosis, apoptosis, or active secretion possibly cause it. cfDNA is predominantly released from hematopoietic cells, while various conditions, including pregnancy, organ transplantation, cancer, surgery, and radiation, can lead to increased cfDNA [67,68]. In cancer patients, cfDNA is originated from cancer cells and the tumor microenvironment, including non-malignant cells [69]. ...
Simple Summary
Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of pancreatic cancers. It is considered one of the deadliest cancers due to its high metastatic potential and drug resistance. This review discusses various targets and nanoparticle-based delivery systems developed, tested, and approved for the effective diagnosis and targeted treatment of PDAC.
Abstract
Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest cancers, presents significant challenges in diagnosis and treatment due to its aggressive, metastatic nature and lack of early detection methods. A key obstacle in PDAC treatment is the highly complex tumor environment characterized by dense stroma surrounding the tumor, which hinders effective drug delivery. Nanotechnology can offer innovative solutions to these challenges, particularly in creating novel drug delivery systems for existing anticancer drugs for PDAC, such as gemcitabine and paclitaxel. By using customization methods such as incorporating conjugated targeting ligands, tumor-penetrating peptides, and therapeutic nucleic acids, these nanoparticle-based systems enhance drug solubility, extend circulation time, improve tumor targeting, and control drug release, thereby minimizing side effects and toxicity in healthy tissues. Moreover, nanoparticles have also shown potential in precise diagnostic methods for PDAC. This literature review will delve into targeted mechanisms, pathways, and approaches in treating pancreatic cancer. Additional emphasis is placed on the study of nanoparticle-based delivery systems, with a brief mention of those in clinical trials. Overall, the overview illustrates the significant advances in nanomedicine, underscoring its role in transcending the constraints of conventional PDAC therapies and diagnostics.
... Each tissue is characterized by an epigenetic signature that allows for the identification of the DNA origin through the analysis of its methylation profile [54]. Advanced molecular analyses as whole-genome bisulfite sequencing allow for the correct identification and quantification of the cfDNA source, enabling the non-invasive monitoring of GVHD [55]. This approach has been tested by Pellan Cheng and colleagues [56], who analyzed a pilot cohort of HCT recipients, and the result of their proof-of-principle study showed the potential of cfDNA to assist in personalizing care after HCT. ...
Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients’ conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients’ quality of life.
