LINE-1 cfDNA Methylation as an Emerging Biomarker in Solid Cancers.

Epigenetic dysregulation is a hallmark of many human malignancies, with DNA methylation being a primary mechanism influencing gene expression and maintaining genomic stability. Genome-wide hypomethylation, characteristic of many cancers, is partly attributed to the demethylation of repetitive elements, including LINE-1, a prevalent non-LTR retrotransposon. The methylation status of LINE-1 is closely associated with overall genomic methylation levels in tumors.

The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization.

Liquid biopsies, in particular the profiling of circulating tumor DNA (ctDNA), have long held promise as transformative tools in cancer precision medicine. Despite a prolonged incubation phase, ctDNA profiling has recently experienced a strong wave of development and innovation, indicating its imminent integration into the cancer management toolbox. Various advancements in mutation-based ctDNA analysis methodologies and technologies have greatly improved sensitivity and specificity of ctDNA assays, such as optimized preanalytics, size-based pre-enrichment strategies, targeted sequencing, enhanced library preparation methods, sequencing error suppression, integrated bioinformatics and machine learning.

A pocket companion to cell-free DNA (cfDNA) preanalytics.

The cumulative pool of cell-free DNA (cfDNA) molecules within bodily fluids represents a highly dense and multidimensional information repository. This "biological mirror" provides real-time insights into the composition, function, and dynamics of the diverse genomes within the body, enabling significant advancements in personalized molecular medicine. However, effective use of this information necessitates meticulous classification of distinct cfDNA subtypes with exceptional precision.

Cell-Free Nucleic Acids: Physico-Chemical Properties, Analytical Considerations, and Clinical Applications.

Human body fluids are rich sources of cell-free nuclear material, which exhibits unique characteristics [...

Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology.

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard KEDTA tubes, but mainly have been tested in healthy individuals.

The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients.

Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests.

Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods.

Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts-among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods.

New Perspectives on the Importance of Cell-Free DNA Biology.

Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms.

Preanalytical Variables in the Analysis of Mitochondrial DNA in Whole Blood and Plasma from Pancreatic Cancer Patients.

Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis.

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line.

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.

Tracing the Origin of Cell-Free DNA Molecules through Tissue-Specific Epigenetic Signatures.

All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease onset or pathological conditions that lead to increased cell death alter the contribution of different tissues to the total pool of cfDNA.

The Utility of Repetitive Cell-Free DNA in Cancer Liquid Biopsies.

Liquid biopsy is a broad term that refers to the testing of body fluids for biomarkers that correlate with a pathological condition. While a variety of body-fluid components (e.g.

Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures.

Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted.

Histone Modifications in Stem Cell Development and Their Clinical Implications.

Human stem cells bear a great potential for multiple therapeutic applications but at the same time constitute a major threat to human health in the form of cancer stem cells. The molecular processes that govern stem cell maintenance or differentiation have been extensively studied in model organisms or cell culture, but it has been difficult to extrapolate these insights to therapeutic applications. Recent advances in the field suggest that local and global changes in histone modifications that affect chromatin structure could influence the capability of cells to either maintain their stem cell identity or differentiate into specialized cell types.

Putative Origins of Cell-Free DNA in Humans: A Review of Active and Passive Nucleic Acid Release Mechanisms.

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules.

Towards systematic nomenclature for cell-free DNA.

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality.

Preanalytical variables that affect the outcome of cell-free DNA measurements.

Fragments of cell-free DNA (cfDNA) in human body fluids often carry disease-specific alterations and are now widely recognized as ideal biomarkers for the detection and monitoring of genomic disorders, especially cancer, that are normally difficult to examine noninvasively. However, the conversion of promising research findings into tools useful in routine clinical testing of cancer has been a slow-moving process. A major reason is that the diagnostic sensitivity and specificity of cfDNA-based clinical assays are negatively impacted by a combination of suboptimal and inter-institutional differences in preanalytical procedures.

Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.

In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy of the method used for DNA purification, and is often a juncture of experimental confusion. Therefore, in this study, we compared six commercially available cell-free DNA isolation kits for the recovery of cell-free DNA from the cell culture supernatant of a human bone cancer cell line (143B), including two magnetic bead-based manual kits, one automated magnetic bead-based extraction method, and three manual spin-column kits.

Harmonizing Cell-Free DNA Collection and Processing Practices through Evidence-Based Guidance.

Circulating cell-free DNA (cfDNA) is rapidly transitioning from discovery research to an important tool in clinical decision making. However, the lack of harmonization of preanalytic practices across institutions may compromise the reproducibility of cfDNA-derived data and hamper advancements in cfDNA testing in the clinic. Differences in cellular genomic contamination, cfDNA yield, integrity, and fragment length have been attributed to different collection tube types and anticoagulants, processing delays and temperatures, tube agitation, centrifugation protocols and speeds, plasma storage duration and temperature, the number of freeze-thaw events, and cfDNA extraction and quantification methods, all of which can also ultimately impact subsequent downstream analysis.

Early detection of cancer using circulating tumor DNA: biological, physiological and analytical considerations.

Early diagnosis of cancer improves the efficacy of curative therapies. However, due to the difficulties involved in distinguishing between small early-stage tumors and normal biological variation, early detection of cancer is an extremely challenging task and there are currently no clinically validated biomarkers for a pan-cancer screening test. It is thus of particular significance that increasing evidence indicates the potential of circulating tumor DNA (ctDNA) molecules, which are fragmented segments of DNA shed from tumor cells into adjacent body fluids and the circulatory system, to serve as molecular markers for early cancer detection and thereby allow early intervention and improvement of therapeutic and survival outcomes.

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant.

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats.

The emerging role of cell-free DNA as a molecular marker for cancer management.

An increasing number of studies demonstrate the potential use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. However, harnessing the full potential of cfDNA requires (i) the optimization and standardization of preanalytical steps, (ii) refinement of current analysis strategies, and, perhaps most importantly, (iii) significant improvements in our understanding of its origin, physical properties, and dynamics in circulation. The latter knowledge is crucial for interpreting the associations between changes in the baseline characteristics of cfDNA and the clinical manifestations of cancer.

Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing.

The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature.

Since the detection of cell-free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non-invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle-induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno- or gene therapy.