Search

Method summary Concentrations of circulating DNA in blood plasma were compared using NanoDrop, Qubit, quantitative PCR and Bioanalyzer, and DNA integrity was evaluated with the Bioanalyzer according to the time of plasma preparation.

Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [+31.4%] vs 3.8% [≤+31.4%] and 50.0% [0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure.

BackgroundBecause circulating DNA (cirDNA) are 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 cirDNAMethodsWe used the synergistic analytical information provided by specifically quantifying DNA by qPCR, and analyzing fragment size analysis by shallow WGS, and capillary electrophoresis to unequivocally study the following: thein vitrokinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) byex vivoactivated neutrophils;in vitroNET degradation in serum. We also performed anin vivostudy in knockout mice, and anin vitrostudy 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).ResultsOur studies reveal that: gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24H 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 hours 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 8h 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.ConclusionsOur work thus describes the mechanisms by which NET and cirDNA are linked, by demonstrating that NET are a major source of mono-N cirDNA independent of apoptosis, and thus establishing a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions, as well as demonstrating a link between immune response and cirDNA.

BACKGROUND: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics. METHODS: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle. RESULTS: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p

We studied the levels of extracellular nuclear and mitochondrial DNA of blood serum and DNA damage in leukocytes of healthy donors of different sex and age groups. The baseline levels of DNA damage in leukocytes and serum DNA levels were shown to vary greatly among different donors. The baseline level of DNA damage in leukocytes was not associated with the presence of chronic deceases or an occupational health risk for elderly donors. It was found that extracellular DNA concentrations were generally higher in men than in women. There is a tendency towards an increase in the relative mitochondrial DNA copy number determined by ΔCt in women but not in men: the relative mtDNA copy number in elderly individuals varies significantly in both sexes, possibly due to age-related physiological changes. It is necessary to consider the gender and age of patients when using an indicator such as the level of extracellular DNA of blood serum for diagnosis and monitoring.

Biomarkers based on DNA methylation have attracted wide attention in biomedical research due to their potential clinical value. Therefore, a sensitive and accurate method for DNA methylation detection is highly desirable for the discovery and diagnostics of human diseases, especially cancers. Here, an integrated, low-cost, and portable point-of-care (POC) device is presented to analyze DNA methylation, which integrates the process of pyrosequencing in a digital microfluidic chip. Without additional equipment and complicated operation, droplets are manipulated by patterned electrodes with individually programmed control. The system exhibited an excellent sensitivity with a limit of detection (LOD) of 10 pg and a comparable checkout down to 5% methylation level within 30 min, which offered a potential substitute for the detection of DNA methylation. With the advantages of portability, ease of use, high accuracy, and low cost, the POC platform shows great potential for the analysis of tumor-specific circulating DNA.

Purpose The RadTox assay measures circulating cell-free DNA released in response to radiotherapy (RT)-induced tissue damage. The primary objectives for this clinical trial were to determine whether cell-free DNA numbers measured by the RadTox assay are (1) correlated with body integral dose, (2) lower with proton RT compared with photon RT, and (3) higher with larger prostate cancer RT fields. Patients and Methods Patients planned to receive proton or photon RT for nonmetastatic prostate cancer in the setting of an intact prostate or postprostatectomy were eligible for the trial. Plasma was collected pre-RT and at 5 additional daily collection points beginning 24 hours after the initiation of RT. Data from 54 evaluable patients were analyzed to examine any correlations among RadTox scores with body-integral dose, RT modality (photon versus proton), and RT field size (prostate or prostate bed versus whole pelvis). Results Body integral dose was significantly associated with the peak post-RT RadTox score (P = .04). Patients who received photon RT had a significant increase in peak post-RT RadTox score (P = .04), average post-RT RadTox score (P = .04), and day-2 RadTox score (all minus the pre-RT values for each patient) as compared with patients who received proton RT. Field size was not significantly associated with RadTox score. Conclusion RadTox is correlated with body integral dose and correctly predicts which patients receive proton versus photon RT. Data collection remains ongoing for patient-reported RT toxicity outcomes to determine whether RadTox scores are correlated with toxicity.

Circulating tumor DNA (ctDNA), carrying tumor-specific sequence mutations, is a promising biomarker for classification, diagnosis and prognosis of cancers. However, there is still a great challenge in discriminating single-base difference between ctDNA and its coexisting analogue (normal circulating DNA, ncDNA) at a serum sample. A locked nucleic acid (LNA) probe combined with α-HL nanopore sensor was designed, which achieved a high signal-to-background ratio (SBR) of ∼8.34 × 103, as well as a significant discrimination capability (∼12.3 times) of single-base difference. The accurate discrimination strategy is label-free, convenient, selective and sensitive, which has great potential in the early diagnosis of diseases and biomedical research fields.

Background Cell-free DNA analysis (cfDNA) holds promise for residual disease or tumor burden quantification in colorectal cancer, with reduced costs and diagnostic equipment compared to gold standard-specific tumor DNA (ctDNA) analysis. Methods This prospective case-control study included 46 colorectal cancer patients and healthy controls to perform cfDNA quantification by fluorometry using Quantus Fluorometer (Promega, Madison, WI) and using cell-free DNA ScreenTape assay (Agilent) and 4200 TapeStation instrument (Agilent Technologies, Inc., Santa Clara, CA, USA). cfDNA quantification results were correlated with stage, clinical and histopathological features. Results 33 localized (8 stage I, 12 stage II, and 13 stage III) and 13 advanced colorectal cancer patients were included. No differences in cfDNA quantification by fluorometry were demonstrated depending on stage or histopathological features in localized disease patients. Differences in cfDNA quantification by fluorometry could be demonstrated in patients with advanced disease depending on the presence of liver metastases and synchronous or metachronous metastatic disease. Differences in cfDNA quantification by fluorometry could be demonstrated between advanced colorectal cancer patients and both localized disease patients and healthy controls. Secondary cfDNA analysis by electrophoresis, although showing more specificity to measure ctDNA in cfDNA values, could not improve the capacity to detect differences between analyzed a groups beyond previously achieved with fluorometry. Conclusion This exploratory analysis of cfDNA based on fluorometry and electrophoresis methods showed promising results discriminating colorectal cancer and non-cancer patients, as well as different colorectal cancer stages and disease profiles. Further studies are needed to increase our knowledge and to help to overcome barriers to broader implementation and applications.

Background: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay—panHPV-detect—in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. Method: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. Results: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70–99%) and 100% (95% CI-30–100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. Conclusions: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort.

Powered by rapid technology developments, biomarkers become increasingly diverse, including those detected at genomic, transcriptomic, proteomic, metabolomic and cellular levels. While diverse sets of biomarkers have been utilized in breast cancer predisposition, diagnosis, prognosis, treatment and management, recent additions derived from lincRNA, circular RNA, circulating DNA together with its methylated and hydroxymethylated forms and immune signatures are likely to further transform clinical practice. Here, we take breast cancer as an example of heterogeneous diseases that require many informed decisions from treatment to care to review the huge variety of biomarkers. By assessing the advantages and limitations of modern biomarkers in diverse use scenarios, this article outlines the prospects and challenges of releasing complimentary advantages by augmentation of multiscale molecular biomarkers.

BACKGROUND Since the discovery of cell-free fetal DNA in maternal blood in 1997, the interplay of basic scientific observations and technological developments have continued to drive new clinical applications in the field. AIMS This commentary discusses a number of examples in this virtuous circle of science, technology and clinical applications. MATERIALS & METHODS: Commentary and literature review. RESULTS One example of technological developments is the detection technologies for detecting circulating DNA, moving from conventional PCR, to real-time PCR, to massively parallel sequencing. One example of basic scientific understanding is the size and fragmentation patterns of circulating DNA. DISCUSSION Beyond creating a global paradigm in prenatal medicine, the development of noninvasive prenatal testing has also impacted other areas such as cancer screening and transplantation monitoring. Finally, the commentary looks forward to what might be in store in the next decade.

The current study sought to explore the significance of copy number variations (CNVs) of MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived [avian]) and ALK (anaplastic lymphoma kinase) genes individually as well as their combined impact on clinical outcome and overall survival of patients with neuroblastoma (NB).A total 71 individuals including healthy controls (n = 11), circulating DNA (n = 11), and primary tumors (n = 49) were evaluated to detect CNVs of MYCN and ALK genes using droplet digital polymerase chain reaction. Data were correlated with univariate and multivariate survival analysis.CNVs of MYCN and ALK were detected in 27% and 18.2% from circulating DNA samples. A statistically significant difference in CNVs was noted between healthy controls and circulating DNA samples for MYCN (P = 0.001) and ALK (P = 0.004) genes. Further, we noted70% concordance in CNVs of MYCN (P = 0.030) and ALK (P = 0.040) from primary tumors and concordant plasma samples of patients with NB. Multivariate survival analysis for disease-free survival (P = 0.031) and overall survival (P = 0.011) showed that CNVs of both genes emerged at step 1 and thus remained as significant markers for predicting early recurrence and shorter survival, respectively, for patients with NB.Our study showed that the analysis of circulating DNA by droplet digital polymerase chain reaction is a helpful technique to identify high-risk patients for aggressive therapy at an early stage of disease. We also concluded that codetection of MYCN and ALK is a more powerful tool for identifying high-risk patients with NB. Thus, this study showed a novel coordinately significant prognostic role of MYCN and ALK CNVs.

Background: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.QTM H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. Samples were also collected longitudinally from 3 dogs undergoing treatment for multicentric lymphoma at TAMU as a pilot study to investigate the pattern of nucleosome concentrations in plasma during treatment. Results: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. Nucleosome concentrations from serially monitored patients were elevated at diagnosis and progression with subsequent decreases in nucleosome concentration that corresponded to clinically detectable responses to therapy. Conclusions: The Nu.QTM H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines. Results from serially monitored patients indicate that nucleosomes could be an objective monitoring tool for remission status in canine lymphoma patients.

While liquid biopsy is still relatively a new concept, the advent of next-generation sequencing (NGS) technologies has recently generated a revolution in the field and will be the focus of this review. Circulating tumor DNA (ctDNA) derives from tumor cells and provides information about the genetic alterations of tumors. However, ctDNA concentration in plasma can be below the level of detection by conventional methods; therefore, screening for actionable genetic information is challenging. Clinical trials exploring targeted and untargeted sequencing to improve the outcomes of ctDNA detection are showing promising results, having reached a limit of detection as low as 0.001% of ctDNA in a background of normal circulating DNA. Most of the challenges related to the sensitivity of detection of ctDNA have been defeated by dint of NGS-based approaches. Despite all the efforts, these methods are still expensive, time-consuming, and require advanced skills for appropriate interpretation. Nevertheless, the technology is rapidly improving, and the expectations for the implementation of liquid biopsy into the clinical practice in the near future are high.

Cancer is one of the leading causes of death worldwide. This life-threatening disease requires novel strategies for the early detection and therapy response prediction. Circulating DNA was first described 70 years ago. However, only the recent evolution in the PCR-based sequencing techniques allow the minimally invasive molecular profiling of circulating mutant DNA from small-volume “liquid biopsies” such as blood, urine, or saliva. In this article, we aim to summarize the fast-growing evidence for cfDNA and exosomal DNA as minimally invasive diagnostic markers in solid tumors and to highlight their opposing diagnostic advantages and disadvantages.

