Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients.

Objectives: Elevated circulating DNA (cirDNA) concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release. We carried out the first prospective, multicenter study of the dynamics of cirDNA and neutrophil extracellular trap (NETs) markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients. Methods: We examined the plasma levels of two NETs protein markers [myeloperoxidase (MPO) and neutrophil elastase (NE)], as well as levels of cirDNA of nuclear (cir-nDNA) and mitochondrial (cir-mtDNA) origin in 29 colon, prostate, and breast cancer patients and in 114 healthy individuals (HI). Results: The synergistic analytical information provided by these markers revealed that: (i) NETs formation contributes to post-surgery conditions; (ii) post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer [r = 0.60 (P < 0.001) and r = 0.53 (P < 0.01), respectively], but not in prostate and breast cancer; (iii) each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics, but overall the pre- and post-surgery median values of cir-nDNA, NE, and MPO were significantly higher in cancer patients than in HI. Conclusion: Taken as a whole, our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period, depending on surgical procedure or cancer type. By contrast, cir-mtDNA is poorly associated with NETs formation in the studied perioperative period, which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

Corrigendum: What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

[This corrects the article DOI: 10.3389/fmed.2023.1268748.].

Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.

BACKGROUND: Research on circulating mitochondrial DNA (cir-mtDNA) based diagnostic is insufficient, as to its function, origin, structural features, and particularly its standardization of isolation. To date, plasma preparation performed in previous studies do not take into consideration the potential bias resulting from the release of mitochondria by activated platelets. METHODS: To tackle this, we compared the mtDNA amount determined by a standard plasma preparation method or a method optimally avoiding platelet activation. MtDNA extracted from the plasma of seven healthy individuals was quantified by Q-PCR in the course of the process of both methods submitted to filtration, freezing or differential centrifugation. RESULTS: 98.7 to 99.4% of plasma mtDNA corresponded to extracellular mitochondria, either free or into large extracellular vesicles. Without platelet activation, the proportion of both types of entities remained preponderant (76-80%), but the amount of detected mtDNA decreased 67-fold. CONCLUSION: We show the high capacity of platelets to release free mitochondria in "in vitro" conditions. This represents a potent confounding factor when extracting mtDNA for cir-mtDNA investigation. Platelet activation during pre-analytical conditions should therefore be avoided when studying cir-mtDNA. Our findings lead to a profound revision of the assumptions previously made by most works in this field. Overall, our data suggest the need to characterize or isolate mtDNA associated various structural forms, as well as to standardize plasma preparation, to better circumscribe cir-mtDNA's diagnostic capacity.

What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

Despite significant progress in dialysis modalities, intermittent renal replacement therapy remains an "unphysiological" treatment that imperfectly corrects uremic disorders and may lead to low-grade chronic inflammation, neutrophil activation, and oxidative stress due to repetitive blood/membrane interactions contributing to the "remaining uremic syndrome" and cardiovascular disease burden of hemodialysis patients. Understanding dialysis bioincompatibility pathways still remains a clinical and biochemical challenge. Indeed, surrogate biomarkers of inflammation including C-reactive protein could not discriminate between all components involved in these complex pathways. A few examples may serve to illustrate the case. Cytokine release during dialysis sessions may be underestimated due to their removal using high-flux dialysis or hemodiafiltration modalities. Complement activation is recognized as a key event of bioincompatibility. However, it appears as an early and transient event with anaphylatoxin level normalization at the end of the dialysis session. Complement activation is generally assumed to trigger leukocyte stimulation leading to proinflammatory mediators' secretion and oxidative burst. In addition to being part of the innate immune response involved in eliminating physically and enzymatically microbes, the formation of Neutrophil Extracellular Traps (NETs), known as NETosis, has been recently identified as a major harmful component in a wide range of pathologies associated with inflammatory processes. NETs result from the neutrophil degranulation induced by reactive oxygen species overproduction via NADPH oxidase and consist of modified chromatin decorated with serine proteases, elastase, bactericidal proteins, and myeloperoxidase (MPO) that produces hypochlorite anion. Currently, NETosis remains poorly investigated as a sensitive and integrated marker of bioincompatibility in dialysis. Only scarce data could be found in the literature. Oxidative burst and NADPH oxidase activation are well-known events in the bioincompatibility phenomenon. NET byproducts such as elastase, MPO, and circulating DNA have been reported to be increased in dialysis patients more specifically during dialysis sessions, and were identified as predictors of poor outcomes. As NETs and MPO could be taken up by endothelium, NETs could be considered as a vascular memory of intermittent bioincompatibility phenomenon. In this working hypothesis article, we summarized the puzzle pieces showing the involvement of NET formation during hemodialysis and postulated that NETosis may act as a disease modifier and may contribute to the comorbid burden associated with dialysis bioincompatibility.

