Cell-free DNA end characteristics enable accurate and sensitive cancer diagnosis.

The fragmentation patterns of cell-free DNA (cfDNA) in plasma can potentially be utilized as diagnostic biomarkers in liquid biopsy. However, our knowledge of this biological process and the information encoded in fragmentation patterns remains preliminary. Here, we investigated the cfDNA fragmentomic characteristics against nucleosome positioning patterns in hematopoietic cells. cfDNA molecules with ends located within nucleosomes were relatively shorter with altered end motif patterns, demonstrating the feasibility of enriching tumor-derived cfDNA in patients with cancer through the selection of molecules possessing such ends. We then developed three cfDNA fragmentomic metrics after end selection, which showed significant alterations in patients with cancer and enabled cancer diagnosis. By incorporating machine learning, we further built high-performance diagnostic models, which achieved an overall area under the curve of 0.95 and 85.1% sensitivity at 95% specificity. Hence, our investigations explored the end characteristics of cfDNA fragmentomics and their merits in building accurate and sensitive cancer diagnostic models.

Application of graft-derived cell-free DNA for solid organ transplantation.

Monitoring the status of grafts and the occurrence of postoperative complications, such as rejection, is crucial for ensuring the success and long-term survival of organ transplants. Traditional histopathological examination, though effective, is an invasive procedure and poses risks of complications, making frequent use impractical. In recent years, graft-derived cell-free DNA (gd-cfDNA) has emerged as a promising non-invasive biomarker. It not only provides early warnings of rejection and other types of graft injury but also offers important information about the effectiveness of immunosuppressive therapy and prognosis. gd-cfDNA shows potential in the monitoring of organ transplants. The early, real-time information on graft injury provided by gd-cfDNA facilitates timely individualized treatment and improves patient outcomes. However, the progress of research on gd-cfDNA varies across different organs. Therefore, this article will comprehensively review the application and findings of gd-cfDNA in monitoring various solid organs, discussing the advantages, limitations, and some future research directions to aid in its clinical application.

DNA demethylation triggers cell free DNA release in colorectal cancer cells.

BACKGROUND: Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. METHODS: We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. RESULTS: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. CONCLUSIONS: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

5-Hydroxymethylated Biomarkers in Cell-Free DNA Predict Occult Colorectal Cancer up to 36 Months Before Diagnosis in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

PURPOSE: Using the prostate, lung, colorectal, and ovarian (PLCO) Cancer Screening Trial samples, we identified cell-free DNA (cfDNA) candidate biomarkers bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that detected occult colorectal cancer (CRC) up to 36 months before clinical diagnosis. MATERIALS AND METHODS: We performed the 5hmC-seal assay and sequencing on ≤8 ng cfDNA extracted from PLCO study participant plasma samples, including n = 201 cases (diagnosed with CRC within 36 months of blood collection) and n = 401 controls (no cancer diagnosis on follow-up). We conducted association studies and machine learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. RESULTS: We successfully obtained 5hmC profiles from these decades-old samples. A weighted Cox model of 32 5hmC-modified gene bodies showed a predictive detection value for CRC as early as 36 months before overt tumor diagnosis (training set AUC, 77.1% [95% CI, 72.2 to 81.9] and validation set AUC, 72.8% [95% CI, 65.8 to 79.7]). Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and race/ethnicity, and significantly outperformed risk factors such as age and obesity (assessed as BMI). Finally, when splitting cases at median weighted prediction scores, Kaplan-Meier analyses showed significant risk stratification for CRC occurrence in both the training set (hazard ratio, [HR], 3.3 [95% CI, 2.6 to 5.8]) and validation set (HR, 3.1 [95% CI, 1.8 to 5.8]). CONCLUSION: Candidate 5hmC biomarkers and a scoring algorithm have the potential to predict CRC occurrence despite the absence of clinical symptoms and effective predictors. Developing a minimally invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient outcomes.

Progress in Noninvasive Surveillance for Acute Rejection in Pediatric Heart Transplant Recipients: A Real-World Analysis of Donor-Derived Cell-Free DNA-Based Surveillance Protocol.