Cell-free DNA (cfDNA) harboring mutations has been found in patients with diseases. Experimental studies have shown that cfDNA can be transmitted, leading to transformations in the host. In the present study, we evaluated whether bone allograft material contains cfDNA and whether this foreign cfDNA can be released into the patient’s blood circulation. Plasma samples were collected preoperatively and postoperatively on the same day, at 5 weeks, and 4 months from 25 women who received bone allograft material (test group) from male donors and from 10 women who were treated with autologous graft (control group, only pre- and postoperative samples were collected). DNA was quantified and characterized in bone material and plasma samples by quantitative PCR with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Y chromosome and gel electrophoresis. DNA in bone material was digested by different concentrations of DNase I. We detected between 1 and 1.8 μg cfDNA fragments at a length around 601 base pairs (bp) and smaller in each 100 mg allograft. Treatment of the allograft with DNase I completely degraded the longer but not the shorter DNA 90-bp fragments. Y-DNA was not detected in the patients’ bloodstream at any time during the treatment and follow-up, but elevated levels of circulating cfDNA could be measured immediately postoperatively. Our results suggest that a transmission of DNA from allografts used for alveolar ridge reconstruction in humans is unlikely. The observed increase in circulating cfDNA in allograft and autograft patients immediately postoperatively may be elicited by the surgical procedure. The results support the safety of allograft materials. The results suggest that human allograft materials seem not to release DNA into the blood since we did not measure Y-DNA with our technique.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village.Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs.Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR.Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.Evaluer trois tests non invasifs pour le diagnostic de la schistosomiase mansoni dans un village égyptien. MÉTHODES: L'urine a été collectée pour la détection de l'antigène cathodique circulant (ACC) et de l’ADN du parasite libéré des cellules (cfpd) par le test en cassette POC (point-of-contact) et par PCR, respectivement. Ces tests ont été comparés au test de Kato Katz (KK) sur frottis fécal épais pour la détection des œufs de Schistosoma mansoni. RÉSULTATS: La prévalence de la maladie par dosage POC-ACC était de 86%; elle était de 39% par PCR contre 27% par KK. Par rapport à KK, la sensibilité du POC-ACC atteignait 100%, mais sa spécificité n’était que de 19,2% avec une précision de 41%. La sensibilité du PCR pour la cfpd était de 55,56% et sa spécificité de 67,12% avec une précision de 64%. Un nouveau seuil a été calculé pour l'analyse combinée des tests KK, POC-ACC et PCR. La sensibilité pour les trois tests était de 52,94%, 90,2% et 76,47%; la spécificité était de 100% pour KK et PCR et de 18,37% pour POC-ACC. La précision calculée pour les trois tests au point seuil était de 76% pour KK, 55% pour le POC-ACC et 88% pour la PCR.Le test PCR conventionnel pour la détection de la cfpd constitue un outil de dépistage potentiel de la schistosomiase intestinale avec une spécificité fiable, une précision raisonnable et un coût financier et technique abordable.

Background: Cell-free circulating DNA (cfDNA), which can be extracted by liquid biopsy, has been studied as a noninvasive biomarker for various diseases. The potential of cfDNA fragment size and level as a marker for low back pain (LBP) has never been studied. We investigated whether cfDNA is a biomarker of LBP severity in patients with a lumbar degenerative disease (LDD). Methods: Blood samples were obtained from patients with LDD (n = 21) before and immediately after spinal surgery. Plasma DNA was isolated and examined for cfDNA fragment size and concentration. A cohort of healthy volunteers (n = 5) constituted the control group.Results: The cfDNA fragment size tended to be shorter in patients than in healthy controls, but this difference was not significant (P = .224). cfDNA level was significantly higher in LDD patients (mean 0.642±0.199 ng/mL, range 0.302–1.150 ng/mL) than in healthy controls (mean 0.429±0.064 ng/mL, range 0.366–0.506 ng/mL) (P = .029). cfDNA level correlated positively with present pain (r = .421, P = .036), maximum pain (r = .419, P = .037), average pain (r = .566, P = .003), low back pain (r = .403, P = .041), leg pain (r = .480, P = .013), and leg numbness (r = .455, P = .020). cfDNA fragment size did not differ from before to after surgery, but cfDNA level increased postoperatively in patients with LDD. Conclusions: This was the first study investigating whether cfDNA fragment size and level are associated with pain, including LBP, in patients with LDD. Our findings suggest that cfDNA level may be an objective indicator of pain and surgical invasiveness in patients with LDD.

The European Society of Gynaecological Oncology (ESGO), the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG), the International Ovarian Tumour Analysis (IOTA) group and the European Society for Gynaecological Endoscopy (ESGE) jointly developed clinically relevant and evidence-based statements on the preoperative diagnosis of ovarian tumors, including imaging techniques, biomarkers and prediction models. ESGO/ISUOG/IOTA/ESGE nominated a multidisciplinary international group, including expert practising clinicians and researchers who have demonstrated leadership and expertise in the preoperative diagnosis of ovarian tumors and management of patients with ovarian cancer (19 experts across Europe). A patient representative was also included in the group. To ensure that the statements were evidence-based, the current literature was reviewed and critically appraised. Preliminary statements were drafted based on the review of the relevant literature. During a conference call, the whole group discussed each preliminary statement and a first round of voting was carried out. Statements were removed when consensus among group members was not obtained. The voters had the opportunity to provide comments/suggestions with their votes. The statements were then revised accordingly. Another round of voting was carried out according to the same rules to allow the whole group to evaluate the revised version of the statements. The group achieved consensus on 18 statements. This Consensus Statement presents these ESGO/ISUOG/IOTA/ESGE statements on the preoperative diagnosis of ovarian tumors and the assessment of carcinomatosis, together with a summary of the evidence supporting each statement.

Although it is widely accepted that cancer derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12 % of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20 % are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Circulating DNA, also named cell-free DNA (cfDNA), is a series of highly fragmented DNA that is present in the blood circulation and other body fluids of human and is free of extracellular. In certain situations, such as cancer patients, pregnant women, patients undergoing organ transplants, etc., a small number of circulating DNA from “heterologous” cells can be used as markers for genetic testing [1]. Large quantity of autologous cell-free DNA reflects abnormal cell death. Autologous cell-free DNA quantification can be used for the diagnosis and monitoring of related diseases. The “liquid biopsy” technology based on circulating DNA detection has received great attention in many aspects such as prenatal diagnosis, tumor screening, early diagnosis, and treatment monitoring, and prognosis evaluation. Because of its extremely low content, a very large number of studies have been done to develop various methods for quantitative detection [1, 2]. At the moment circulating DNA quantitative detection still faces challenges such as the lack of specification, precision, and standardization in detection processes.

It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that exosomes from healthy females (HF) or breast cancer patients (BCP) plasma contain less than 1.4 × 10−8 pg of DNA. Thus, only a minor part of cirDNA is attached to the outer side of the exosome as part of the vesicle crown: the share of exosomal DNA does not exceed 0.025% HF plasma DNA and 0.004% BCP plasma DNA. Treatment of plasma exosomes with DNase I with subsequent dot immunoassay reveals that H2a, H2b, and H3 histones are not part of the exosomal membrane, but are part of the cirDNA–protein macromolecular complex associated with the surface of the exosome either through interaction with DNA-binding proteins or with histone-binding proteins. Using bioinformatics approaches after identification by MALDI-TOF mass spectrometry, 16 exosomal DNA-binding proteins were identified. It was shown that four proteins—AIFM1, IGHM, CHD5, and KCNIP3—are candidates for DNA binding on the outer membrane of exosomes; the crown of exosomes may include five DNA-binding proteins: H2a, H2b, H3, IGHM, and ALB. Of note, AIFM1, IGHM, and CHD5 proteins are found only in HF plasma exosomes; KCNIP3 protein is identified only in BCP plasma exosomes; and H2a, H2b, H3, and ALB are revealed in all samples of plasma exosomes. Two histone-binding proteins, CHD5 and KDM6B, have been found in exosomes from HF plasma. The data obtained indicate that cirDNA preferentially binds to the outer membrane of exosomes by association with DNA-binding proteins.

Simple Summary Prostate cancer is a major cause of cancer-related death in men worldwide. There is an urgent clinical need for improved prognostic biomarkers to better predict the likely outcome and course of the disease and thus inform the clinical management of these patients. Currently, clinically recognised prognostic markers lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised, intermediate grade prostate cancer. Thus, there is major interest in identifying new molecular biomarkers to complement existing standard clinicopathological markers. DNA methylation is a frequent alteration in the cancer genome and offers potential as a reliable and robust biomarker. In this review, we provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis. We highlight advances in this field that have enabled the discovery of novel prognostic genes and discuss the potential of methylation biomarkers for noninvasive liquid-biopsy testing. Abstract There is a major clinical need for accurate biomarkers for prostate cancer prognosis, to better inform treatment strategies and disease monitoring. Current clinically recognised prognostic factors, including prostate-specific antigen (PSA) levels, lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised intermediate grade prostate cancer. There has therefore been a major focus on identifying molecular biomarkers that can add prognostic value to existing markers, including investigation of DNA methylation, which has a known role in tumorigenesis. In this review, we will provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis, and highlight the advances that have been made in this field. We cover the numerous studies into well-established candidate genes, and explore the technological transition that has enabled hypothesis-free genome-wide studies and the subsequent discovery of novel prognostic genes.