A New Paradigm of the Origins of Circulating DNA in Patients with Cancer.

SUMMARY: By shedding light on the cellular origins of circulating DNA (cirDNA), this research provides important insights into the mechanisms of cirDNA production in cancer. Contrary to expectations, the increased cirDNA in patients with cancer was not derived predominantly from neoplastic cells or surrounding nonneoplastic epithelial cells; rather, the excess cirDNA originated primarily from leukocytes, implying a systemic impact of cancer on cell turnover or DNA clearance. See related article by Mattox et al., p. 2166 (1).

Circulating DNA reveals a specific and higher fragmentation of the Y chromosome.

Chromosome stability is a key point in genome evolution, particularly that of the Y chromosome. The Y chromosome loss in blood and tumor cells is well established. Through processes that are common to other chromosomes too, the Y chromosome undergoes degradation and fragmentation in the blood stream before elimination. This process gives rise to circulating DNA (cirDNA) fragments, whose examination may provide potential insight into the role of DNA fragmentation in blood for the Y chromosome elimination. In this study, we employed shallow whole genome sequencing (sWGS) to comprehensively assess the total cirDNA and the individual chromosome fragment size profiles in the plasma of healthy male individuals. Here, we show that (i) the fragment size profiles of total circulating DNA (cirDNA) and DNA fragments originating from autosomes and the X chromosome in blood plasma are homogeneous, and have a remarkably low variability (mean CV = 7%) among healthy individuals, (ii) the Y chromosome has a distinct fragment size profile with the accumulation of the fragment < 145 bp and depletion of the dinucleosome-associated fragments (290-390 bp), and its fragment fraction in blood decreases with age. These results indicate a higher fragmentation of the Y chromosome compared to other chromosomes and this in turn might be due to its increased susceptibility to degradation. Our findings pave the way for an elucidation of the impact of chromosomal origin on DNA degradation and the Y chromosome biology.

Mitochondria-derived cell-to-cell communication.

In addition to their intracellular mobility, mitochondria and their components can exist outside the cells from which they originate. As a result, they are capable of acting on non-parental distant cells and mediate intercellular communication in physiological conditions and in a variety of pathologies. It has recently been demonstrated that this horizontal transfer governs a wide range of biological processes, such as tissue homeostasis, the rescue of injured recipient cells, and tumorigenesis. In addition, due to mitochondria's bacterial ancestry, they and their components can be recognized as damage-associated molecular patterns (DAMPs) by the immune cells, leading to inflammation. Here, we provide an overview of the most current and significant findings concerning the different structures of extracellular mitochondria and their by-products and their functions in the physiological and pathological context. This account illustrates the ongoing expansion of our understanding of mitochondria's biological role and functions in mammalian organisms.

Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA.

Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

A straightforward method to quantify circulating mRNAs as biomarkers of colorectal cancer.

Optimizing the biomarker combination to be analyzed in liquid biopsies should improve personalized medicine. We developed a method to purify circulating cell-free mRNAs from plasma samples and to quantify them by RT-qPCR. We selected three candidate colorectal cancer biomarkers (B2M, TIMP-1, and CLU). Their mRNA levels were significantly higher in plasma of patients with metastatic colorectal cancer patients (mCRC) (n = 107) than in healthy individuals (HI) (n = 53). To increase the discriminating performance of our method, we analyzed the sum of the three mRNA levels (BTC index). The area under the ROC curve (AUC) to estimate the BTC index capacity to discriminate between mCRC and HI plasma was 0.903. We also determined the optimal BTC index cut-off to distinguish between plasma samples, with 82% of sensitivity and 93% of specificity. By using mRNA as a novel liquid biopsy analytical parameter, our method has the potential to facilitate rapid screening of CRCm.

Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients.

In the early phase of the pandemic, we were among the first to postulate that neutrophil extracellular traps (NETs) play a key role in COVID-19 pathogenesis. This exploratory prospective study based on 279 individuals showed that plasma levels of neutrophil elastase, myeloperoxidase and circulating DNA of nuclear and mitochondrial origins in nonsevere (NS), severe (S) and postacute phase (PAP) COVID-19 patients were statistically different as compared to the levels in healthy individuals, and revealed the high diagnostic power of these NETs markers in respect to the disease severity. The diagnostic power of NE, MPO, and cir-nDNA as determined by the Area Under Receiver Operating Curves (AUROC) was 0.95, 097, and 0.64; 0.99, 1.0, and 0.82; and 0.94, 1.0, and 0.93, in NS, S, and PAP patient subgroups, respectively. In addition, a significant fraction of NS, S as well as of PAP patients exhibited aCL IgM/IgG and anti-B2GP IgM/IgG positivity. We first demonstrate persistence of these NETs markers in PAP patients and consequently of sustained innate immune response imbalance, and a prolonged low-level pro-thrombotic potential activity highlighting the need to monitor these markers in all COVID-19 PAP individuals, to investigate postacute COVID-19 pathogenesis following intensive care, and to better identify which medical resources will ensure complete patient recovery.

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA.

BACKGROUND: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. METHODS: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). RESULTS: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. CONCLUSIONS: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer.

We postulate that a significant part of circulating DNA (cirDNA) originates in the degradation of neutrophil extracellular traps (NETs). In this study, we examined the plasma level of two markers of NETs (myeloperoxidase (MPO) and neutrophil elastase (NE)), as well as cirDNA levels in 219 patients with a metastatic colorectal cancer (mCRC), and in 114 healthy individuals (HI). We found that in patients with mCRC the content of these analytes was (i) highly correlated, and (ii) all statistically different (p < 0.0001) than in HI (N = 114). These three NETs markers may readily distinguish between patients with mCRC from HI, (0.88, 0.86, 0.84, and 0.95 AUC values for NE, MPO, cirDNA, and NE + MPO + cirDNA, respectively). Concomitant analysis of anti-phospholipid (anti-cardiolipin), NE, MPO, and cirDNA plasma concentrations in patients with mCRC might have value for thrombosis prevention, and suggested that NETosis may be a critical factor in the immunological response/phenomena linked to tumor progression.

SARS-CoV-2 and Implantation Window: Gene Expression Mapping of Human Endometrium and Preimplantation Embryo.

Understanding whether SARS-CoV-2 could infect cells and tissues handled during ART is crucial for risk mitigation, especially during the implantation window when either endometrial biopsies are often practiced for endometrial receptivity assessment or embryo transfer is performed. To address this question, this review analyzed current knowledge of the field and retrospectively examined the gene expression profiles of SARS-CoV-2-associated receptors and proteases in a cohort of ART candidates using our previous Affymetrix microarray data. Human endometrial tissue under natural and controlled ovarian stimulation cycles and preimplantation embryos were analyzed. A focus was particularly drawn on the renin-angiotensin system, which plays a prominent role in the virus infection, and we compared the gene expression levels of receptors and proteases related to SARS-CoV-2 infection in the samples. High prevalence of genes related to the ACE2 pathway during both cycle phases and mainly during the mid-secretory phase for ACE2 were reported. The impact of COS protocols on endometrial gene expression profile of SARS-CoV-2-associated receptors and proteases is minimal, suggesting no additional potential risks during stimulated ART procedure. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level. Specimens from female genital tract should be considered as potential targets for SARS-CoV-2, especially during the implantation window.

Guidelines for pre-analytical conditions for assessing the methylation of circulating cell-free DNA.

Methylation analysis of circulating cell-free DNA (cirDNA), as a liquid biopsy, has a significant potential to advance the detection, prognosis, and treatment of cancer, as well as many genetic disorders. The role of epigenetics in disease development has been reported in several hereditary disorders, and epigenetic modifications are regarded as one of the earliest and most significant genomic aberrations that arise during carcinogenesis. Liquid biopsy can be employed for the detection of these epigenetic biomarkers. It consists of isolation (pre-analytical) and detection (analytical) phases. The choice of pre-analytical variables comprising cirDNA extraction and bisulfite conversion methods can affect the identification of cirDNA methylation. Indeed, different techniques give a different return of cirDNA, which confirms the importance of pre-analytical procedures in clinical diagnostics. Although novel techniques have been developed for the simplification of methylation analysis, the process remains complex, as the steps of DNA extraction, bisulfite treatment, and methylation detection are each carried out separately. Recent studies have noted the absence of any standard method for the pre-analytical processing of methylated cirDNA. We have therefore conducted a comprehensive and systematic review of the important pre-analytical and analytical variables and the patient-related factors which form the basis of our guidelines for analyzing methylated cirDNA in liquid biopsy.

Association of COVID-19 Lockdown With the Tumor Burden in Patients With Newly Diagnosed Metastatic Colorectal Cancer.

Importance: The COVID-19 pandemic has been associated with substantial reduction in screening, case identification, and hospital referrals among patients with cancer. However, no study has quantitatively examined the implications of this correlation for cancer patient management. Objective: To evaluate the association of the COVID-19 pandemic lockdown with the tumor burden of patients who were diagnosed with metastatic colorectal cancer (mCRC) before vs after lockdown. Design, Setting, and Participants: This cohort study analyzed participants in the screening procedure of the PANIRINOX (Phase II Randomized Study Comparing FOLFIRINOX + Panitumumab vs FOLFOX + Panitumumab in Metastatic Colorectal Cancer Patients Stratified by RAS Status from Circulating DNA Analysis) phase 2 randomized clinical trial. These newly diagnosed patients received care at 1 of 18 different clinical centers in France and were recruited before or after the lockdown was enacted in France in the spring of 2020. Patients underwent a blood-sampling screening procedure to identify their RAS and BRAF tumor status. Exposures: mCRC. Main Outcomes and Measures: Circulating tumor DNA (ctDNA) analysis was used to identify RAS and BRAF status. Tumor burden was evaluated by the total plasma ctDNA concentration. The median ctDNA concentration was compared in patients who underwent screening before (November 11, 2019, to March 9, 2020) vs after (May 14 to September 3, 2020) lockdown and in patients who were included from the start of the PANIRINOX study. Results: A total of 80 patients were included, of whom 40 underwent screening before and 40 others underwent screening after the first COVID-19 lockdown in France. These patients included 48 men (60.0%) and 32 women (40.0%) and had a median (range) age of 62 (37-77) years. The median ctDNA concentration was statistically higher in patients who were newly diagnosed after lockdown compared with those who were diagnosed before lockdown (119.2 ng/mL vs 17.3 ng/mL; P < .001). Patients with mCRC and high ctDNA concentration had lower median survival compared with those with lower concentration (14.7 [95% CI, 8.8-18.0] months vs 20.0 [95% CI, 14.1-32.0] months). This finding points to the potential adverse consequences of the COVID-19 pandemic and related lockdown. Conclusions and Relevance: This cohort study found that tumor burden differed between patients who received an mCRC diagnosis before vs after the first COVID-19 lockdown in France. The findings of this study suggest that CRC is a major area for intervention to minimize pandemic-associated delays in screening, diagnosis, and treatment.

Monitoring levels of circulating cell-free DNA in patients with metastatic colorectal cancer as a potential biomarker of responses to regorafenib treatment.

Circulating cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA), which can be obtained from serial liquid biopsies to enable tumor genome analysis throughout the course of treatment. We investigated cfDNA and mutant ctDNA as potential biomarkers to predict the best outcomes of regorafenib-treated metastatic colorectal cancer (mCRC) patients. We analyzed longitudinally collected plasma cfDNA of 43 mCRC patients prospectively enrolled in the phase II TEXCAN trial by IntPlex qPCR. Qualitative (KRAS, NRAS, BRAFV600E mutations) and quantitative (total cfDNA concentration, mutant ctDNA concentration, mutant ctDNA fraction) parameters were correlated with overall survival (OS) and progression-free survival (PFS). When examined as classes or continuous variables, the concentrations of total cfDNA, mutant ctDNA, and, partly, mutant ctDNA fraction prior to regorafenib treatment correlated with OS. Patients with baseline cfDNA > 26 ng·mL-1 had shorter OS than those with cfDNA value below this threshold (4.0 vs 6.9 months; log-rank P = 0.0366). Patients with baseline mutant ctDNA > 2 ng·mL-1 had shorter OS than those with mutant ctDNA below this threshold (log-rank P = 0.0154). We show that pretreatment cfDNA and mutant ctDNA levels may identify mCRC patients that may benefit from regorafenib treatment.

Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics.

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

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.