PURPOSE: Acute cellular (ACR) and antibody-mediated (AMR) rejection are risk factors for allograft loss in heart transplant (HT) recipients. Endomyocardial biopsy (EMB), although considered the gold standard for rejection surveillance, is invasive and has high interobserver variability. Noninvasive donor-derived cell-free DNA (dd-cfDNA) sampling has a high negative predictive value (NPV) for rejection in adults and is increasingly used in pediatrics. This single center study aimed to test the performance of dd-cfDNA in screening for acute rejection (AR) and donor-specific antibodies (DSAs) in pediatric HT recipients. METHODS: Blood samples for dd-cfDNA were obtained per clinical protocol for all eligible HT recipients in our center from July 1, 2022 to December 31, 2023. Primary endpoints were episodes of AR, pathology grading of EMBs temporally related to ddcfDNA sampling (0-150 days), and presence of DSAs. RESULTS: There were 471 interpretable samples, in 192 unique patients. Of those, 199 dd-cfDNA tests were paired with EMB ± DSA in 152 patients. Abnormal dd-cfDNA (> 0.2%) was found in 77 samples (median 0.48%, range 0.21%-11%) and led to EMB, where one sample was positive for ACR (grade 2R), 13 for AMR, yielding an NPV of 97% for AMR. After excluding abnormal ddcfDNA testing associated with AR, 65 abnormal dd-cfDNA tests were paired with DSA testing. The NPV of the test for detection of DSAs was 93%. CONCLUSION: Implementation of noninvasive rejection surveillance with dd-cfDNA in a pediatric cohort demonstrates high NPV for AR and high DSAs, making it an ideal screening tool for long-term monitoring of allograft health in pediatrics.

Clinical Utility of Donor-Derived Cell-Free DNA in Heart Transplant Recipients With Multi-Organ Transplants.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) has emerged as a reliable, noninvasive method for the surveillance of allograft rejection in heart transplantation (HT) patients, but its utility in multi-organ transplants (MOT) is unknown. We describe our experience using dd-cfDNA in simultaneous MOT recipients. METHODS: A single-center retrospective review of all HT recipients between 2018 and 2022 that had at least one measurement of dd-cfDNA collected. Patients who had simultaneous MOT were identified and included in this study. Levels of dd-cfDNA were paired with endomyocardial biopsies (EMB) performed within 1 month of blood testing if available. Acute cellular rejection (ACR) was defined as ISHLT (International Society for Heart and Lung Transplantation) grade ≥ 2R. and antibody-mediated rejection (AMR) was defined as pAMR grade > 0. The within-patient variability score of the dd-cfDNA was calculated by the variance/average. RESULTS: The study included 25 multiorgan transplant recipients: 13 heart-kidney (H-K), 8 heart-liver (H-Li), and 4 heart-lung (H-Lu). The median age was 55 years, 44% were female; the median time from HT until the first dd-cfDNA measurement was 4.5 months (IQR 2, 10.5). The median dd-cfDNA level was 0.18% (IQR 0.15%, 0.27%) for H-K, 1.15% (IQR 0.77%, 2.33%) for H-Li, and 0.69% (IQR 0.62%, 1.07%) for H-Lu patients (p < 0.001). Prevalence of positive dd-cfDNA tests (threshold of 0.20%) were 42.2%, 97.3%, and 92.3% in the H-K, H-Li, and H-Lu groups, respectively. The within-patient variability score was highest in the H-Li group (median of 0.45 [IQR 0.29, 0.94]) and lowest in the H-K group (median of 0.09 [IQR 0.06, 0.12]); p = 0.002. No evidence of cardiac ACR or AMR was found. Three patients experienced renal allograft ACR and/or AMR, two patients experienced rejection of the liver allograft, and one patient experienced an episode of AMR-mediated lung rejection. One person in the H-K group experienced an episode of cardiac allograft dysfunction that was not associated with biopsy-confirmed rejection. CONCLUSION: Dd-cfDNA is chronically elevated in most MOT recipients. There is a high degree of within-patient variability in levels (particularly for H-Li and H-Lu recipients), which may limit the utility of this assay in monitoring MOT recipients.

Clinical Examples of the Additive Value of Absolute Quantification of Cell-Free DNA After Heart Transplantation.

OBJECTIVE: Cell-free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR-based approach allowing us to separately quantify absolute values of dd-cfDNA and recipient-derived cfDNA (rd-cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. METHODS: We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false-negative and false-positive rejections in six patients (five adults, one child). RESULTS: There was a substantial discrepancy between relative (DF) and absolute cfDNA-levels in several clinical situations. Rd- and dd-cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. CONCLUSIONS: Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection.