MicroDNAs are a type of extrachromosomal circular DNAs found both in cell nuclei and as cell-free circulating DNA, with links to cancer and genetic mosaicism. Research suggests that microDNAs originate from chromosomal DNA. To better understand the evolutionary role of microDNAs, it is of interest to determine if and how they interact with the chromosomal DNA. In particular, do microDNAs re-integrate back into the chromosomal genome? Given their circular form, if they do, this will lead to a specific form of repeat in the genome, which we term circular repeat. Due to the presence of mutations, these repeats are expected to be approximate. Motivated by this question, we develop an efficient ab initio algorithm for finding approximate circular repeats in a given genome. The algorithm consists of two main components. First, it performs a two-stage search to locate candidate circular repeat patterns by identifying their substrings. Second, it checks the validity of each candidate by inspecting the flanking sequences of the substrings. By applying our method to human genome chromosomes 21, 22, and Y, we find hundreds of approximate circular repeats. Our simulation shows that the patterns found are unlikely to be purely the result of inherent repetitive structure of the genome, thus suggesting that microDNAs reintegrate back into the genome.

Circulating DNA can be released in the biological fluids by a physiological process and by different pathological conditions. The first reports detecting circulating DNA in human plasma date from the late 40s. Even when specific pathological conditions were analyzed, the clinical importance of circulating DNA remained unclear. After PCR introduction, genetic and epigenetic alterations in circulating DNA gained more prominence for understanding the mechanisms of cancer development and progression. Nowadays, the circulating DNA assays are highlighted for their clinical relevance for cancer screening in liquid biopsy. In this review, we described the landscape of studies on circulating DNA isolated from human plasma or serum and the molecular tools used to obtain these findings throughout the 20th century and the current application in cancer.

Background To detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment. Methods The study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (κ) was used to estimate the agreement between these diagnostic tests compared to a combined “gold standard” of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall’s Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points. Results By using the combined “gold standard” of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time (which were determined by the serum-based real-time PCR) showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later. Conclusions The data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.

Short fragments of cell-free DNA are released into the plasma when cells die. In patients with cancer some of this circulating DNA is released by tumour cells; in pregnant women some is derived from the fetus; and increased amounts are found in many pathological conditions associated with cell death. In each of these circumstances, analysis of cell-free DNA can provide useful clinical information (e.g. detection or monitoring of cancer, determination of mutation status of a fetus). With further improvement in analytical technologies and developments of new markers, it is likely that the application of circulating cell-free DNA and cell-free RNA species in molecular diagnostics will increase in the future.

Background Endocrine therapy is recommended as a first-line treatment for hormone receptor-positive metastatic breast cancer (HR+MBC) patients. No biomarker has been validated to predict tumor progression in that setting. We aimed to prospectively compare the risk of early progression according to circulating ESR1 mutations, CA-15.3, and circulating cell-free DNA in MBC patients treated with a first-line aromatase inhibitor (AI). Methods Patients with MBC treated with a first-line AI were prospectively included. Circulating biomarker assessment was performed every 3 months. The primary objective was to determine the risk of progression or death at the next follow-up visit (after 3 months) in case of circulating ESR1 mutation detection among patients treated with a first-line AI for HR+MBC. Results Overall, 103 patients were included, and 70 (68%) had progressive disease (PD). Circulating ESR1 mutations were detected in 22/70 patients with PD and in 0/33 patients without progression (p ESR1-mutated patients, 18/22 had a detectable mutation prior to progression, with a median delay of 110 days from first detection to PD. The detection of circulating ESR1 mutations was associated with a 4.9-fold (95% CI 3.0–8.0) increase in the risk of PD at 3 months. Using a threshold value of 25% or 100%, a CA-15.3 increase was also correlated with progression (p p = 0.003, respectively). In contrast to ESR1, the CA-15.3 increase occurred concomitantly with PD in most cases, in 27/47 (57%) with a 25% threshold and in 21/25 (84%) with a 100% threshold. Using a threshold value of either 25% or 100%, cfDNA increase was not correlated with progression. Conclusion The emergence of circulating ESR1 mutations is associated with a 4.9-fold increase in the risk of early PD during AI treatment in HR+MBC. Our results also highlighted that tracking circulating ESR1 mutations is more relevant than tracking CA-15.3 or cfDNA increase to predict progression in this setting. Trial registration ClinicalTrials.gov, NCT02473120. Registered 16 June 2015—retrospectively registered after one inclusion (first inclusion 1 June 2015)

