cfDNA Isolation Research Articles

CfDNA of feto-placental origin is not detectable in plasma of pregnant mares by optimised sexing-qPCR.

Non-invasive prenatal testing is a cell-free-DNA (cfDNA)-based genetic screening, well-established in pregnant women, utilising cfDNA of feto-placental origin (cffDNA) isolated from maternal plasma (Christiaens et al, Prenatal Diagnosis. 2021;41:1316-1323). Fetal aneuploidy is described as causative for equine pregnancy loss (Shilton et al, Scientific Reports. 2020;10:13314), suggesting cffDNA-screening in mares is diagnostically warranted. The aims of the current study were to (i) optimise isolation methodology and examine presence of cffDNA in maternal plasma and (ii) develop a non-invasive cffDNA-based test to determine fetal sex in pregnant mares. Peripheral blood (10ml) from pregnant mares, carrying fetuses of confirmed sex (D70-80:n=4; last trimester:n=15), non-pregnant mares (n=2) and geldings (n=4) was collected into Streck®tubes. Samples were double-centrifuged within 24hrs, and supernatant transferred into Eppendorf® DNA-low-bind-tubes. CfDNA was isolated from multiple plasma aliquots per individual (QIAamp, ccfDNA-kit), applying two different temperatures (56°C, 21°C) for the magnetic-bead-binding step of the protocol. CfDNA was quantified by fluorometry (Qubit®fluorometer, 1XdsDNA-HS-assay). Control DNA was isolated from adult peripheral blood mononuclear cells (PBMC) and fetal tissue. CfDNA-isolation resulted in a median yield of 0.99ng/ml plasma (n=80 isolations) and did not differ (P>0.05) between aliquots of one individual or gestational stages but was significantly higher (P=0.0037) with an incubation temperature of 56°C (n=40), compared to 21°C (n=40). Quantification of cfDNA (n=6) by automatedelectrophoresis (cfDNA-Screentape, Agilent® TapeStation) was in alignment with fluorometry. A qPCR was optimized and validated for GAPDH and the Y-sequence TSPY utilizing 0.1ng and 0.2ng of DNA (indicated by cfDNA-concentrations, isolated above) from adult PBMC (n=41) and fetal tissue (n=6) of confirmed sex. A correct female / male classification was achieved for 95.1% (0.2ng) and 87.2% (0.1ng) of adult and 100% of fetal (0.1ng) DNA. Sensitivity (adult:0.2ng:100%; 0.1ng:100%), specificity (adult:0.2ng:100%; 0.1ng:95.2%) and PPV (adult:0.2ng:100%; 0.1ng:92.8%) were high. Next, qPCR accuracy was examined utilizing plasma cfDNA of non-pregnant mares (n=2) and geldings (n=4). Sensitivity, specificity and positive predictive value (PPV) for Y-chromosomal detection were 100%. In women, a minimum of 2-4% cffDNA is deemed diagnostic for prenatal testing. To determine TSPY detection at this ratio, male DNA was diluted in female DNA and TSPY accurately found at 4-10% but not 2%-dilutions. Finally, cfDNA from the plasma of pregnant mares (n=19) was analysed. All samples amplified GAPDH, however all clinically confirmed male pregnancies (6 / 19) were TSPY-negative. In conclusion, cfDNA was isolated from equine plasma and the yield improved by incubation at 56°C during the magnetic-bead-binding. A highly sensitive sexing-qPCR was validated for equine adult, fetal and cfDNA, but cffDNA was not detected in plasma of pregnant mares carrying male fetuses. CffDNA was not clearly observed by electrophoresis, suggesting isolation of cfDNA was of maternal but not feto-placental origin.

Mutation detection of urinary cell-free DNA via catch-and-release isolation on nanowires for liquid biopsy

Cell-free DNA (cfDNA) and extracellular vesicles (EVs) are molecular biomarkers in liquid biopsies that can be applied for cancer detection, which are known to carry information on the necessary conditions for oncogenesis and cancer cell-specific activities after oncogenesis, respectively. Analyses for both cfDNA and EVs from the same body fluid can provide insights into screening and identifying the molecular subtypes of cancer; however, a major bottleneck is the lack of efficient and standardized techniques for the isolation of cfDNA and EVs from clinical specimens. Here, we achieved catch-and-release isolation by hydrogen bond-mediated binding of cfDNA in urine to zinc oxide (ZnO) nanowires, which also capture EVs by surface charge, and subsequently we identified genetic mutations in urinary cfDNA. The binding strength of hydrogen bonds between single-crystal ZnO nanowires and DNA was found to be equal to or larger than that of conventional hydrophobic interactions, suggesting the possibility of isolating trace amounts of cfDNA. Our results demonstrated that nanowire-based cancer screening assay can screen cancer and can identify the molecular subtypes of cancer in urine from brain tumor patients through EV analysis and cfDNA mutation analysis. We anticipate our method to be a starting point for more sophisticated diagnostic models of cancer screening and identification.

Abstract 3378: Rapid and efficient sample preparations for cell free DNA

Abstract The analysis of cell-free DNA (cfDNA) as a sensitive biomarker for cancer prognosis, diagnosis and monitoring resulted in a need for efficient and fast cfDNA isolation. The traditional cfDNA purification methods using chaotropic solvent to separate nucleic acids from plasma through column or magnetic bead purification processes are usually laborious and time consuming, involving multiple washing steps, and the residual organic solvent could be carried over to the elution solution that could cause downstream PCR inhibition. Herein, we developed a novel particle surface chemistry that can biochemically bind rapidly (<1min) and release nucleic acids rapidly (<1min) under different buffer conditions without needing chaotropic solvent. We further optimized the binding and elution buffer solutions that allow to isolate cfDNA from plasma within 5-10 minutes in the sample volumes from 1 ml to 4 ml. The real-time quantitative PCR indicated DNA extraction yields were comparable or slightly better than the traditional methods for both small and large volumes. The novel bead chemistry and the optimized binding and elution buffers are not only compatible to high throughput format but could also be applied to a variety sample types for rapid sample preparations. Citation Format: Zhiyang Zeng, Andrew Taft, Kasen Riemersma, Connor Fitzgerald, Mike Scurria, Spenccer Hermanson, Wenhui Zhou. Rapid and efficient sample preparations for cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3378.

Abstract 998: A blood-based extracellular vesicles DNA methylation signature for colorectal cancer screening

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer related deaths, accounting for an estimated 1.8 million new cancer diagnoses and over 880,000 deaths in 2018. Furthermore, CRC patients who are diagnosed at the most advanced (metastatic) stage is estimated to have the 5-year survival rate of 14%. Dramatically, studies shows that if it can be detected at an early stage when surgical removal is feasible, the 5-year survival rate significantly increases to 90%. Therefore, early detection for CRC with curative treatment intent is crucially important to significantly reduce the mortality and morbidity and even cure CRC. Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles of sub-micrometer size that are secreted by virtually all cell types. They contain hundreds of different proteins, thousands of intact RNA species, and double-stranded DNA (dsDNA) fragments that sample the entire human genome. Recent findings further corroborate the potential role of EVs in early cancer screening, cancer diagnosis, treatment selection and monitoring. In this study, we used lipid nanoprobe (LNP) for EV isolation and methylation-specific qPCR assay for detecting SEPT9 methylation to develop a minimal invasive CRC screening test. Firstly, we have successfully detected promoter methylation of cancer specific genes SEPT9 in EVs derived from both genomic DNA and EV DNA of the CRC cell line. Secondly, we isolated EV using LNP from plasma samples in a cohort of 19 CRC patients and 9 healthy donor controls. In parallel, we isolated circulating free DNA (cfDNA) from the same cohort of the plasma samples using commercially cfDNA isolation Kit. Then, SEPT9 methylation signature was analyzed by our quantitative real-time PCR assay in these 28 EV DNA samples and 28 cfDNA samples, respectively. In the 19 CRC samples, we identified 10 SEPT9 gene methylation samples from LNP-isolated EV DNA and cfDNA, respectively. In 9 healthy control samples, no SEPT9 methylation was detection in the EV-DNA samples, while one false-positive was detected with cfDNA. Thus, the sensitivity and specificity of EV-DNA in the detection of SEPT9 gene methylation from CRC were determined to be 53% and 100% respectively. In comparison, the sensitivity and specificity of cfDNA are 53% and 89%, respectively from this preliminary pilot study. This indicated that EV-DNA might be superior to cfDNA in detecting SEPT9 gene methylation for CRC screening. Citation Format: Sheng Zhang, Faming Wang, Mingxi Chen, Jeongyun Kim, Mengrou Lu, Hongzhang He, Siyang Zheng. A blood-based extracellular vesicles DNA methylation signature for colorectal cancer screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 998.

Rationale and Design of a Single-Arm, Phase 2, Multi-Center Study of Chemo-Immunotherapy Followed by Hypo-Fractionated RT and Maintenance Immunotherapy in Patients With Unresectable Stage III NSCLC: The DEDALUS Trial

Introduction/BackgroundThis single-arm, phase 2, multi-center, study aims to assess the safety and efficacy of a regimen of induction chemo-immunotherapy followed by de-intensified, hypo-fractionated thoracic radiotherapy (RT) given concurrently with durvalumab and maintenance durvalumab in patients with unresectable, stage III NSCLC. Material and methodswe will enroll 45 patients with unresectable stage III NSCLC, any PD-L1, deemed ineligible for concurrent CRT by a thoracic oncology multidisciplinary team, and candidate to sequential chemoradiation followed by durvalumab. ResultsPrimary endpoint is safety, defined by the incidence of grade 3 and 4 possibly related adverse events (PRAEs) within 6 months from the initiation of treatment. The secondary objectives are PFS and OS (median and 12 months). Ancillary endpoints are molecular response evaluated by cfDNA isolation baseline, after chemo-immuno RT and at progression, and radiomics analysis on CT scans at baseline and before maintenance. ConclusionDEDALUS phase 2 trial explores the safety and efficacy of a novel sequence of chemo-radiation (with de-intensified RT) plus the anti-PD-L1 agent durvalumab in patients with stage III unresectable NSCLC who are candidates to sequential chemoradiation plus maintenance immunotherapy.

Hypertensive disorders of pregnancy share common cfDNA methylation profiles

Hypertensive disorders of pregnancy (HDP) contribute substantially to perinatal morbidity and mortality. Epigenetic changes point towards cardio-metabolic dysregulation for these vascular disorders. In early pregnancy, epigenetic changes using cell free DNA (cfDNA) are largely unexplored. We aimed to investigate these in HDP between 11 and 14 weeks of gestation by analysis of cfDNA methylation profiles in patients with hypertensive disorders. We identified patients without chronic hypertension but with subsequent development of preeclampsia (PE) (n = 11), with chronic hypertension (HT) but without PE development (n = 14), and lacking both PE and HT (n = 422). We matched patients according to PE risk factors into three groups (n = 5 each group): (1) PE: no HT but PE development, (2) HT: chronic hypertension but no PE and (3) Control: no PE or HT. We successfully optimized our cfDNA isolation process prior to whole genome bisulfite sequencing. Analysis of cfDNA methylation changes indicate a common predisposition in PE and HT groups, chiefly of maternal origin. Assessment of significant differentially methylated regions and annotated genes point towards a common cardiovascular predisposition in preeclampsia and hypertension groups in the first trimester. We postulate the pivotal role of the maternal cardiovascular system in HDP, which is already evident in the first trimester.

Identifying the minimum concentrations of cell-free fetal DNA in maternal blood required for bovine fetal sexing using PCR

We evaluated the feasibility of cffDNA extraction from the maternal blood samples regarding the threshold concentrations required for fetal sexing in pregnant cattle by PCR. In four trials, we 1) compared the extraction efficiency of seven methods using freshly harvested plasma/blood of cows carrying male fetii (150–240 d gestation) bovine amelogenin (bAML) and Y-specific gene sequences, 2) identified the minimum amounts of spiked cffDNA needed for a PCR for fetal sexing, 3) determined the most optimal protocol among three commercial kits for cffDNA extraction from neat and spiked plasma samples (181–240 d gestation) for PCR detection of Y-specific sequence and 4) tested Y-specific sequence PCR on pregnant cows at different stages of gestation (60–150 versus 151–240 d pregnant). In these experiments, blood samples from unbred dairy heifers (Canadian Holstein, n = 10), pregnant dairy cows (Canadian Holstein, 60–240 d gestation, n = 25 with male fetii), and aborting beef cows (Angus cross, n = 5, 100–150 d pregnant) were used for DNA extraction, spiking, and PCR. Extracted DNA from the blood samples of unbred heifers (n = 5) and bull calves (n = 5) served as controls in all trials. In the first trial, DNeasy Blood and Tissue, Qiagen DSP Virus, and NucleoMag cfDNA isolation kits were relatively successful among seven methods to isolate cffDNA from freshly harvested maternal plasma/blood of pregnant cows. In trial 2, using serial dilutions of cffDNA from male fetii spiked in cow plasma samples, a positive and unambiguous detection by PCR targeting Y-specific sequence and bAML gene was observed when spiked cffDNA concentration in plasma was >31.3 pg/ml and >2 ng/ml, respectively. In the third trial, the DNeasy Blood and Tissue kit was most successful in extracting cffDNA spiked at the minimum concentrations in maternal plasma and subsequent PCR for Y-specific sequence. In our fourth trial, more cows in the second half (9/10) of gestation showed a positive Y-specific PCR outcome than those in the first half (3/9, Fischer's exact test; P < 0.05, 90%; CI: 55.5–99.75 vs 33%; CI: 7.5–70.1). In conclusion, we observed variability between different DNA extraction methodologies and stages of gestation results in the PCR for prenatal sexing. Thus, the current PCR methodologies are unreliable for detecting cffDNA in pregnant cows. Additionally, ≥10 (≥31.3 pg/ml of cffDNA) and ≥648 (≥2 ng/ml of cffDNA) copies of the whole fetal genome in bovine maternal plasma are needed for Y-specific PCR and bAML PCR, respectively.

The lncRNA gene copy number variation as a low-invasive marker of rectal tumor cell sensitivity to radiotherapy.

e15536 Background: Radiotherapy (RT) is a key component of rectal cancer (RC) treatment, however, cases of non-response to preoperative RT in patients are very common, which is associated with radioresistance of tumor cells, mediated by their molecular characteristics, including the expression level of long non-coding RNA (lncRNA). lncRNAs are longer than 200 bp, show specific expression to various incentives, regulate transcription by acting as molecular traps, and can serve as biomarkers. The number of genes encoding lncRNA is 2 times greater than the number of protein coding genes. However, the rapid degradation of lncRNA molecules in the extracellular environment makes this indicator unsuitable for low invasive diagnostics. The problem can be solved by switching to a more stable marker - gene copy number variation (CNV) of lncRNA, which can be determined in extracellular DNA (cfDNA) of blood plasma. Therefore, the aim of this study was to identify the relationship between CNV of lncRNA genes in cfDNA of blood plasma and the RT effectiveness for rectal tumors. Methods: We used cfDNA from blood plasma obtained before RT from 200 RC patients and from the blood plasma of 50 apparently healthy donors (without cancer, AHD). RT was carried out on Novalis TX linear accelerator (TFD = 54 Gy). Blood samples were separated into plasma and cell fraction by centrifugation. Isolation of cfDNA from plasma was carried out by the phenol-chloroform extraction method. Determination of lncRNA genes CNV ( XIST, HELLPAR, NEAT1, AC008124.1, AC016717.2, AC093297.2, AC097634.1, BX890604.1, CASC9, IQCH-AS1, LINC00662, LINC00707, LINC01089, LINC01468, LINC0154, MALAT0154, MUC20-OT1, PVT1, VASH1-AS1) was performed by Real-Time qPCR. Differences were assessed using the Mann-Whitney test (Bonferroni correction was used). Results: An analysis of RT results in 200 patients allowed dividing them into 2 groups: complete tumor regression (group 1, n = 120) and minor or no tumor regression (group 2, n = 80). In cfDNA of patients in group 1, a decrease (p &lt; 0.05) in the CNV of lncRNA XIST, HELLPAR, NEAT1, AC008124.1 and AC016717.2 genes was found by 2.5, 3.3, 2.0, 2.0, and 5.0 times, respectively, relative to the AHD. In cfDNA of patients in group 2, an increase (p &lt; 0.05) in the CNV of lncRNA XIST, HELLPAR, NEAT1, AC008124.1, LINC01089, LINC01547 and VASH1-AS1 genes by 1.9, 2.9, 3.5, 3.7, 2.6, 4.7 and 1.9 times was found respectively relative to the AHD. Differences (p &lt; 0.05) by 4.8, 9.7, 7.0, 7.4, 5.0, 3.1, 4.8 and 2.2 times were observed between 2 groups of patients in terms of CNV of XIST, HELLPAR, NEAT1, AC008124.1, AC016717.2, LINC01089, LINC01547 and VASH1-AS1 genes, respectively. Conclusions: Increased CNV of XIST, HELLPAR, NEAT1, AC008124.1, LINC01089, LINC01547 and VASH1-AS1 genes in blood plasma cfDNA is associated with low RT efficiency.

DELFI-L101: Development of a blood-based assay that evaluates cell-free DNA fragmentation patterns to detect lung cancer.

TPS3164 Background: Despite longstanding national recommendations, uptake of lung cancer screening in the US remains low. Barriers include access to lung cancer screening, costs, and concerns over potential harms like false-positives and radiation exposure. The DELFI technology evaluates fragmentation patterns of cfDNA using supervised machine learning to distinguish cancer from non-cancer [PMID30943338; 34417454; 31142840]. Methods: The DELFI-L101 is a case-control observational study (NCT04825834) prospectively enrolling at academic and community sites. Eligible participants (≥50 years of age) are individuals who currently smoke or previously smoked, with smoking histories of ≥20 pack-years. Individuals are ineligible if they had cancer treatment in the prior year or a history of hematologic malignancies or myelodysplasia. Cases are individuals with pathologically confirmed cancers (group A – lung, group C – non-lung). Controls are those without cancer (group B) as determined by low-dose computed tomography screening completed within 6 weeks of enrollment. Cases and controls are identified among enrollees from all participating sites. Total enrollment is estimated to be ̃2500 participants across all groups. Blood samples are collected at enrollment for DELFI analyses, which involves cfDNA isolation from plasma, low-coverage, whole-genome, next-generation sequencing, and machine learning methods. Clinical data (medical history, demographics, and diagnostic, surgery, imaging, and pathology reports, and/or other diagnostic information) are collected at enrollment and at 12 months post-enrollment. The primary objective is to train and test a classifier for the detection of lung cancer using the DELFI technology with other biomarkers and clinical features. Secondary objectives include the evaluation of classifiers to distinguish lung cancer from other cancers, modeling benefits and harms using performance estimates of these classifiers, and description of the analytical performance (eg, repeatability and reproducibility) of the DELFI technology and classifiers. The primary endpoint is the accuracy of lung cancer detection as measured by sensitivity, specificity, and the area under the receiver operating characteristic curve. Secondary endpoints include accuracy of tissue of origin classification, and adverse events associated with blood sample collection. The training and performance characterization of a DELFI classifier will further the development of an affordable, accessible, blood-based cancer detection tool with potential to overcome barriers to lung cancer screening. Clinical trial information: NCT04825834.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

Bronchoalveolar Lavage Fluid (BALF) as a Compartment for Donor-Derived Cell-Free DNA (dd-cfDNA) Assessment in Lung Recipients

<h3>Purpose</h3> Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute lung rejection. It is unknown whether BALF may represent a more sensitive compartment for dd-cfDNA analysis in lung recipients. Additionally, the relative contribution of the recipient or donor to the BALF cfDNA content remains uncertain. We sought to understand the feasibility of distinguishing dd-cfDNA in the BALF and determine the greatest contributor of BALF cfDNA. <h3>Methods</h3> The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with paired BALF and plasma samples (n=320 each). Total cfDNA was isolated from ∼1mL of sample using automated nucleic acid purification. The % cfDNA contributed by the donor vs. recipient was distinguished in plasma and BALF samples using a next generation sequencing (NGS) assay that measures single nucleotide polymorphisms (SNPs) followed by bioinformatics algorithms. Because these algorithms were developed on plasma, for each BALF sample the SNP typing from the paired plasma sample was used to verify the accuracy of the donor vs. recipient cfDNA contribution in the BALF. <h3>Results</h3> Applying standard QC filters, 168 BALF samples "passed" and generated a dd-cfDNA result while 96 "failed" and did not generate a dd-cfDNA result. 45 samples yielded insufficient total cfDNA to support NGS while 11 generated a potentially invalid dd-cfDNA result. To understand reasons underlying inability to estimate BALF dd-cfDNA, we compared the volume of BALF used for total cfDNA isolation and the total cfDNA yield from the BALF in passed vs. failed samples. The BALF volume was similar between these groups, however the total cfDNA yield was notably less among samples that failed (median total cfDNA 0.38ng/uL in failed vs. 1.42ng/uL in passed samples). Considering only passed BALF samples, the median (25^th, 75^th percentile) fraction of cfDNA contributed by the donor was 6.95% (2.40%, 14.00%), with the remainder contributed by the recipient. <h3>Conclusion</h3> Our data point to challenges in consistently generating dd-cfDNA data from the BALF and suggest the dilute nature of BALF may be a contributing factor. Additionally, these results indicate that the recipient, rather than the donor, is the largest contributor to the BALF cfDNA content potentially reflecting intragraft recipient immune cells.

Fluidic system with movable layers for all-in-one assay of cell-free DNA in blood

Detection of circulating cell-free DNA (cfDNA) requires multiple processes, including plasma separation, cfDNA isolation, and high-sensitivity detection. However, implementing such processes in a single system in a simple and effective manner is nontrivial. Here, we present a fluidic system for the isolation and detection of cfDNA from whole blood performed in a fully automated and effective manner. The system utilizes vertically mobile top and rotational bottom parts with multiple disposable chambers for sequential processes. The key components for plasma filtration are a sedimentation chamber that enables the dextran-enhanced sedimentation of red blood cells and a plunger chamber with a porous filter and a check valve. cfDNA in the plasma is captured by silica-coated magnetic beads in the plunger chamber, washed via the motion of the top and bottom parts, and then detected by real-time on-chip polymerase chain reaction. We vary several parameters, including the plunger speed, dextran concentration, and hematocrit concentration, to optimize the time and efficiency of the assay. The total assay time is 1.5 h with a detection limit of 42 ag/mL for a sample volume of 1.5 mL. Plasma and cfDNA recoveries are 53% and 28%, respectively. Our system is a useful tool for all-in-one assays in liquid biopsies of cancers and other diseases detectable by cfDNA.

Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material

The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.

Peptide-Affinity Isolation of Extracellular Vesicles and Cell-Free DNA From Human Plasma.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that carry DNA, RNA, and protein cargoes and are found in a variety of biofluids. EVs, along with cell-free DNA (cfDNA), have attracted interest as a source of biomarker material for liquid biopsy, a process in which a sample of body fluid is used for the detection or monitoring of disease. The Vn96 synthetic peptide facilitates the isolation of both EVs and cfDNA from multiple body fluids, including human plasma, placing it as a versatile tool for the capture of multiple biomarker materials for disease detection and/or treatment monitoring. In this chapter, we describe an optimized protocol for Vn96-mediated isolation of EVs and cfDNA from human plasma samples, as well as downstream methods for EV enumeration and DNA, RNA, and protein extraction from the material captured by Vn96 for use in biomarker discovery or detection.

Comparative Evaluation of Diagnostic Performance of Circulating free DNA by Conventional vs. Real Time PCR in Gallbladder Cancer

Background: Circulating free DNA (cfDNA) in serum/plasma has been studied as a promising biomarker in various pathologies, including cancer. However, there is no standardized method for the isolation and quantification of serum cfDNA. An effective and reliable method for isolation and quantification is of utmost importance before any clinical decision. The current study compares the conventional and real-time PCR methods to find any differences and concordance in cfDNA levels between the two methods and the diagnostic accuracy of cfDNA by each method. Methods: Serum sample was collected from 67 subjects, including 17 normal healthy individuals (control, n=17), 19 disease controls (cholecystitis, n=19), and 31 Gallbladder cancer patients (cancer, n=31) before any treatment for cfDNA quantification. Results: The cfDNA level did not differ significantly between two methods in both control and cholecystitis groups. In cancer group, cfDNA level was significantly (P &lt; 0.001) different and higher in real time PCR as compared to conventional PCR. There was no significant correlation between two methods in control (r=0.02, P = 0.937), cholecystitis (r=0.10, P = 0.697), cancer (r=-0.08, P = 0.657) and total cases (control + cholecystitis + cancer) (r=0.06, P = 0.622). The diagnostic accuracy of two methods was found similar (P &gt; 0.05) when assessed between control vs. cholecystitis (Z=0.85, P = 0.397), and control vs. total cases (Z=1.35, P = 0.177). However, the diagnostic accuracy of real time PCR was found significantly different and higher as compared to conventional PCR when assessed between control vs. cancer (Z=2.98, P = 0.003), and cholecystitis vs. cancer (Z=4.41, P &lt; 0.001). Conclusion: Quantitative real-time PCR method is of high accuracy, reproducibility, and time-effectiveness. The diagnostic accuracy of real-time PCR was higher compared to conventional PCR.

Comparison of Methods of Isolation and Quantification of Cell Free DNA from Plasma of Patients with Breast Cancer.

Objective&#x0D; Cell-free DNA (cfDNA) released in response to necrosis in cancer patients The objective of this study was to compare the efficiency of two commercial and two manual methods for cell-free DNA extraction. as well as to search for a method that is easy to extract the DNA from the plasma and cost-effective.&#x0D; Methods&#x0D; Plasma samples seven in number of patients of Breast Cancer was taken. We evaluated DNA quantity and its subsequent amplification obtained by four different cfDNA isolation methods; Modified Phenol Chloroform Isoamyl, Triton Heat Phenol, EpiQUik Circulating Cell-Free DNA isolation Easy kit” (EpiGentek) and “Nucleospin cfDNA kit”. Extracted DNA was quantified using Qubit and quantitative real-time PCR.&#x0D; Results&#x0D; Quantity of cf DNA varied between different extractions methods of a total of seven samples analyzed. The highest quantity was found from the samples extracted from the Nucleospin XS kit and the extraction efficiency was significantly higher in a pairwise comparison with the other three methods (p-value &lt;0.0001). The concentration of the cfDNA obtained by all four methods was assessed on a Qubit fluorometer. The concentration was higher for the Nucleospin ?MPC?THP?Epiquik kit The qPCR values were consistently higher for Nucleospin XS as compared to all others. This indicates good amplifiability of Nucleospin XS&#x0D; Conclusion&#x0D; We tested four methods of cf DNA extraction. In our hands, Nucleospin XS gave the best yield and amplifiability. It is a quick and cost-effective method and sensitive for quantification of cfDNA on Real-time PCR. Therefore, it is highly recommended for clinical use of plasma as a liquid biopsy.&#x0D;

High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR.ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.ResultsWe aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.ConclusionsWe propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

Longitudinal analysis of individual cfDNA methylome patterns in metastatic prostate cancer

BackgroundDisease progression and therapeutic resistance are hallmarks of advanced stage prostate cancer (PCa), which remains a major cause of cancer-related mortality around the world. Longitudinal studies, coupled with the use of liquid biopsies, offer a potentially new and minimally invasive platform to study the dynamics of tumour progression. Our aim was to investigate the dynamics of personal DNA methylomic profiles of metastatic PCa (mPCa) patients, during disease progression and therapy administration.ResultsForty-eight plasma samples from 9 mPCa patients were collected, longitudinally, over 13–21 months. After circulating cell-free DNA (cfDNA) isolation, DNA methylation was profiled using the Infinium MethylationEPIC BeadChip. The top 5% most variable probes across time, within each individual, were utilised to study dynamic methylation patterns during disease progression and therapeutic response. Statistical testing was carried out to identify differentially methylated genes (DMGs) in cfDNA, which were subsequently validated in two independent mPCa (cfDNA and FFPE tissue) cohorts. Individual cfDNA global methylation patterns were temporally stable throughout the disease course. However, a proportion of CpG sites presented a dynamic temporal pattern that was consistent with clinical events, including different therapies, and were prominently associated with genes linked to immune response pathways. Additionally, study of the tumour fraction of cfDNA identified > 2000 DMGs with dynamic methylation patterns.ConclusionsLongitudinal assessment of cfDNA methylation in mPCa patients unveiled dynamic patterns associated with disease progression and therapy administration, thus highlighting the potential of using liquid biopsies to study PCa evolution at a methylomic level.

Association of genes copy number in extracellular DNA in patients with prostate cancer and sensitivity to radiotherapy.

e15014 Background: Radiotherapy (RT) is one of the main treatments for prostate cancer (PC). The effectiveness of such therapy depends on the initial radioresistance of tumor cells, which is ensured by their certain molecular features, which include the genes copy number variation (CNV). Model experiments on cell cultures (obtained from surgical material) have shown that CNVs have high potential as predictors of RT sensitivity. However, this potential is limited by the high level of invasiveness in obtaining biomaterials. A possible solution to this problem lies in the transition to CNV study in the extracellular DNA (cfDNA) of blood plasma. The aim of the study was to screen predictors of radioresistant PC based on the genes CNV in cfDNA. Methods: The study included 400 patients with diagnosed PC (T2a-3bN0M0, st. II-III), 40 of them after RT had a state of biochemical relapse (RT was performed on a Novalis TX linear accelerator (Varian, USA) (TFDisoeff = 75 Gr), mean time to biochemical relapse 7.5 months). Blood samples were separated into plasma and cell fraction by centrifugation. Isolation of cfDNA from blood plasma was performed using a set of reagents “DNA-Plasma-M” (Russia). Determination of the relative CNV of 13 genes (CDK1, CCND3, CDKN1B, TP53, PTEN, BCL2, XRCC4, BAX, RBBP8, H2AX, BRCA2, RAD50, EP300) was performed using the Real-Time qPCR method. Differences were assessed using the Mann-Whitney test; the Benjamin-Hochberg correction was used to correct multiple comparisons. Results: In the group with biochemical relapse (n = 40), the CNV of genes CDK1, CDKN1B, RBBP8, XRCC4, BRCA2 and RAD50 was statistically significantly (p &lt; 0.05) higher by 2.0 times, 2.3 times, 2.1 times, 1.4 times, 2.4 times and 2.8 times, respectively, relative to the CNV of these genes in the cfDNA of the group without relapse (n = 360). Conclusions: Thus, it was found that the CNV of 6 genes (CDK1, CDKN1B, RBBP8, XRCC4, BRCA2 and RAD50) may be a potential molecular marker of radiosensitivity of prostate tumors. Based on the obtained data, a low invasive method for determining the prostate tumors sensitivity to RT has been developed.