Differential methylation of circulating free DNA assessed through cfMeDiP as a new tool for breast cancer diagnosis and detection of BRCA1/2 mutation.

BACKGROUND: Recent studies have highlighted the importance of the cell-free DNA (cfDNA) methylation profile in detecting breast cancer (BC) and its different subtypes. We investigated whether plasma cfDNA methylation, using cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq), may be informative in characterizing breast cancer in patients with BRCA1/2 germline mutations for early cancer detection and response to therapy. METHODS: We enrolled 23 BC patients with germline mutation of BRCA1 and BRCA2 genes, 19 healthy controls without BRCA1/2 mutation, and two healthy individuals who carried BRCA1/2 mutations. Blood samples were collected for all study subjects at the diagnosis, and plasma was isolated by centrifugation. Cell-free DNA was extracted from 1 mL of plasma, and cfMeDIP-seq was performed for each sample. Shallow whole genome sequencing was performed on the immuno-precipitated samples. Then, the differentially methylated 300-bp regions (DMRs) between 25 BRCA germline mutation carriers and 19 non-carriers were identified. DMRs were compared with tumor-specific regions from public datasets to perform an unbiased analysis. Finally, two statistical classifiers were trained based on the GLMnet and random forest model to evaluate if the identified DMRs could discriminate BRCA-positive from healthy samples. RESULTS: We identified 7,095 hypermethylated and 212 hypomethylated regions in 25 BRCA germline mutation carriers compared to 19 controls. These regions discriminate tumors from healthy samples with high accuracy and sensitivity. We show that the circulating tumor DNA of BRCA1/2 mutant breast cancers is characterized by the hypomethylation of genes involved in DNA repair and cell cycle. We uncovered the TFs associated with these DRMs and identified that proteins of the Erythroblast Transformation Specific (ETS) family are particularly active in the hypermethylated regions. Finally, we assessed that these regions could discriminate between BRCA positives from healthy samples with an AUC of 0.95, a sensitivity of 88%, and a specificity of 94.74%. CONCLUSIONS: Our study emphasizes the importance of tumor cell-derived DNA methylation in BC, reporting a different methylation profile between patients carrying mutations in BRCA1, BRCA2, and wild-type controls. Our minimally invasive approach could allow early cancer diagnosis, assessment of minimal residual disease, and monitoring of response to therapy.

Liquid biopsy for CNS lymphoma: CSF exosomes and CSF exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b are promising biomarkers for diagnosis.

BACKGROUND: Central nervous system lymphoma (CNSL) is a devastating disease with a poor prognosis. Early diagnosis, monitoring of the treatment response, and outcome prediction carry the utmost importance in the management of patients with CNSL. Surgical biopsy is the gold standard for tissue diagnosis, however, this procedure has potential complications. Therefore, there is a need for a method that provides information about diagnosis and patient monitoring to avoid surgical risks. The study aimed to investigate potential diagnostic biomarkers for patients with CNSL. METHODS AND RESULTS: Patients with secondary CNSL were included in this study. Serum and cerebrospinal fluid (CSF) samples were collected before treatment and after completion of the treatment. Cell-free DNA (cfDNA), exosomes, free and exosomal microRNA (miR)-15a, miR-21, miR-155, miR-210, and miR-19b in both serum and CSF were examined, and they were compared with the controls. Also, their levels before and after treatment were compared. Nine patients with the diagnosis of secondary CNSL were reviewed. cfDNA, miR-15a, and miR-155 in serum, and exosome in CSF were found to be significantly higher in CNSL patients compared to the controls. Exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b in CSF were found to be significantly higher in CNSL patients compared to controls, whereas their levels in serum were not significantly high. CONCLUSIONS: Our findings suggested that exosomes and exosomal miR-15a, miR-21, miR-155, miR-210 and miR-19b in CSF would be promising biomarkers for the diagnosis of patients with CNSL. Further studies are needed to confirm our findings.

Distinct methylome profile of cfDNA in AMI patients reveals significant alteration in cAMP signaling pathway genes regulating cardiac muscle contraction.

BACKGROUND: The role of epigenetics in cardiovascular diseases has paved the way for innovative therapeutic approaches. Investigating epigenetic changes using cell-free DNA (cfDNA) holds substantial promise beyond mere diagnostics, especially for heart-related conditions like acute myocardial infarction (AMI), where obtaining tissue samples is a challenge. This study explores the methylation patterns of cfDNA in AMI patients and compares them with genomic DNA (gDNA) from the same individuals, aiming to evaluate the effectiveness of cfDNA as a valuable resource for studying heart-related diseases. METHODOLOGY: We generated global methylome profiles of cfDNA and gDNA from 25 AMI patients using EM-Seq. Tissue deconvolution analysis was performed to estimate tissue specificity based on the methylation patterns. Differentially methylated loci were identified and explored to understand AMI pathophysiology. RESULTS: Comparative analysis of cfDNA and gDNA methylation patterns in AMI patients reveals cfDNA holds more significance than gDNA. Principal component analysis revealed distinct clusters for cfDNA and gDNA, indicating distinct methylome profiles. cfDNA originated from multiple sources, predominantly from neutrophils (~ 75%) and about 10% from the left atrium, highlighting cardiac-specific changes. In contrast, immune cells are the major source of gDNA, indicative of inflammatory responses. Gene set enrichment analysis (GSEA) associates cfDNA methylation patterns with pathways related to cardiac muscle contraction, inflammation, hypoxia, and lipid metabolism. The affected genes include G protein-coupled receptors (GHSR, FFAR2, HTR1A, and VIPR2) that are part of the cAMP signaling pathway. CONCLUSION: Epigenetic changes in cfDNA are more specific to cardiac tissue compared to those in gDNA, providing better insights into the molecular mechanisms involved in AMI. Genes that are differentially methylated in cfDNA and regulate core pathways, such as cAMP signaling, could be targeted for clinical applications, including the development of effective biomarkers and therapeutic targets.

Cancer treatment monitoring using cell-free DNA fragmentomes.

Circulating cell-free DNA (cfDNA) assays for monitoring individuals with cancer typically rely on prior identification of tumor-specific mutations. Here, we develop a tumor-independent and mutation-independent approach (DELFI-tumor fraction, DELFI-TF) using low-coverage whole genome sequencing to determine the cfDNA tumor fraction and validate the method in two independent cohorts of patients with colorectal or lung cancer. DELFI-TF scores strongly correlate with circulating tumor DNA levels (ctDNA) (r = 0.90, p < 0.0001, Pearson correlation) even in cases where mutations are undetectable. DELFI-TF scores prior to therapy initiation are associated with clinical response and are independent predictors of overall survival (HR = 9.84, 95% CI = 1.72-56.10, p < 0.0001). Patients with lower DELFI-TF scores during treatment have longer overall survival (62.8 vs 29.1 months, HR = 3.12, 95% CI 1.62-6.00, p < 0.001) and the approach predicts clinical outcomes more accurately than imaging. These results demonstrate the potential of using cfDNA fragmentomes to estimate tumor burden in cfDNA for treatment response monitoring and clinical outcome prediction.

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines. METHODS: Four primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared. RESULTS: In all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA. CONCLUSIONS: This data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Histone modifications of circulating nucleosomes are associated with changes in cell-free DNA fragmentation patterns.

The analysis of tissues of origin of cell-free DNA (cfDNA) is of research and diagnostic interest. Many studies focused on bisulfite treatment or immunoprecipitation protocols to assess the tissues of origin of cfDNA. DNA loss often occurs during such processes. Fragmentomics of cfDNA molecules has uncovered a wealth of information related to tissues of origin of cfDNA. There is still much room for the development of tools for assessing contributions from various tissues into plasma using fragmentomic features. Hence, we developed an approach to analyze the relative contributions of DNA from different tissues into plasma, by identifying characteristic fragmentation patterns associated with selected histone modifications. We named this technique as FRAGmentomics-based Histone modification Analysis (FRAGHA). Deduced placenta-specific histone H3 lysine 27 acetylation (H3K27ac)-associated signal correlated well with the fetal DNA fraction in maternal plasma (Pearson's r = 0.96). The deduced liver-specific H3K27ac-associated signal correlated with the donor-derived DNA fraction in liver transplantation recipients (Pearson's r = 0.92) and was significantly increased in patients with hepatocellular carcinoma (HCC) (P < 0.01, Wilcoxon rank-sum test). Significant elevations of erythroblasts-specific and colon-specific H3K27ac-associated signals were observed in patients with ß-thalassemia major and colorectal cancer, respectively. Furthermore, using the fragmentation patterns from tissue-specific H3K27ac regions, a machine learning algorithm was developed to enhance HCC detection, with an area under the curve (AUC) of up to 0.97. Finally, genomic regions with H3K27ac or histone H3 lysine 4 trimethylation (H3K4me3) were found to exhibit different fragmentomic patterns of cfDNA. This study has shed light on the relationship between cfDNA fragmentomics and histone modifications, thus expanding the armamentarium of liquid biopsy.

Fragmentomics of plasma mitochondrial and nuclear DNA inform prognosis in COVID-19 patients with critical symptoms.

BACKGROUND: The mortality rate of COVID-19 patients with critical symptoms is reported to be 40.5%. Early identification of patients with poor progression in the critical cohort is essential to timely clinical intervention and reduction of mortality. Although older age, chronic diseases, have been recognized as risk factors for COVID-19 mortality, we still lack an accurate prediction method for every patient. This study aimed to delve into the cell-free DNA fragmentomics of critically ill patients, and develop new promising biomarkers for identifying the patients with high mortality risk. METHODS: We utilized whole genome sequencing on the plasma cell-free DNA (cfDNA) from 33 COVID-19 patients with critical symptoms, whose outcomes were classified as survival (n = 16) and death (n = 17). Mitochondrial DNA (mtDNA) abundance and fragmentomic properties of cfDNA, including size profiles, ends motif and promoter coverages were interrogated and compared between survival and death groups. RESULTS: Significantly decreased abundance (~ 76% reduction) and dramatically shorter fragment size of cell-free mtDNA were observed in deceased patients. Likewise, the deceased patients exhibited distinct end-motif patterns of cfDNA with an enhanced preference for "CC" started motifs, which are related to the activity of nuclease DNASE1L3. Several dysregulated genes involved in the COVID-19 progression-related pathways were further inferred from promoter coverages. These informative cfDNA features enabled a high PPV of 83.3% in predicting deceased patients in the critical cohort. CONCLUSION: The dysregulated biological processes observed in COVID-19 patients with fatal outcomes may contribute to abnormal release and modifications of plasma cfDNA. Our findings provided the feasibility of plasma cfDNA as a promising biomarker in the prognosis prediction in critically ill COVID-19 patients in clinical practice.

5-Hydroxymethylcytosine in circulating cell-free DNA as a potential diagnostic biomarker for SLE.

BACKGROUND: SLE is a complex autoimmune disease with heterogeneous manifestations and unpredictable outcomes. Early diagnosis is challenging due to non-specific symptoms, and current treatments only manage symptoms. Epigenetic alternations, including 5-Hydroxymethylome (5hmC) modifications, are important contributors to SLE pathogenesis. However, the 5hmC modification status in circulating cell-free DNA (cfDNA) of patients with SLE remains largely unexplored. We investigated the distribution of 5hmC in cfDNA of patients with SLE and healthy controls (HCs), and explored its potential as an SLE diagnosis marker. METHODS: We used 5hmC-Seal to generate genome-wide 5hmC profiles of plasma cfDNA and bioinformatics analysis to screen differentially hydroxymethylated regions (DhMRs). In vitro mechanistic exploration was conducted to investigate the regulatory effect of CCCTC-binding factor (CTCF) in 5hmC candidate biomarkers. RESULTS: We found distinct differences in genomic regions and 5hmC modification motif patterns between patients with SLE and HCs, varying with disease progression. Increased 5hmC modification enrichment was detected in SLE. Additionally, we screened 151 genes with hyper-5hmC, which are significantly involved in SLE-related processes, and 5hmC-modified BCL2, CD83, ETS1 and GZMB as SLE biomarkers. Our findings suggest that CTCF regulates 5hmC modification of these genes by recruiting TET (ten-eleven translocation) protein, and CTCF knockdown affected the protein expression of these genes in vitro. CONCLUSIONS: Our findings demonstrate the increased 5hmC distribution in plasma cfDNA in different disease activity in patients with SLE compared with HCs and relating DhMRs involved in SLE-associated pathways. Furthermore, we identified a panel of SLE relevant biomarkers, and these viewpoints could provide insight into the pathogenesis of SLE.

The Next Frontier for Colorectal Cancer Screening: Blood-Based Tests.

