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Background: Noninvasive prenatal testing (NIPT) is increasingly used to screen for fetal chromosomal aneuploidy by analyzing cell-free DNA (cfDNA) in peripheral maternal blood. The method provides an opportunity for early detection of large genetic abnormalities without an increased risk of miscarriage due to invasive procedures. Commercial applications for use at clinical laboratories often take advantage of DNA sequencing technologies and include the bioinformatic workup of the sequence data. The interpretation of the test results and the clinical report writing, however, remains the responsibility of the diagnostic laboratory. In order to facilitate this step, we developed NIPTviewer, a web-based application to visualize and guide the interpretation of NIPT data results. Results: NIPTviewer has a database functionality to store the NIPT results and a web interface for user interaction and visualization. The application has been implemented as part of a novel analysis pipeline for NIPT in a diagnostic laboratory at Uppsala University Hospital. The validation data set included 84 previously analyzed plasma samples with known results regarding chromosomes 13, 18, 21, X and Y. They were sequenced in six different experiments, uploaded to NIPTviewer and assigned to a clinical laboratory geneticist for interpretation. The results of all previously analyzed samples were replicated. Conclusion: NIPTviewer facilitates NIPT results interpretation and has been implemented as part of a NIPT analysis routine that was accredited by the national accreditation body for Sweden (Swedac). [ABSTRACT FROM AUTHOR]

Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we develop a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We apply this platform to engineer amide synthetases by evaluating substrate preference for 1217 enzyme variants in 10,953 unique reactions. We use these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Over these nine compounds, ML-predicted enzyme variants demonstrate 1.6- to 42-fold improved activity relative to the parent. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel. While machine learning shows promise in expanding protein engineering efforts, its potential is limited by the challenge of gathering large datasets of sequence-function relationships. Here, authors introduce a platform that integrates cell-free DNA assembly and gene expression to accelerate enzyme engineering. [ABSTRACT FROM AUTHOR]

The emergence of liquid biopsy technologies holds great promise in the cancer setting, including in pediatric central nervous system (CNS) tumors. In contrast to broad lower-depth sequencing, commonly referred to as low pass whole genome sequencing (WGS), targeted platforms with a higher depth of coverage have also been established. Here, we review targeted liquid biopsy techniques with applicability to pediatric CNS tumors. These include polymerase chain reaction (PCR), both droplet digital PCR and reverse transcription-based PCR, Sanger sequencing, and next-generation sequencing approaches that incorporate amplicon- and hybrid capture-based methods. The goal of this paper is to facilitate an understanding of these targeted techniques and provide a context for clinical relevance within disease categories, as well as a discussion on optimizing real-world implementation for pediatric CNS tumors. [ABSTRACT FROM AUTHOR]

Purpose: Renal cell carcinoma (RCC) is a common malignancy, with patients frequently diagnosed at an advanced stage due to the absence of sufficiently sensitive detection technologies, significantly compromising patient survival and quality of life. Advances in cell-free DNA (cfDNA) methylation profiling using liquid biopsies offer a promising non-invasive diagnostic option, but robust biomarkers for early detection are current not available. This study aimed to identify methylation biomarkers for RCC and establish a DNA methylation signature-based prognostic model for this disease. Methods: High-throughput methylation sequencing was performed on peripheral blood samples obtained from 49 primarily Stage I RCC patients and 44 healthy controls. Comparative analysis and Least Absolute Shrinkage and Selection Operator (LASSO) regression methods were employed to identify RCC methylation signatures.Subsequently, methylation markers-based diagnostic and prognostic models for RCC were independently trained and validated using random forest and Cox regression methodologies, respectively. Results: Comparative analysis revealed 864 differentially methylated CpG islands (DMCGIs), 96.3% of which were hypermethylated. Using a training set from The Cancer Genome Atlas (TCGA) dataset of 443 early-stage RCC tumors and matched normal tissues, we applied LASSO regression and identified 23 methylation signatures. We then constructed a random forest-based diagnostic model for early-stage RCC and validated the model using two independent datasets: a TCGA set of 460 RCC tumors and controls, and a blood sample set from our study of 15 RCC cases and 29 healthy controls. For Stage I RCC tissue, the model showed excellent discrimination (AUC-ROC: 0.999, sensitivity: 98.5%, specificity: 100%). Blood sample validation also yielded commendable results (AUC-ROC: 0.852, sensitivity: 73.9%, specificity: 89.7%). Further analysis using Cox regression identified 7 of the 23 DMCGIs as prognostic markers for RCC, allowing the development of a prognostic model with strong predictive power for 1-, 3-, and 5-year survival (AUC-ROC > 0.7). Conclusions: Our findings highlight the critical role of hypermethylation in RCC etiology and progression, and present these identified biomarkers as promising candidates for diagnostic and prognostic applications. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) lacks a specific biomarker, but is defined by relatively selective toxicity to motor neurons (MN). As others have highlighted, this offers an opportunity to develop a sensitive and specific biomarker based on detection of DNA released from dying MN within accessible biofluids. Here we have performed whole genome bisulfite sequencing (WGBS) of iPSC-derived MN from neurologically normal individuals. By comparing MN methylation with an atlas of tissue methylation we have derived a MN-specific signature of hypomethylated genomic regions, which accords with genes important for MN function. Through simulation we have optimised the selection of regions for biomarker detection in plasma and CSF cell-free DNA (cfDNA). However, we show that MN-derived DNA is not detectable via WGBS in plasma cfDNA. In support of our experimental finding, we show theoretically that the relative sparsity of lower MN sets a limit on the proportion of plasma cfDNA derived from MN which is below the threshold for detection via WGBS. Our findings are important for the ongoing development of ALS biomarkers. The MN-specific hypomethylated genomic regions we have derived could be usefully combined with more sensitive detection methods and perhaps with study of CSF instead of plasma. Indeed we demonstrate that neuronal-derived DNA is detectable in CSF. Our work is relevant for all diseases featuring death of rare cell-types. [ABSTRACT FROM AUTHOR]

Introduction: Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development. Methods and analysis: Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30–60). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases. Discussion: This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers. [ABSTRACT FROM AUTHOR]

Plasma cell‐free DNA (cfDNA) analysis to track estrogen receptor 1 (ESR1) mutations is highly beneficial for the identification of tumor molecular dynamics and the improvement of personalized treatments for patients with metastatic breast cancer (MBC). Plasma‐cfDNA is, up to now, the most frequent liquid biopsy analyte used to evaluate ESR1 mutational status. Circulating tumor cell (CTC) enumeration and molecular characterization analysis provides important clinical information in patients with MBC. In this study, we investigated whether analysis of CTCs and circulating tumor DNA (ctDNA) provide similar or complementary information for the analysis of ESR1 mutations. We analyzed both plasma‐cfDNA (n = 90) and paired CTC‐derived genomic DNA (gDNA; n = 42) from 90 MBC patients for seven ESR1 mutations. Eight out of 90 (8.9%) plasma‐cfDNA samples tested using the ddPLEX Mutation Detection Assay (Bio‐Rad, Hercules, CA, USA), were found positive for one ESR1 mutation, whereas 11/42 (26.2%) CTC‐derived gDNA samples were found positive for at least one ESR1 mutation. Direct comparison of paired samples (n = 42) revealed that the ESR1 mutation rate was higher in CTC‐derived gDNA (11/42, 26.2%) than in plasma‐cfDNA (6/42, 14.3%) samples. Our results, using this highly sensitive ddPLEX assay, reveal a higher percentage of mutations in CTC‐derived gDNAs than in paired ctDNA in patients with MBC. CTC‐derived gDNA analysis should be further evaluated as an important and complementary tool to ctDNA for identifying patients with ESR1 mutations and for guiding individualized therapy. [ABSTRACT FROM AUTHOR]

PURPOSE: Circulating tumor DNA (ctDNA) analysis is an alternative to tissue biopsy for genotyping in various cancers. We aimed to establish a plasma ctDNA sequencing assay, then evaluate its clinical utility in advanced urothelial cancer (UC). MATERIALS AND METHODS: This study included 82 patients with muscle-invasive or metastatic UC. A total of 158 blood samples and 73 tumor tissue samples were sequenced using our designed panel covering 53 UC-relevant genes and TERT promoter. We investigated the association between the proportion of ctDNA in total cell-free DNA (ctDNA fraction) and response to pembrolizumab treatment. ctDNA dynamics were explored during systemic therapy. RESULTS: In paired tissue-ctDNA samples with estimated tumor fraction, half (50.2%) of the somatic mutations were shared between both sources, while some (17.6%) mutations were found only in ctDNA. In the metastatic setting, on-treatment increase in ctDNA fraction during pembrolizumab treatment was significantly associated with a poor response rate (18.7% v 76.1%; P =.001) and progression-free survival (median 2.8 v 9.8 months; P <.001). A comparison of cancer cell fraction, the fraction of tumor cells carrying somatic mutations, between pembrolizumab initiation and on-treatment showed a strong correlation among patients where ctDNA fraction increased during treatment (r = 0.73). By contrast, no correlation was observed in patients without ctDNA fraction increase (r = 0.09). Most mutations newly detected at pembrolizumab resistance have already been identified in tumor tissues at earlier stages. CONCLUSION: Our newly established assay is suitable for assessing the genomic profile of ctDNA in patients with advanced UC. Our data may support future analyses of prognostic or predictive biomarkers for UC. [ABSTRACT FROM AUTHOR]

Enhancing the effectiveness of utilizing circulating cell‐free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost‐efficiency, and patient adherence. We present here LAMP‐MS, an innovative technology that integrates linear amplification with single‐base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5‐methylcytosine (5 mC) status for colorectal cancer (CRC) screening. LAMP‐MS enables unbiased amplification of as little as 1 ng of cfDNA, site‐specifically quantify methylation levels of multiple 5 mC sites, thereby facilitating cost‐effective, high‐resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra‐low input materials and a visually interpretable output, LAMP‐MS stands out as a promising method for real‐world applications in clinics and laboratories for nucleic acid methylation detection and quantification. [ABSTRACT FROM AUTHOR]

Background: A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Questions/purposes: Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? Methods: This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. Results: This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. Conclusion: In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. Clinical Relevance: These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose. [ABSTRACT FROM AUTHOR]

Objective: To report the diagnostic accuracy of cell‐free fetal DNA (cfDNA) in detecting fetal chromosomal anomalies in women experiencing miscarriage. Methods: PubMed, MEDLINE, EMBASE and Cochrane databases were searched from inception to June 2024. The inclusion criteria were women experiencing miscarriage (defined as pregnancy loss before 20 weeks of gestation) who underwent cfDNA screening for trisomies 21, 18 and 13, other autosomal aneuploidies, sex‐chromosome aneuploidies and/or copy‐number variants (CNVs). The index test was cfDNA screening for each of the chromosomal anomalies listed. The reference standard was cytogenetic analysis of pregnancy tissue. The quality of the studies was assessed using the revised tool for the quality assessment of diagnostic accuracy studies. Summary estimates of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio were computed using the hierarchical summary receiver‐operating‐characteristics model. Results: Seven studies (887 women) were included in the systematic review and meta‐analysis. cfDNA had a sensitivity and specificity of 100% (95% CI, 81.5–100%) and 100% (95% CI, 99.1–100%), respectively, for trisomy 21, 100% (95% CI, 54.1–100%) and 100% (95% CI, 99.0–100%), respectively, for trisomy 18, and 88.9% (95% CI, 51.8–99.7%) and 100% (95% CI, 99.1–100%), respectively, for trisomy 13. The respective values for other autosomal trisomies were 75.8% (95% CI, 65.7–84.2%) and 99.4% (95% CI, 97.9–99.9%), while those for CNVs were 60.0% (95% CI, 14.7–94.7%) and 100% (95% CI, 97.4–100%). Failure of cfDNA testing was reported in 7.3% (95% CI, 5.7–9.2%) of cases. Conclusion: cfDNA has high diagnostic accuracy in detecting fetal trisomies 21, 18 and 13 in women experiencing miscarriage, while its accuracy for other autosomal aneuploidies and CNVs is only moderate. © 2024 International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Objective Low fetal fraction (FF) on cell-free DNA (cfDNA)-based noninvasive prenatal screening (NIPS) is a common etiology for indeterminate results. As maternal Black race is implicated as a risk factor for low FF and more indeterminate results, we sought to evaluate this association. Study Design This was a single-institution, retrospective cohort study of cfDNA-based NIPS performed between May 2017 and May 2022 with complete clinical data abstraction. We compared FF, indeterminate rates, and total cfDNA concentration among self-reported Black, White, and Other groups from NIPS results from 2017 to 2022 with full clinical data abstraction. Using linear regression and interaction testing, we evaluated associations between self-reported race, FF, indeterminate rate, and total cfDNA concentration. Results In total, 1,591 participants met the inclusion criteria; 70.8% (n = 1,126) self-identified as White, 6.9% (n = 110) as Black, and 22.3% (n = 355) self-identified with another race. Mean FF was not different between the White, Black, or Other groups (11.8 vs. 11.2 vs. 11.7%, respectively, p = 0.52). This remained true after adjusting for body mass index (BMI), gestational age (GA) at draw, and fetal sex (all p > 0.17). Interaction testing for FF and total cfDNA by race with BMI, GA at draw, and fetal sex demonstrated no effect modification. Conclusion In our population, maternal self-identified race, particularly Black race, does not affect FF. Biological plausibility for race-based differences on clinical tests requires ongoing thoughtful consideration. Key Points NIPS is widely used to screen for fetal aneuploidy. FF is an important test metric, and low FF is associated with adverse outcomes, like aneuploidy. In existing studies, Black race is implicated as a risk factor for lower FF. Our study found no differences in FF between groups by self-reported race. Biological plausibility for race-based differences on clinical tests requires ongoing consideration. [ABSTRACT FROM AUTHOR]

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5′-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing a rapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors. [ABSTRACT FROM AUTHOR]

Background/Aim: Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy characterized by genomic heterogeneity. KRAS mutations play a significant role in influencing patient prognosis and guiding therapeutic decision-making. This study aimed to determine the prevalence and prognostic significance of KRAS mutations in CCA, asses the detection of KRAS G12/G13 mutations in plasma cell-free DNA (cfDNA), and evaluate the prognostic value of KRAS G12/G13 mutant allele frequency (MAF) in cfDNA in relation to clinicopathological data and patient survival. Materials and Methods: A retrospective analysis of 937 CCA patients was performed using data from cBioPortal to examine KRAS mutation profiles and their association with survival. Plasma from 101 CCA patients was analyzed for KRAS G12/G13 mutations in the cfDNA using droplet digital PCR, and the results were compared with tissuebased sequencing from 78 matched samples. Results: KRAS driver mutations were found in 15.6% of patients, with common variants being G12D (37.0%), G12V (24.0%) and Q61H (8.2%). Patients harboring KRAS mutations exhibited decreased overall and recurrence-free survival. KRAS G12/G13 mutations were detected in 14.9% of cfDNA samples, showing moderate concordance with tissue sequencing, and achieving 80% sensitivity and 93% specificity. Elevated KRAS G12/G13 MAF in cfDNA, combined with high CA19-9 levels, correlated with poorer survival outcomes. Conclusion: The presence of KRAS mutations was associated with poor survival in CCA, underscoring the importance of KRAS mutations as prognostic markers. The detection of KRAS mutations in cfDNA demonstrated potential as a promising non-invasive alternative for mutation detection and, when combined with CA19-9 levels, may improve prognostic efficacy in CCA. [ABSTRACT FROM AUTHOR]

Heart failure (HF) remains a major global health challenge, and more effective and comprehensive plasma biomarkers are needed to effectively treat HF patients. Multiomics studies have shown that DNA fragments, noncoding RNAs, proteins, and metabolites may be potential plasma biomarkers for HF. However, comprehensive reviews that focus on research on plasma biomarkers for HF from an omics perspective are lacking. This review summarizes the applications of various omics approaches in the exploration of biomarkers related to the risk assessment, diagnosis, subtype classification, medical management, and prognosis prediction of HF. Moreover, as heart transplantation and left ventricular assistant device (LVAD) implantation are terminal therapies for end-stage HF patients, this review also discusses the role of cell-free DNA as a biomarker for cardiac transplant rejection and omics studies of plasma biomarkers in patients who respond to LVAD therapy. Our findings suggest that future omics research on HF biomarkers should employ integrated multiomics methods and expand the sample size to increase the robustness of the results and that the identified biomarkers should be further validated in large cohorts. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) poses a severe risk for individuals with systemic lupus erythematosus (SLE), prompting extensive research into targeted delivery systems capable of modulating immune responses and clearing cell-free DNA (cfDNA). Here, we propose a novel renal homing nanogel that acts as a cfDNA scavenger and a dexamethasone (DXM) delivery carrier for LN treatment. Based on the generation 3 polylysine dendrimers, the created cationic nanogels (G3DSP) exhibit minimal toxicity and outstanding DXM loading efficiency. Our studies confirm that these nanogels can competitively bind with anionic cfDNA in vitro, leading to the suppression of toll-like receptor 9 (TLR9) activation. When administered systemically to MRL/lpr mice, the nanogels preferentially localize to and are retained in the inflamed kidneys, releasing their payload in response to reactive oxygen species (ROS), therefore effectively ameliorating SLE symptoms. Consequently, G3DSP nanogels emerge as a promising effective combined therapy for LN, minimizing cfDNA accumulation in vital organs and delivering immunomodulatory benefits through DXM.

This study investigates serum cell-free DNA fluctuations in patients with herpes zoster or post-herpetic neuralgia, offering insight into the tissue damage and inflammatory dynamics associated with these conditions. A single-centre combined cross-sectional and longitudinal study was conducted with 59 patients to assess cell-free DNA levels in herpes zoster and post-herpetic neuralgia. Cell-free DNA was extracted from blood samples of patients with herpes zoster or post-herpetic neuralgia and compared with healthy controls. The findings demonstrated elevated cell-free DNA levels in patients with herpes zoster, which remained elevated for 3 months or longer following treat-ment. These results suggest the presence of a subacute inflammatory state after herpes zoster infection. Furthermore, patients who developed post-herpetic neuralgia did not show elevated cell-free DNA levels, while those who did not develop post-herpetic neuralgia exhibited increased levels. This indicates that post-herpetic neuralgia is likely a localized response to prior nerve damage rather than a systemic inflammatory process with acute tissue damage.

Hemangiosarcoma is a highly malignant tumor commonly affecting canines, originating from endothelial cells that line blood vessels, underscoring the importance of early detection. This canine cancer is analogous to human angiosarcoma, and the development of liquid biopsies leveraging cell-free DNA (cfDNA) represents a promising step forward in early cancer diagnosis. In this study, we utilized Whole Genome Sequencing (WGS) to analyze fragment sizes and copy number alterations (CNAs) in cfDNA from 21 hemangiosarcoma-affected and 36 healthy dogs, aiming to enhance early cancer detection accuracy through machine learning models. Our findings reveal that similar to trends in human oncology, hemangiosarcoma samples exhibited shorter DNA fragment sizes compared to healthy controls, with a notable leftward shift in the primary peak. Interestingly, canine hemangiosarcoma DNA fragment sizes demonstrated eight distinct periodic patterns diverging from those typically observed in human angiosarcoma. Additionally, we identified seven novel genomic gains and nine losses in the hemangiosarcoma samples. Applying machine learning to the cfDNA fragment size distribution, we achieved an impressive average Area Under the Curve (AUC) of 0.93 in 10-fold cross-validation, underscoring the potential of this approach for precise early-stage cancer classification. This study confirms distinctive cfDNA fragment size and CNA patterns in hemangiosarcoma-affected vs. healthy dogs and demonstrates the promise of these biomarkers in canine cancer screening, early detection, and monitoring via liquid biopsies. These findings establish a foundation for broader research on cfDNA analysis in various canine cancers, integrating methodologies from human oncology to enhance early detection and diagnostic precision in veterinary medicine.

Background: Cell-free DNA (cfDNA) is present in healthy individuals but is elevated in those undergoing physical exertion, trauma, sepsis, and certain cancers. Maintaining cfDNA concentrations is vital for immune homeostasis and preventing inflammatory responses. Understanding cfDNA release and clearance is essential for using cfDNA as a biomarker in clinical diagnostics. We focused on the fragment size of cfDNA and investigated cfDNA dynamics and half-life, particularly the 100–250 base pair fragments. Methods: Healthy, adult men (n = 5; age 40 ± 4.1 years) were subjected to a 30 min treadmill exercise. Blood samples were collected at 0, 5, 10, 15, 30, and 60 min post-exercise using PAXgene® Blood ccfDNA tubes to stabilize and prevent nuclease-mediated cfDNA degradation and minimize genomic DNA contamination risk. The cfDNA concentration was measured using an electrophoresis-based technique (4150 TapeStation system) to quantify the concentration based on cfDNA fragment size. Results: The results showed a cfDNA half-life of 24.2 min, with a transient increase in 100–250 base pair cfDNA fragments post-exercise, likely due to nuclease activity. These levels rapidly reverted to the baseline within an hour. Conclusions: The rapid clearance of cfDNA underscores its potential as a biomarker for real-time disease monitoring and the evaluation of treatment efficacy. This study is expected to standardize cfDNA investigations, enhancing diagnosis and treatment monitoring across various disease conditions.

• Alzheimer's disease (AD) causes significant cognitive and behavioral decline. • Liquid biopsy advancements are vital for AD research. • Cell-free DNA methylation emerges as a novel, non-invasive biomarker for diagnosing Alzheimer's disease. • The review reveals a strong link between altered cfDNA methylation and AD disease progression. • The review outlines key areas for future research to optimize the use of these biomarkers in clinical practice. Due to an aging population, Alzheimer's disease (AD), a neurodegenerative disorder, has affected more than 40 million people worldwide, a figure predicted to significantly increase in the coming decades. Despite much effort to understand AD pathogenesis, effective diagnosis and treatment remain a challenge. However, the development of liquid biopsy including the analysis of cell-free DNA (cfDNA) and methylation thereof has provided an alternative source of investigation to further explore the pathophysiology of AD. Herein, we discuss the research progress to date and highlight clinical applications of methylated cfDNA in AD. [ABSTRACT FROM AUTHOR]

• Liquid biopsy shows potential for diagnosing individuals with breast cancer effectively. • cfDNA assays diagnose breast cancer with high sensitivity and specificity. • CTCs, miRNAs, and miR-21 offer moderate to high accuracy for breast cancer diagnosis. • QUADAS-2 identifies 10 systematic reviews with high risk of bias and 4 with unclear applicability. • AMSTAR 2 rates 9 systematic reviews critically low, 1 low, and 1 with moderate confidence. Breast cancer (BC) is the leading type of cancer affecting women globally and remains a significant cause of death. The diagnostic accuracy of liquid biopsy (LB) in the diagnosis of BC has not been well established. This overview synthesizes and critically evaluates the diagnostic test accuracy (DTA) of LB biomarkers in individuals with BC. Of 433 systematic reviews, eleven were included, assessing Fourier transform infrared (FTIR) spectroscopy, circulating tumor cells (CTCs), cell-free DNA (cfDNA), and microRNAs (miRNAs). The overall methodological quality of most of the reviews included was rated as critically low (n = 9, 81.8 %), and the remaining reviews were ranked as low and moderate. Key findings include CTCs with moderate sensitivity (0.50, 95 % confidence interval (CI) 0.48–0.52) and high specificity (0.93, 95 % CI: 0.92–0.95) with moderate certainty; cfDNA assays with high sensitivity (0.71–0.86) and specificity (0.88) with high certainty; FTIR assays with high sensitivity (0.97, 95 % CI: 0.94–0.96) and specificity (0.92, 95 % CI: 0.88–0.95) but low certainty. The miRNAs showed moderate to high sensitivity, while miR-21 had high specificity. Our overview indicates that identified liquid biopsies could serve as valuable tools for the diagnosis of breast cancer. [ABSTRACT FROM AUTHOR]

• WGBS alters cfDNA fragmentation patterns in nuclear and mitochondrial DNA. • WGBS reduces genome coverage and increases GC content in cfDNA. • WGBS samples show increased abundance and length of plasma mtDNA. • WGBS accurately reflects gene expression levels through TSS coverages. • WGBS underestimates fetal cfDNA fraction by ∼ 7 % compared to WGS. Cell-free DNA (cfDNA) is non-randomly fragmented in human body fluids. Analyzing such fragmentation patterns of cfDNA holds great promise for liquid biopsy. Whole-genome bisulfite sequencing (WGBS) is widely used for cfDNA methylation profiling. However, its applicability for studying fragmentomic characteristics remains largely unexplored. We performed paired WGBS and whole-genome sequencing (WGS) on 66 peripheral plasma samples from 58 pregnant women. Then, we systematically compared the fragmentation patterns of cell-free nuclear DNA and mitochondrial DNA (mtDNA) sequenced from these two approaches. Additionally, we evaluated the extent of the size shortening in fetal-derived cfDNA and estimated the fetal DNA fraction in maternal plasma using both sequencing methods. Compared to WGS samples, WGBS samples demonstrated a significantly lower genome coverage and higher GC content in cfDNA. They also showed a significant decrease in the size of cell-free nuclear DNA, along with alterations in the end motif pattern that were specifically associated with CpG and "CC" sites. While there was a slight shift in the inferred nucleosome footprint from cfDNA coverages in WGBS samples, the cfDNA coverage patterns in CTCF and TSS regions remained highly consistent between these two sequencing methods. Both methods accurately reflected gene expression levels through their TSS coverages. Additionally, WGBS samples exhibited an increased abundance and longer length of mtDNA in plasma. Furthermore, we observed the size shortening of fetal cfDNA in plasma consistently, with a highly correlated fetal DNA fraction inferred by cfDNA coverage between WGBS and WGS samples (r = 0.996). However, the estimated fetal cfDNA fraction in WGBS samples was approximately 7 % lower than in WGS samples. We confirmed that WGBS can introduce artificial breakages to cfDNA, leading to altered fragmentomic patterns in both nuclear and mitochondrial DNA. However, WGBS cfDNA remains suitable for analyzing certain cfDNA fragmentomic characteristics, such as coverage in genome regulation regions and the essential characteristics of fetal DNA in maternal plasma. [ABSTRACT FROM AUTHOR]

To investigate whether the methylation of circulating cell-free DNA (cfDNA) differentiates active tuberculosis (TB) from latent TB infection (LTBI). Patients with pulmonary TB, contacts with LTBI, and healthy controls were enrolled (2018–2021). Plasma cfDNA was extracted, and using a 5-methylcytosine (5mC) DNA ELISA kit, the global methylation of cfDNA (5mC-cfDNA) was measured. 59 TB, 63 LTBI, 39 healthy controls were included. The 5mC-cfDNA level was higher in TB (6.4 %) than LTBI (4.1 %) and healthy controls (4.9 %) (both p<0.05). Independent TB factors were 5mC-cfDNA ≥6.6 % and CRP ≥0.32 mg/dL (adjusted odds ratio (aOR) 4.594 [95 % CI:1.628–12.965], p=0.004 and 5.338 [1.659–17.176], p=0.005). Having one or both factors increased TB odds 8- and 16-fold (aOR 8.688 [3.229–23.378], p <0.001 and 16.080 [3.092–83.632], p =0.001). The global cfDNA methylation level was higher in TB than contacts without TB and helped differentiate patients with TB from contacts with LTBI. [ABSTRACT FROM AUTHOR]

Circulating cell free DNA (cfDNA) is a valuable source of biomarkers for a range of medical applications including detection and monitoring of diseases. Currently, cfDNA sequence analysis must take place in a laboratory setting, due to the multiple steps required for processing including collection, purification, amplification, and analysis. Developing a point-of-care test system that combines these steps would simplify DNA processing thereby increasing diagnostic screening accessibility and enabling real-time monitoring for individual patients. Here, we have developed a system that combines multiple cfDNA processing steps into a single microfluidic-based device. This includes cfDNA collection directly from undiluted human plasma followed by purification and on chip amplification. A microelectrode array embedded within the microfluidic chip collected cfDNA through the creation of dielectrophoretic (DEP) forces followed by a wash to achieve purification. DEP utilizes differences in dielectric properties between cfDNA and plasma to preferentially induce a force on cfDNA. We then achieved on-chip amplification of collected DNA by designing a thermal cycling system to enable polymerase chain reaction (PCR) directly on the chip. This successfully consolidated the most labor-intensive steps of collection, purification, and amplification into a single device. Compared to elution of cfDNA for off-chip amplification, our on-chip PCR method improved the lower limit of detection by 3-fold and improved the total DNA yield by 5-fold. Furthermore, we demonstrate its clinical diagnostic potential by detecting KRAS mutations from a pancreatic ductal adenocarcinoma patient using only 60 µL of plasma. This paves the way for future development of a fully self-contained system facilitating the rapid detection of mutations in cfDNA. • Successful integration of DNA collection and amplification into a single device. • Electrode microarray collected cell free DNA from plasma using dielectrophoresis. • Collected DNA was PCR amplified by thermal cycling of the chip. • Pancreatic cancer-related KRAS mutations were detected in the amplified DNA. • This simplifies the most labor intensive steps for DNA analysis. [ABSTRACT FROM AUTHOR]

The presence of excess cell-free DNA (cfDNA) can aggravate acute kidney injury (AKI) and activate oxidative stress-related signaling pathways, damaging the organs of patients. The hemodialysis membranes-based cfDNA and reactive oxygen species (ROS) scavenging strategy remains a research gap, and its pathological mechanism needs to be studied urgently. Herein, the flower-like Nb 2 CT x /WS 2 heterojunctions were synthesized to coat polyvinylidene difluoride (PVDF) membranes. Owing to the prominent antioxidant capacity of Nb 2 CT x /WS 2 nanoflowers, the functionalized membranes could scavenge multiple ROS, inhibit lipid peroxidation, and regulate intracellular antioxidant enzyme activities, therefore reducing oxidative damage. The improved hemodialysis membranes (M 2 membranes) demonstrated outstanding uremic toxin scavenging and cfDNA adsorption due to the distinctive nanoflower stacking structure. Furthermore, the Nb 2 CT x /WS 2 functionalized membranes showed outstanding hemocompatibility and cytocompatibility, which could meet the biocompatibility demands of hemodialysis membranes. The Nb 2 CT x /WS 2 nanoflower functionalized membranes constructed in this study have excellent ability of cfDNA adsorption and oxidative stress intervention while maintaining high dialysis performance, which can effectively enhance patient prognosis and may provide a new idea into the future development of multifunctional hemodialysis membranes for AKI treatment. [Display omitted] • Nb 2 CT x /WS 2 nanoflowers were successfully created utilizing the solvothermal method. • "cfDNA + oxidative stress" intervention hemodialysis membrane was prepared. • The functionalized membranes had outstanding cfDNA adsorption capability. • The functionalized membranes demonstrated excellent clearance of uremic toxins. • The functionalized membranes could inhibit AGEs and possessed good biocompatibility. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cardiac allograft vasculopathy (CAV) leads to impaired myocardial blood flow (MBF), increasing the risk of cardiovascular death or retransplant among heart transplantation (HT) recipients. Data on elevation in donor-derived cell-free DNA (dd-cfDNA) and CAV in the absence of rejection are mixed. We sought to test the hypothesis that CAV with reduced MBF (RMBF) is associated with elevated dd-cfDNA. METHODS: A retrospective review was conducted on HT recipients at a high-volume center who underwent dd-cfDNA testing between September 2019 and November 2022. Inclusion criteria included undergoing CAV screening with cardiac positron emission tomography scans and coronary angiograms. Patients were grouped by the presence of angiographic CAV diagnosis and MBF reserve evaluated through cardiac positron emission tomography. The latter was subdivided into normal MBF or RMBF, with RMBF defined as an MBF reserve ≤2. Elevated dd-cfDNA was defined as ≥0.12%. RESULTS: Two hundred fifty-six HT recipients were included (median age, 55 years; 27.6% female; median, 8 years [interquartile range (IQR), 5–14] post-HT). Ischemic etiology of heart failure was more prevalent in the RMBF group (36%) compared with the normal MBF group (20%; P =0.02). The prevalence and magnitude of a positive dd-cfDNA test with angiographic CAV (29%; median, 0.26% [IQR, 0.15%–0.62%]) were not significantly different from those without CAV (30%; P =0.94; median, 0.31% [IQR, 0.17%–0.71%]; P =0.38). However, RMBF patients exhibited significantly higher dd-cfDNA prevalence and levels (51%; median, 0.81% [IQR, 0.48%–1.11%]) compared with normal MBF patients (27%; P <0.001; median, 0.25% [IQR, 0.15%–0.52%]; P <0.001). CONCLUSIONS: HT recipients with angiographic CAV had similar dd-cfDNA levels and rates as those without. Notably, dd-cfDNA levels and rates were significantly elevated in patients with RMBF assessed by positron emission tomography compared with those with normal MBF. [ABSTRACT FROM AUTHOR]