[Comparison of two superparamagnetic purification magnetic beads-based screening and enrichment techniques for isolating cell-free fetal DNA from maternal plasma for non-invasive prenatal screening].

OBJECTIVE: To assess the efficiency of modified enrichment method for cell-free fetal DNA (cffDNA) through purified superparamagnetic beads during non-invasive prenatal testing (NIPT). METHODS: A total of 26 252 pregnant women undergoing NIPT at the Maternal and Child Health Care Hospital of Haidian District from December 2017 to September 2022 were recruited and randomly assigned into the conventional group (n = 10 573) and the modified enrichment group (n = 15 679), who were then subjected to the screening and enrichment of the cffDNA using a conventional and a modified technique, respectively. High-risk pregnant women detected by NIPT were subjected to invasive prenatal diagnosis. All women were followed up for their pregnancy outcomes, and the detection efficacy of the two methods was compared in terms of fragment size, concentration of cffDNA, duplicate detection rate, and indices of clinical laboratory tests. RESULTS: The fragment size of the main peak of the cell-free DNA library of the modified enrichment group was significantly lower than that of the conventional group [267 (264, 269) bp vs. 294 (292, 296) bp, P < 0.01], while the concentration of cffDNA was significantly higher [21.86% (17.61%, 26.36%) vs. 9.08% (6.87%, 11.87%), P < 0.01]. In addition, the duplicate detection rate (0.740% vs. 2.02%, X2 = 83.90, P < 0.01) and detection failure rate (0.006% vs. 0.057%, P < 0.05) in the modified enrichment group were significantly lower than those of the conventional group. The combined positive predictive value (PPV) in both high-risk (64.3% vs. 76.1%) and low-risk (35.3% vs. 45.5%) pregnant women from the modified enrichment group was slightly lower than those from the conventional group, though no significant difference was detected. There was one false negative case for trisomy 21 among the high-risk pregnant women from the conventional group, and no false negative case was found in the modified enrichment group. CONCLUSION: The modified technique to screen and enrich the cffDNA has significantly enhanced the relative concentration of cffDNA and reduced the failure and duplication detection rate of NIPT, which has significantly reduced the incidence of false negative cases due to the low concentration of cffDNA, and greatly increased the overall detection efficacy of NIPT.

Tissue of origin detection for cancer tumor using low-depth cfDNA samples through combination of tumor-specific methylation atlas and genome-wide methylation density in graph convolutional neural networks.

BACKGROUND: Cell free DNA (cfDNA)-based assays hold great potential in detecting early cancer signals yet determining the tissue-of-origin (TOO) for cancer signals remains a challenging task. Here, we investigated the contribution of a methylation atlas to TOO detection in low depth cfDNA samples. METHODS: We constructed a tumor-specific methylation atlas (TSMA) using whole-genome bisulfite sequencing (WGBS) data from five types of tumor tissues (breast, colorectal, gastric, liver and lung cancer) and paired white blood cells (WBC). TSMA was used with a non-negative least square matrix factorization (NNLS) deconvolution algorithm to identify the abundance of tumor tissue types in a WGBS sample. We showed that TSMA worked well with tumor tissue but struggled with cfDNA samples due to the overwhelming amount of WBC-derived DNA. To construct a model for TOO, we adopted the multi-modal strategy and used as inputs the combination of deconvolution scores from TSMA with other features of cfDNA. RESULTS: Our final model comprised of a graph convolutional neural network using deconvolution scores and genome-wide methylation density features, which achieved an accuracy of 69% in a held-out validation dataset of 239 low-depth cfDNA samples. CONCLUSIONS: In conclusion, we have demonstrated that our TSMA in combination with other cfDNA features can improve TOO detection in low-depth cfDNA samples.

Promoter profiles in plasma CfDNA exhibits a potential utility of predicting the efficacy of neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Gene expression profiles in breast tissue biopsies contain information related to chemotherapy efficacy. The promoter profiles in cell-free DNA (cfDNA) carrying gene expression information of the original tissues may be used to predict the response to neoadjuvant chemotherapy in breast cancer as a non-invasive biomarker. In this study, the feasibility of the promoter profiles in plasma cfDNA was evaluated as a novel clinical model for noninvasively predicting the efficacy of neoadjuvant chemotherapy in breast cancer. METHOD: First of all, global chromatin (5 Mb windows), sub-compartments and promoter profiles in plasma cfDNA samples from 94 patients with breast cancer before neoadjuvant chemotherapy (pCR = 31 vs. non-pCR = 63) were analyzed, and then classifiers were developed for predicting the efficacy of neoadjuvant chemotherapy in breast cancer. Further, the promoter profile changes in sequential cfDNA samples from 30 patients (pCR = 8 vs. non-pCR = 22) during neoadjuvant chemotherapy were analyzed to explore the potential benefits of cfDNA promoter profile changes as a novel potential biomarker for predicting the treatment efficacy. RESULTS: The results showed significantly distinct promoter profile in plasma cfDNA of pCR patients compared with non-pCR patients before neoadjuvant chemotherapy. The classifier based on promoter profiles in a Random Forest model produced the largest area under the curve of 0.980 (95% CI: 0.978-0.983). After neoadjuvant chemotherapy, 332 genes with significantly differential promoter profile changes in sequential cfDNA samples of pCR patients was observed, compared with non-pCR patients, and their functions were closely related to treatment response. CONCLUSION: These results suggest that promoter profiles in plasma cfDNA may be a powerful, non-invasive tool for predicting the efficacy of neoadjuvant chemotherapy breast cancer patients before treatment, and the on-treatment cfDNA promoter profiles have potential benefits for predicting the treatment efficacy.

Extracellular Vesicle DNA Extraction and Sequencing in Ancient Serum Samples From Patients With Breast Cancer.

BACKGROUND/AIM: Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS: A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS: Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION: EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

Accurate Early Detection and EGFR Mutation Status Prediction of Lung Cancer Using Plasma cfDNA Coverage Patterns: A Proof-of-Concept Study.

Lung cancer is a major global health concern with a low survival rate, often due to late-stage diagnosis. Liquid biopsy offers a non-invasive approach to cancer detection and monitoring, utilizing various features of circulating cell-free DNA (cfDNA). In this study, we established two models based on cfDNA coverage patterns at the transcription start sites (TSSs) from 6X whole-genome sequencing: an Early Cancer Screening Model and an EGFR mutation status prediction model. The Early Cancer Screening Model showed encouraging prediction ability, especially for early-stage lung cancer. The EGFR mutation status prediction model exhibited high accuracy in distinguishing between EGFR-positive and wild-type cases. Additionally, cfDNA coverage patterns at TSSs also reflect gene expression patterns at the pathway level in lung cancer patients. These findings demonstrate the potential applications of cfDNA coverage patterns at TSSs in early cancer screening and in cancer subtyping.

Deep learning model integrating cfDNA methylation and fragment size profiles for lung cancer diagnosis.

Detecting aberrant cell-free DNA (cfDNA) methylation is a promising strategy for lung cancer diagnosis. In this study, our aim is to identify methylation markers to distinguish patients with lung cancer from healthy individuals. Additionally, we sought to develop a deep learning model incorporating cfDNA methylation and fragment size profiles. To achieve this, we utilized methylation data collected from The Cancer Genome Atlas and Gene Expression Omnibus databases. Then we generated methylated DNA immunoprecipitation sequencing and genome-wide Enzymatic Methyl-seq (EM-seq) form lung cancer tissue and plasma. Using these data, we selected 366 methylation markers. A targeted EM-seq panel was designed using the selected markers, and 142 lung cancer and 56 healthy samples were produced with the panel. Additionally, cfDNA samples from healthy individuals and lung cancer patients were diluted to evaluate sensitivity. Its lung cancer detection performance reached an accuracy of 81.5% and an area under the receiver operating characteristic curve of 0.87. In the serial dilution experiment, we achieved tumor fraction detection of 1% at 98% specificity and 0.1% at 80% specificity. In conclusion, we successfully developed and validated a combination of methylation panel and a deep learning model that can distinguish between patients with lung cancer and healthy individuals.

Exploring the translational landscape of the long noncoding RNA transcriptome in acute respiratory distress syndrome: it is a long way to the top.

Acute respiratory distress syndrome (ARDS) poses a significant and widespread public health challenge. Extensive research conducted in recent decades has considerably improved our understanding of the disease pathophysiology. Nevertheless, ARDS continues to rank among the leading causes of mortality in intensive care units and its management remains a formidable task, primarily due to its remarkable heterogeneity. As a consequence, the syndrome is underdiagnosed, prognostication has important gaps and selection of the appropriate therapeutic approach is laborious. In recent years, the noncoding transcriptome has emerged as a new area of attention for researchers interested in biomarker development. Numerous studies have confirmed the potential of long noncoding RNAs (lncRNAs), transcripts with little or no coding information, as noninvasive tools for diagnosis, prognosis and prediction of the therapeutic response across a broad spectrum of ailments, including respiratory conditions. This article aims to provide a comprehensive overview of lncRNAs with specific emphasis on their role as biomarkers. We review current knowledge on the circulating lncRNAs as potential markers that can be used to enhance decision making in ARDS management. Additionally, we address the primary limitations and outline the steps that will be essential for integration of the use of lncRNAs in clinical laboratories. Our ultimate objective is to provide a framework for the implementation of lncRNAs in the management of ARDS.

Early detection and stratification of colorectal cancer using plasma cell-free DNA fragmentomic profiling.

Timely accurate and cost-efficient detection of colorectal cancer (CRC) is of great clinical importance. This study aims to establish prediction models for detecting CRC using plasma cell-free DNA (cfDNA) fragmentomic features. Whole-genome sequencing (WGS) was performed on cfDNA from 620 participants, including healthy individuals, patients with benign colorectal diseases and CRC patients. Using WGS data, three machine learning methods were compared to build prediction models for the stratification of CRC patients. The optimal model to discriminate CRC patients of all stages from healthy individuals achieved a sensitivity of 92.31% and a specificity of 91.14%, while the model to separate early-stage CRC patients (stage 0-II) from healthy individuals achieved a sensitivity of 88.8% and a specificity of 96.2%. Additionally, the cfDNA fragmentation profiles reflected disease-specific genomic alterations in CRC. Overall, this study suggests that cfDNA fragmentation profiles may potentially become a noninvasive approach for the detection and stratification of CRC.

Systematic biases in reference-based plasma cell-free DNA fragmentomic profiling.

Plasma cell-free DNA (cfDNA) fragmentation patterns are emerging directions in cancer liquid biopsy with high translational significance. Conventionally, the cfDNA sequencing reads are aligned to a reference genome to extract their fragmentomic features. In this study, through cfDNA fragmentomics profiling using different reference genomes on the same datasets in parallel, we report systematic biases in such conventional reference-based approaches. The biases in cfDNA fragmentomic features vary among races in a sample-dependent manner and therefore might adversely affect the performances of cancer diagnosis assays across multiple clinical centers. In addition, to circumvent the analytical biases, we develop Freefly, a reference-free approach for cfDNA fragmentomics profiling. Freefly runs ∼60-fold faster than the conventional reference-based approach while generating highly consistent results. Moreover, cfDNA fragmentomic features reported by Freefly can be directly used for cancer diagnosis. Hence, Freefly possesses translational merit toward the rapid and unbiased measurement of cfDNA fragmentomics.

Serial Cell-Free DNA Sequencing in ROS1 Fusion-Positive Lung Cancers During Treatment With Entrectinib.

PURPOSE: Patients with metastatic ROS1 fusion-positive non-small cell lung cancer (NSCLC) are effectively treated with entrectinib, a multikinase inhibitor. Whether serial targeted gene panel sequencing of cell-free DNA (cfDNA) can identify response and progression along with mechanisms of acquired resistance to entrectinib is underexplored. METHODS: In patients with ROS1 fusion-positive NSCLC, coclinical trial plasma samples were collected before treatment, after two cycles, and after progression on entrectinib (global phase II clinical trial, ClinicalTrials.gov identifier: NCT02568267). Samples underwent cfDNA analysis using MSK-ACCESS. Variant allele frequencies of detectable alterations were correlated with objective response per RECIST v1.1 criteria. RESULTS: Twelve patients were included, with best response as partial response (n = 9, 75%), stable disease (n = 2, 17%), and progressive disease (PD; n = 1, 8%). A ROS1 fusion was variably detected in cfDNA; however, patients without a ROS1 fusion in cfDNA had no other somatic alterations detected, indicative of possible low cfDNA shedding. Clearance of the enrolling ROS1 fusion or concurrent non-ROS1 alterations (TP53, CDH1, NF1, or ARID1A mutations) was observed in response to entrectinib therapy. Radiologic PD was accompanied by redemonstration of a ROS1 fusion or non-ROS1 alterations. On-target resistance was rare; only one patient acquired ROS1 G2032R at the time of progression. Several patients acquired new off-target likely oncogenic alterations, including a truncating alteration in NF1. CONCLUSION: Serial cfDNA monitoring may complement radiographic assessments as determinants of response and resistance to entrectinib in ROS1 fusion-positive lung cancers in addition to detecting putative resistance mechanisms on progression.

Noninvasive prenatal screening in a pregnant woman with a history of stem cell transplant from a male donor: A case report and literature review.

BACKGROUND: As a screening method, inaccuracies in noninvasive prenatal screening (NIPS) exist, which are often attributable to biological factors. One such factor is the history of transplantation. However, there are still limited reports on such NIPS cases. METHODS: We report an NIPS case of a pregnant woman who had received a stem cell transplant from a male donor. To determine the karyotype in the woman's original cell, we performed chromosome microarray analysis (CMA) on her postnatal blood and oral mucosa. To comprehensively estimate the cell-free DNA (cfDNA) composition, we further performed standard NIPS procedures on the postnatal plasma. Moreover, we reviewed all published relevant NIPS case reports about pregnant women with transplantation history. RESULTS: NIPS showed a low-risk result for common trisomies with a fetal fraction of 65.80%. CMA on maternal white blood cells showed a nonmosaic male karyotype, while the oral mucosa showed a nonmosaic female karyotype. The proportion of donor's cfDNA in postnatal plasma was 94.73% based on the Y-chromosome reads ratio. The composition of cfDNA in maternal plasma was estimated as follows: prenatally, 13.60% maternal, 65.80% donor, and 20.60% fetal/placental, whereas postnatally, 5.27% maternal and 94.73% donor. CONCLUSIONS: This study expanded our understanding of the influence of stem cell transplantation on NIPS, allowing us to optimize NIPS management for these women.

Exploring the Role of Cell-Free Nucleic Acids and Peritoneal Dialysis: A Narrative Review.

INTRODUCTION: Cell-free nucleic acids (cf-NAs) represent a promising biomarker of various pathological and physiological conditions. Since its discovery in 1948, cf-NAs gained prognostic value in oncology, immunology, and other relevant fields. In peritoneal dialysis (PD), blood purification is performed by exposing the peritoneal membrane. Relevant sections: Complications of PD such as acute peritonitis and peritoneal membrane aging are often critical in PD patient management. In this review, we focused on bacterial DNA, cell-free DNA, mitochondrial DNA (mtDNA), microRNA (miRNA), and their potential uses as biomarkers for monitoring PD and its complications. For instance, the isolation of bacterial DNA in early acute peritonitis allows bacterial identification and subsequent therapy implementation. Cell-free DNA in peritoneal dialysis effluent (PDE) represents a marker of stress of the peritoneal membrane in both acute and chronic PD complications. Moreover, miRNA are promising hallmarks of peritoneal membrane remodeling and aging, even before its manifestation. In this scenario, with multiple cytokines involved, mtDNA could be considered equally meaningful to determine tissue inflammation. CONCLUSIONS: This review explores the relevance of cf-NAs in PD, demonstrating its promising role for both diagnosis and treatment. Further studies are necessary to implement the use of cf-NAs in PD clinical practice.

Prenatal Genome-Wide Cell-Free DNA Screening: Three Years of Clinical Experience in a Hospital Prenatal Diagnostic Unit in Spain.

Genome-wide prenatal cell-free DNA (cfDNA) screening can be used to screen for a wide range of fetal chromosomal anomalies in pregnant patients. In this study, we describe our clinical experience with a genome-wide cfDNA assay in screening for common trisomies, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RAAs), and copy-number variations (CNVs) in about 6000 patients over a three-year period at our hospital's Prenatal Diagnostic Unit in Spain. Overall, 204 (3.3%) patients had a high-risk call, which included 76 trisomy 21, 21 trisomy 18, 7 trisomy 13, 29 SCAs, 31 RAAs, 31 CNVs, and 9 cases with multiple anomalies. The diagnostic outcomes were obtained for the high-risk cases when available, allowing for the calculation of positive predictive values (PPVs). Calculated PPVs were 95.9% for trisomy 21, 77.8% for trisomy 18, 66.7% for trisomy 13, 10.7% for RAAs, and 10.7% for CNVs. Pregnancy and birth outcomes were also collected for the majority of RAA and CNV cases. Adverse perinatal outcomes for some of these cases included preeclampsia, fetal growth restriction, preterm birth, reduced birth weight, and major congenital structural abnormalities. In conclusion, our study showed strong performance for genome-wide cfDNA screening in a large cohort of pregnancy patients in Spain.

The impact of confined placental mosaicism on prenatal cell-free DNA screening: Insights from a monocentric study of 99 cases.

INTRODUCTION: Confined placental mosaicism (CPM) is thought to be one of the main sources of false-positive prenatal cell-free DNA (cfDNA) screening results, but extensive and systematic studies to prove this statement are limited. We evaluate the contribution of CPM to false-positive prenatal cfDNA screening results in the largest cohort published to date. METHOD: We systematically offered postnatal analysis on placenta and umbilical cord to women who had a negative amniocentesis following a positive prenatal cfDNA screening result. A standardized protocol was used in which (when available) biopsies were taken at five locations in the placenta and umbilical cord. RESULTS: We analyzed a series of 99 placentas. CPM could be confirmed in 32.3 % of cases (32/99). CPM was detected across all subtypes of chromosomal aberrations (common and rare autosomal trisomies, sex chromosome abnormalities, copy number variations and autosomal monosomies). A lower detection rate was present in umbilical cord biopsies in comparison with placental biopsies. When comparing different sections of the placenta, no clear difference could be observed with regard to the probability of CPM being present nor to the grade of mosaicism. DISCUSSION: We confirm an important role for CPM in explaining false-positive prenatal cfDNA screening results. Placental regional differences are common. Given its limited clinical relevance, we do however not advocate placental studies in a diagnostic setting.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

PURPOSE: Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. METHODS: CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. RESULTS: Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200 µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. CONCLUSIONS: Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Ovarian Cancer Diagnosis and Prognosis Based on Cell-Free DNA Methylation.

Background: Ovarian cancer stands as the deadliest malignant tumor within the female reproductive tract. As a result of the absence of effective diagnostic and monitoring markers, 75% of ovarian cancer cases are diagnosed at a late stage, leading to a mere 50% survival rate within five years. The advancement of molecular biology is essential for accurate diagnosis and treatment of ovarian cancer. Methods: A review of several randomized clinical trials, focusing on the ovarian cancer, was undertaken. The advancement of molecular biology and diagnostic methods related to accurate diagnosis and treatment of ovarian cancer were examined. Results: Liquid biopsy is an innovative method of detecting malignant tumors that has gained increasing attention over the past few years. Cell-free DNA assay-based liquid biopsies show potential in delineating tumor status heterogeneity and tracking tumor recurrence. DNA methylation influences a multitude of biological functions and diseases, especially during the initial phases of cancer. The cell-free DNA methylation profiling system has emerged as a sensitive and non-invasive technique for identifying and detecting the biological origins of cancer. It holds promise as a biomarker, enabling early screening, recurrence monitoring, and prognostic evaluation of cancer. Conclusions: This review evaluates recent advancements and challenges associated with cell-free DNA methylation analysis for the diagnosis, prognosis monitoring, and assessment of therapeutic responses in the management of ovarian cancers, aiming to offer guidance for precise diagnosis and treatment of this disease.

Ovarian cancer stands as the deadliest malignant tumor within the female reproductive tract. As a result of the absence of effective diagnostic and monitoring markers, 75% of ovarian cancer cases are diagnosed at a late stage, leading to a mere 50% survival rate within five years. Nearly 80% of advanced stages have a poor prognosis or recurrence within five years. Ovarian cancer is linked to a grim long-term prognosis attributable to its elevated mortality and recurrence rates. The advancement of molecular biology and diagnostic methods is essential for accurate diagnosis and treatment of ovarian cancer. Liquid biopsy is an innovative method of detecting malignant tumors that has gained increasing attention over the past few years. Cell-free DNA assay-based liquid biopsies show potential in delineating tumor status heterogeneity and tracking tumor recurrence. DNA methylation represents a prevalent epigenetic modification. DNA methylation influences a multitude of biological functions and diseases, especially during the initial phases of cancer. The cell-free DNA methylation profiling system has emerged as a sensitive and non-invasive technique for identifying and detecting the biological origins of cancer. This review assesses recent progress and obstacles linked to cell-free DNA methylation analysis for diagnosing, prognostic monitoring, and evaluating therapeutic responses in managing ovarian cancers.

DNA Sizing with pg/µL Sensitivity for Cell-Free DNA Analysis with the µLAS Technology.

The µLAS technology enables in-line DNA concentration and separation in a microchannel. Here, we describe its operation to analyze the size profile of cell-free DNA (cfDNA) extracted from blood plasma. Operated on commercial systems for capillary electrophoresis, we provide the size distribution of healthy individuals or patients using an input of 10 µL.

On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples.

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).

Exploring mitochondrial DNA copy number in circulating cell-free DNA and extracellular vesicles across cardiovascular health status: A prospective case-control pilot study.

Cardiovascular disease (CVD) is a leading global cause of mortality, difficult to predict in advance. Evidence indicates that the copy number of mitochondrial DNA (mtDNAcn) in blood is altered in individuals with CVD. MtDNA released into circulation may act as a mediator of inflammation, a recognized factor in the development of CVD, in the long distance. This pilot study aims to test if levels of mtDNAcn in buffy coat DNA (BC-mtDNA), in circulating cellfree DNA (cf-mtDNA), or in DNA extracted from plasma extracellular vesicles (EV-mtDNA) are altered in CVD patients and if they can predict heart attack in advance. A group of 144 people with different CVD statuses (50 that had CVD, 94 healthy) was selected from the LifeLines Biobank according to the incidence of new cardiovascular event monitored in 6 years (50 among controls had heart attack after the basal assessment). MtDNAcn was quantified in total cf-DNA and EV-DNA from plasma as well as in buffy coat. EVs have been characterized by their size, polydispersity index, count rate, and zeta potential, by Dynamic Light Scattering. BC-mtDNAcn and cf-mtDNAcn were not different between CVD patients and healthy subjects. EVs carried higher mtDNAcn in subject with a previous history of CVD than controls, also adjusting the analysis for the EVs derived count rate. Despite mtDNAcn was not able to predict CVD in advance, the detection of increased EV-mtDNAcn in CVD patients in this pilot study suggests the need for further investigations to determine its pathophysiological role in inflammation.