Genetic testing based on the analysis of circulating cell-free DNA (cfDNA) in body fluids, especially blood plasma, is raising interest for the management and follow-up of many diseases, including cancer. Because the concentration of cfDNA is low and its composition mostly degraded, this material can only be assayed with the most sensitive nucleic acid processing technologies. cfDNA analysis therefore constitutes a model target and a driving force for innovation in microfluidic biotechnologies. Here, we overview the main physico-chemical characteristics of cfDNA, and provide a critical review on the different methods for its processing out of blood samples. Then, we describe recent microfluidic developments for high sensitivity DNA analysis, evaluate their practical relevance for cfDNA analysis, and identify a few challenges for technologists in the near future. Keywords: Cell-free circulating DNA, Body fluid nucleic acid biomarkers, DNA separation, DNA concentration

The advent of targeted therapy in non-small-cell lung cancer (NSCLC) has made the routine molecular diagnosis of EGFR mutations crucial for optimal patient management. Obtaining tumour tissue for biomarker testing, especially in the setting of re-biopsy, can present many challenges. A potential alternative source of tumour dna is circulating cell-free tumour-derived DNA (CTDNA). Although CTDNA is present in low quantities in plasma, the convenience of sample acquisition and the increasing reliability of detection methods make this approach a promising one. The various performance characteristics of both digital and nondigital platforms are still variable, and a standardized approach is needed that will make those platforms reliable clinical tools for the detection of EGFR sensitizing mutations and resistance mutations, including the T790M resistance mutation. Information derived from ctdna can be used to assess tumour burden, to identify genomic-based resistance mechanisms, and to track dynamic changes during therapy.

We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice.

Background Patients with BC have a higher amount of cell-free circulating DNA (CFCDNA) in the blood and urine than healthy people. We aimed to verify if the Z-Scan method could analyze the concentrations of uDNA (urinary) and pDNA (plasma) in relation to the time of collection during treatment for patients with bladder cancer. Methods Peripheral blood and urine samples were obtained from 30 patients with BC at the time of diagnosis, 45, 90 and 180 days after initiating treatment; 5 μL of k-DNA (k = u or p) was added in 250 μL a solution of 1:1000 Ethidium Bromide dye (EtBr) in water. Continum laser Nd:YVO4, wavelength λ = 532 nm was used. Samples of uDNA and pDNA in water were submitted to the laser with an incident power of 84.5 mW and an exposure time of 30 ms. Results There was a different concentration of pDNA and uDNA during the treatment of patients using both optical techniques. However, the reaction rate of pDNA and uDNA was similar with spectrophotometry, whereas the z-scan technique presented different values. Conclusion Z-scan technique has potential for use in the differentiation of pDNA and uDNA concentrations, which are distinct in patients with BC and healthy people.

// Jessica Garcia 1, 2, 3, 4, ** , Eric Dusserre 1, 3, 4, ** , Valerie Cheynet 3, 5 , Pierre Paul Bringuier 6 , Karen Brengle-Pesce 3, 5 , Anne-Sophie Wozny 1, 7 , Claire Rodriguez-Lafrasse 1, 3, 4, 7 , Gilles Freyer 4, 8, 9 , Marie Brevet 4, 6, 9 , Lea Payen 1, 2, 3, 4, 7, * and Sebastien Couraud 4, 9, 10, * 1 Laboratoire de Biochimie et Biologie Moleculaire, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 69310, Pierre Benite, France 2 Centre de Recherche en Cancerologie de Lyon, INSERM 1052, CNRS 5286, Universite Claude Bernard Lyon 1, Lyon, 69003, France 3 Laboratoire Commun de Recherche Hospices Civils de Lyon – BioMerieux, Centre Hospitalier Lyon Sud, 69310, Pierre Benite, France 4 Institut de Cancerologie des Hospices Civils de Lyon, CIRculating CANcer Program (CIRCAN), 69002 Lyon, France 5 Medical Diagnostic Discovery Department, BioMerieux, 69290 Craponne, France 6 Service d’Anatomie et de Cytologie Pathologiques, Groupement Hospitalier Est, Hospices Civils de Lyon, 69500, Bron, France 7 Faculte de Pharmacie de Lyon (IPSB), Universite de Lyon1, Lyon, 69008, France 8 Service d’Oncologie Medicale, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France 9 EMR 3738 Ciblage Therapeutique en Oncologie, Faculte de Medecine Lyon Sud, Universite Lyon 1, 69600, Oullins, France 10 Service de Pneumologie Aigue Specialisee et Cancerologie Thoracique, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France * Authors share co-senior authorship ** Authors share co-first authorshop Correspondence to: Lea Payen, email: lea.payen-gay@chu-lyon.fr Keywords: lung cancer, EGFR mutation, circulating-free DNA, liquid biopsy, digital PCR Received: December 22, 2016 Accepted: June 26, 2017 Published: September 21, 2017 ABSTRACT Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.

// Jie Cheng 1, 2 , Katarina Cuk 1, 2 , Jorg Heil 3 , Michael Golatta 3 , Sarah Schott 3 , Christof Sohn 3 , Andreas Schneeweiss 3, 4 , Barbara Burwinkel 1, 2, * and Harald Surowy 1, 2, * 1 Division of Molecular Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, University of Heidelberg, Heidelberg, Germany 3 Department of Gynecology and Obstetrics, University Women’s Clinic, Heidelberg, Germany 4 National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany * These authors have share the last authorship Correspondence to: Barbara Burwinkel, email: B.Burwinkel@dkfz.de Keywords: breast cancer, recurrence, circulating DNA integrity, biomarker Received: October 11, 2016 Accepted: March 26, 2017 Published: April 24, 2017 ABSTRACT Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.

Since introducing the concept of epigenome-wide association studies (EWAS) in 2011, there has been a vast increase in the number of published EWAS studies in common diseases, including in cancer. These studies have increased our understanding of epigenetic events underlying carcinogenesis and have enabled the discovery of cancer-specific methylation biomarkers. In this mini-review, we have focused on the state of the art in EWAS applied to cell-free circulating DNA for epigenetic biomarker discovery in cancer and discussed associated technical advances and challenges, and our expectations for the future of the field.

DNA methylation is a well-studied mechanism of epigenetic regulation, which plays an important role in oncogenesis and tumor progression. Even at very early stage, cancer genome exhibits aberrant methylation patterns, such as hypermethylation and hypomethylation at different scales. The detection of abnormal methylation patterns with whole-genome bisulfite sequencing (WGBS) using circulating DNA from plasma has become a promising method for cancer diagnosis. In this study, Boruta, an extension of the random forest, was used to select important features (variables). Those selected features were used to establish a support vector machine (SVM) classifier for liver cancer diagnosis. As the results, a WGBS data set from hepatocellular carcinoma (HCC) patients was employed to show the improved performance of the proposed method for diagnosis.

Cetuximab (CTX) has been used to treat metastatic colorectal cancer (mCRC) with wild-type (wt) RAS and BRAF genes. Meanwhile HER2 amplification reportedly denoted CTX-resistant mCRC tumors. We investigated whether monitoring of HER2 amplification in circulating DNA allowed early detection of mCRC progression and CTX resistance. We analyzed HER2 amplification in circulating DNA at 8-week intervals using ddPCR from 36 patients with RASwt/BRAFwt mCRC, who progressed after CTX treatments between July 2015 and January 2018. Of the 36 patients, 5 (13.9%) exhibited dynamic fluctuations of HER2 amplification in plasma in the course of CTX treatment, of whom 2 were positive for HER2 amplification in matched tumor specimens at baseline (per FISH). All 5 primary sites were left side: 3 rectums and 2 descending colon. HER2 ratio fluctuations in circulating DNA not only reflected changes in tumor volume, but their obvious increases presaged CT-documented progress by an average lead time of 2 months. Interestingly, progression-free survival did not significantly differ between these 5 patients and those without HER2 amplification (HR 1.06, 95% CI 0.40–2.77, P = 0.909). Plasma HER2 amplification detected by ddPCR changed over time and predicted resistance to CTX, by an average lead time of 2 months. Further study is needed to validate our findings.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.