Colorectal cancer is associated with substantial morbidity and mortality worldwide. Detection of early colorectal cancer is possible through approved screening tests but overall adherence is quite low. Implementation of effective noninvasive options could increase screening uptake and prevent a significant number of deaths. Noninvasive early cancer detection can potentially be achieved using a liquid biopsy. In this issue of Cancer Research, Cao and colleagues report on a novel multidimensional fragmentomics assay, named FRAGTECT, for colorectal cancer detection in circulating cell-free DNA with promising results. See related article by Cao et al., p. 3286.

A promising application of kidney-specific cell-free DNA methylation markers in real-time monitoring sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (SI-AKI) is a common clinical syndrome that is associated with high mortality and morbidity. Effective timely detection may improve the outcome of SI-AKI. Kidney-derived cell-free DNA (cfDNA) may provide new insight into understanding and identifying SI-AKI. Plasma cfDNA from 82 healthy individuals, 7 patients with sepsis non-acute kidney injury (SN-AKI), and 9 patients with SI-AKI was subjected to genomic methylation sequencing. We deconstructed the relative contribution of cfDNA from different cell types based on cell-specific methylation markers and focused on exploring the association between kidney-derived cfDNA and SI-AKI.Based on the deconvolution of the cfDNA methylome: SI-AKI patients displayed the elevated cfDNA concentrations with an increased contribution of kidney epithelial cells (kidney-Ep) DNA; kidney-Ep derived cfDNA achieved high accuracy in distinguishing SI-AKI from SN-AKI (AUC = 0.92, 95% CI 0.7801-1); the higher kidney-ep cfDNA concentrations tended to correlate with more advanced stages of SI-AKI; strikingly, SN-AKI patients with potential kidney damage unmet by SI-AKI criteria showed higher levels of kidney-Ep derived cfDNA than healthy individuals. The autonomous screening of kidney-Ep (n = 24) and kidney endothelial (kidney-Endo, n = 12) specific methylation markers indicated the unique identity of kidney-Ep/kidney-Endo compared with other cell types, and its targeted assessment reproduced the main findings of the deconvolution of the cfDNA methylome. Our study first demonstrates that kidney-Ep- and kidney-Endo-specific methylation markers can serve as a novel marker for SI-AKI emergence, supporting further exploration of the utility of kidney-specific cfDNA methylation markers in the study of SI-AKI.

Evaluation of a plasma cell-free DNA methylation test for colorectal cancer diagnosis: a multicenter clinical study.

BACKGROUND: A blood-based diagnostic test is a promising strategy for colorectal cancer (CRC). The MethyDT test (IColohunter), which detects methylation levels of NTMT1 and MAP3K14-AS1, exhibited potential in discriminating CRC, but its clinical performance needs to be validated in large-scale populations. METHODS: A multicenter, double-blinded, cross-sectional study that enrolled 1194 participants was performed. Plasma samples were collected to detect methylation levels of NTMT1 and MAP3K14-AS1 using quantitative methylation-specific PCR with the MethyDT test, and the accuracy was further evaluated by Sanger sequencing. RESULTS: The sensitivities of the MethyDT test for detecting CRC, early stages of CRC (I and II), advanced adenoma (AA), and high-grade intraepithelial neoplasia (HGIN) were 91.2% (95% confidence interval [CI], 88.4-94.0), 87.4% (95% CI, 82.5-92.2), 43.5% (95% CI, 35.7-51.4), and 72.7% (95% CI, 57.5-87.9), respectively. The specificities for participants with non-AA, interfering diseases (ID), and no evidence of disease (NED) were 85.0% (95% CI, 78.8-91.3), 93.7% (95% CI, 91.4-95.9) and 97.3% (95% CI, 90.5-99.7), respectively, and its overall specificity for all-controls was 92.4% (95% CI, 90.3-94.4). The consistency of the MethyDT test with pathology for CRC was high with a kappa value of 0.830 (95% CI, 0.795-0.865). Additionally, the MethyDT test was comparable to Sanger sequencing for detecting methylation with kappa values > 0.97. CONCLUSIONS: The MethyDT test demonstrates excellent sensitivity and specificity for CRC and high consistency with Sanger sequencing for methylation, suggesting it may serve as a potential noninvasive diagnostic tool for the detection of CRC. TRIAL REGISTRATION: This clinical trial has been registered in ClinicalTrials.gov (NCT05508503).

Impact of sample processing delays on plasma markers of inflammation, chemotaxis, cell death, and blood coagulation.

BACKGROUND: Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics. OBJECTIVE: To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1ß), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples. METHODS: Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay. RESULTS: Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation. CONCLUSIONS: Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity.