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 K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.

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. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications.

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. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research.

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. Because cfDNA molecules retain cell-type specific epigenetic features, it is possible to infer tissue-of-origin from epigenetic characteristics. Recent research efforts demonstrated that analysis of, e.g., methylation patterns, nucleosome occupancy, and fragmentomics determined the cell- or tissue-of-origin of individual cfDNA molecules. This novel tissue-of origin-analysis enables to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies towards a wide range of pathologies and early diagnosis. In this review, we summarize the currently available tissue-of-origin approaches and point out the next steps towards clinical implementation.

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.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1-3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing.

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. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.

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. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids.

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. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA.

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. The most prominent factors include: (i) a poor understanding of the biological factors that determine the characteristics of the cfDNA population in a biospecimen prior to collection, (ii) inattention to how cfDNA with different structures and physical properties are affected differently by a given preanalytical step, and (iii) the sheer number of possible conditions that can be selected from for each preanalytical step along with a continually expanding menu of commercial products that often show varying degrees of bias and efficiency. The convergence of these variables makes it difficult for research groups and institutions to reach a consensus on optimal preanalytical procedures and a challenging task to establish widely applied standards, which ultimately hamper the development of cfDNA assays that are fit for broad clinical implementation. In this review, we follow a systematic approach to explore the most confounding preanalytical factors that affect the outcome of cfDNA measurements.

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. Based on cell-free DNA quantitation and sizing, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods showed significant variability concerning recovery, reproducibility, and size discrimination. These findings highlight the importance of selecting a cell-free DNA extraction method that is appropriate for the aims of a study. For example, mutational analysis of cell-free DNA may be enhanced by a method that favors a high yield or is biased toward the isolation of short cell-free DNA fragments. In contrast, quantitative analysis of cell-free DNA in a comparative setting (e.g. measuring the fluctuation of cell-free DNA levels over time) may require the selection of a cell-free DNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.

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. This is possible because ctDNA molecules bear cancer-specific fragmentation patterns, nucleosome depletion motifs, and genetic and epigenetic alterations, as distinct from plasma DNA originating from non-cancerous tissues/cells. Compared to traditional biomarkers, ctDNA analysis therefore presents the distinctive advantage of detecting tumor-specific alterations. However, based on a thorough survey of the literature, theoretical and empirical evidence suggests that current ctDNA analysis strategies, which are mainly based on DNA mutation detection, do not demonstrate the necessary diagnostic sensitivity and specificity that is required for broad clinical implementation in a screening context. Therefore, in this review we explain the biological, physiological, and analytical challenges toward the development of clinically meaningful ctDNA tests. In addition, we explore some approaches that can be implemented in order to increase the sensitivity and specificity of ctDNA assays.

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. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

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. In this review we explore recent advancements and highlight the current gaps in our knowledge concerning each point of contact between cfDNA analysis and the different stages of cancer management.

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. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

New insights into the catalytic mechanism of human glycine N-acyltransferase.

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism.